Evaluation of Soluble Suppression of Tumorigenesis-2 (Sst2) & Endothelin -

1 in Patients with Metabolic Syndrome

By

Shahed Munther Mahmoud

Supervisor

Dr. Khaled Abdul-Aziz Ahmed

Co. Supervisor

Dr. Husni Shukri Farah

This Thesis was Submitted in Partial Fulfillment of the

Requirements for the master’s degree of Pharmaceutical Sciences

Deanship of Graduate Studies and Scientific Research

Amman Al-Ahliyya University

Faculty of Pharmacy

Amman, Jordan

August,2023

i
 Committee Decision

This Thesis entitled “Evaluation of Soluble Suppression of Tumorigenesis-2 (Sst2) &

Endothelin -1 in Patients with Metabolic Syndrome” was successfully defended by

Shahed Munther Mahmoud, Reg. No. 202027130 and approved on from the examination

committee listed as below:

Dr. Khaled Abdul-Aziz Ahmed

Supervisor and Head of the Committee

Faculty of Pharmacy, Al-Ahliyya Amman University

Dr. ………………………………….

External Examiner

-------------------------

……………………………………………………………………………….

Dr. ………………………………….

Internal Examiner

---------------------------

…………………………………………………………………………………………………

Al-Ahliyya Amman University

August,2023

ii
 DEDICATION

I would love to dedicate this work to my peers and pharmaceutical Researchers

who non-stop have been challenging all impossibilities to cure incurable ailments.

A special feeling of gratitude to my supervisor Dr. Khaled Abdul-Aziz Ahmed who

contributed to my learnings enormously.

Very special thanks to my hardworking Co. Supervisor Dr. Husni Shukri Farah who

never deprived me of her consistent instructions.

iii
 ACHNOWLEDGMENT

I would love to express my gratitude to my supervisor Prof. Dr. Khaled Abdul-Aziz Ahmed

who his presence gave me resilience and solace and never deprived me of his

feedbacks and points of views.

Many thanks to my CO. Supervisor Dr. Dr. Husni Shukri Farah who with patience guided

and encouraged me at times, I thought I would not be able to progress further.

To my family, thank you for motivating me in all of my goals and cheering me to pursue my

dreams. I am especially grateful to my parents, who supported me emotionally and

financially.

iv
 TABLE OF CONTENT

Committee Decision..................................................................................................................ii

DEDICATION.........................................................................................................................iii

ACHNOWLEDGMENT.........................................................................................................iv

TABLE OF CONTENT............................................................................................................v

LIST OF TABLES...................................................................................................................vi

LIST OF FIGURES................................................................................................................vii

LIST OF ABBRIVIATIONS................................................................................................viii

ABSTRACT..............................................................................................................................xi

PREFACE................................................................................................................................xii

CHAPTER ONE......................................................................................................................13

INTRODUCTION...................................................................................................................13

1.0 Historical Background of Metabolic Syndrome.............................................................14

1.1 Concept of Metabolic Syndrome from Different Health-Related Bodies....................16

1.2 Metabolic Syndrome International Incidence and Prevalence.....................................18

1.3 Prevalence of Metabolic syndrome in Middle East & Kingdom of Jordan.................21

1.4 Problem Statement............................................................................................................23

1.5 Significance of the Study..................................................................................................24

1.6 Study Aim..........................................................................................................................24

v
1.7 Study Objectives................................................................................................................24

1.8 Hypothesis/ Research Question........................................................................................25

CHAPTER TWO....................................................................................................................26

LITRATURE REVIEW.........................................................................................................26

2.0 Over view on Cardio-Vascular Disease (CVD)..............................................................27

2.1 Biology of Suppression of Tumorigenicity (ST2) & its Relationship with Heart........29

2.2 sST2 and its Biomarker Relationship with Cardiovascular Disorders........................33

2.3 Biomarker of ST2 in Other Clinical Settings.................................................................39

2.4 Biology of Endothelin-1 (ET-1) and its Relationship with its Receptors.....................39

2.5 ET-1 Relationship with Heart Diseases...........................................................................43

2.6 Endothelin- 1 and Hypertension......................................................................................45

2.7 Association between Hypertension and Metabolic Syndrome......................................47

CHAPTER THREE................................................................................................................52

MATERIALS AND METHODS...........................................................................................52

3.1 Purpose of this Research..................................................................................................53

3.2Methods of Application......................................................................................................53

3.3 Sample Preparation..........................................................................................................55

3.3.1 Serum Preparation.........................................................................................................55

3.3.2 Plasma Preparation........................................................................................................55

3.3.3 Urine Samples.................................................................................................................55

3.3.4 Cell Sampling..................................................................................................................55

3.3.5 Tissue Sampling..............................................................................................................55

vi
3.4 Sample Distribution Procedures......................................................................................56

3.5 Criteria and Lab Principles..............................................................................................57

3.6 How to Calculate Results..................................................................................................58

3.7 Precision Involvement.......................................................................................................58

CHAPTER FOUR...................................................................................................................59

RESULTS.................................................................................................................................59

4.1 Participants’ Socio-Clinical Characteristics...................................................................60

4.2 Correlation Analysis of Thyroid Function Tests, Lipids Profile, Fast Blood Sugar

and Chronic Diseases with ET-1 and Suppression of Tumorigenesis-2.............................62

4.3 Clinical Parameters Mean Difference Comparison between Healthy and Metabolic

Participants..............................................................................................................................64

CHAPTER FIVE.....................................................................................................................66

DISCUSSION..........................................................................................................................66

CHAPTER SIX........................................................................................................................75

CONCLUSION........................................................................................................................75

CHAPTER SEVEN.................................................................................................................78

LIMITATIONS & FUTURE STUDIES...............................................................................78

REFERENCES........................................................................................................................80

APPENDEXES......................................................................................................................104

‫الملخص‬......................................................................................................................................110

vii
 LIST OF TABLES

Table.1: Cells-Releasing ET-1 and ETA, ETB Receptors Expression………………...44

Table.2: Stimuli and Suppressors behind ET-1 Release…………………....…….…….47

Table.3 Equipment Provided Alongside the Kit………………….………...…….....

…...58

Table.4: Different Standards……………………………....………………………...

…...60

Table 5. Participants’ Socio and Clinical Characteristics……………………..……….65

Table 6. Correlation Analysis of Thyroid Function Tests, Lipids Profile, FBS and

Chronic Diseases with ET-1 and sST2-2……………………………...…………….

…….67

Table7. mean differences of clinical parameters between healthy and metabolic

participants ………………………………………………………………………………..69

viii
 LIST OF FIGURES

Figure (1.1) Mechanisms Manifesting Metabolic Syndrome Pathophysiology…….….20

Figure (2.1): Interaction of IL-33 with the Transmembrane Receptor of ST2L……...35

Figure (2.2): Pulmonary Arterial Hypertension (PAH)………………….…..

……….....41

Figure (2.3): ETA Receptor Located on VSMC……………………..

…………………...46

Figure (2.4): The Chief Mechanism Which Connects Insulin Resistance to

Hypertension and Hypertension to Metabolic Syndrome………………….………..

………………….55

Figure (3.1): Standard Diluent Distribution……………...…...……..…………….........60

ix
 LIST OF ABBRIVIATIONS

Acute Heart failure ……………………………………………………………..……(AHF)

American National Health and Nutrition Examination Survey…………..…

(ANHNES)

Body Mass Index…………………………………...…...…………………….………

(BMI)

Brain Natriuretic Peptide ………………………….....….………………….……….

(BNP)

Coronary Artery Disease …………………………………………………….………

(CAD)

Center of Disease Control and Prevention ………………………………..………...

(CDC)

Chronic Heart Failure …………………………………………...…..………………(CHF)

Conicity Index ……………………………………………..…………………..……….(CI)

Chronic Obstructive Pulmonary Disease ……………………..………………….

(COPD)

C-Reactive Protein…………………………………………………..………………. (CRP)

Central Tendency Index……………………..…………..…………………………... (CTI)

Cardio-Vascular Disease……………………..……………………………...……… (CVD)

Cardio vascular system …………………………………………...…………..……...(CVS)

Ejection Fraction……………………………...…………………..…………………... (EF)

Endothelin-1 ……………………………………………………………..……...……(ET-1)

Fast Blood Sugar ………………………………………….....………………………. (FBS)

x
Gastro-Intestinal Tract …………………………………………………….…………

(GIT)

G protein-coupled receptors ………………………………...………………..…...(GPCR)

Growth Stimulated Fibroblasts ……………………………………………………...

(GSF)

High Density Lipoprotein ……………………………………………………………

(HDL)

Heart Failure………...………………………...………………………………………. (HF)

International Diabetic Federation ………………………………………...….……...(IDF)

Interferon Gamma…………………………………..………...…………………… (IF-Nγ)

Ischemic Heart Disease………………………………......…………………………...

(IHD)

Idiopathic Pulmonary Fibrosis………………………………………………………. (IPF)

Left Ventricular ……………………………………………………..…...…………….

(LV)

Lipo-Poly-Saccharide …………...…………………………………………….……...(LPS)

Mid-Regional pro-Adreno-Medellin……………………………..……… (MR-pro-

ADM)

National Cholesterol Education Program’s Adult Treatment Panel III.

……………………………………………………………………………..(NCEP: ATP III)
Non-Esterified Fatty Acids ……………………………………………….…...…...(NEFA)

National Heart, Lung and Blood Institute……………………………………… (NHLBI)

Nitric Oxide……...………………………………………..…………………………... (NO)

N-Terminal pro-Brain Natriuretic Peptide………………………………. (NT-pro-

BNP)

Optimal Density ……………………………………………………….....….…………

xi
(OD)

Oxidized Low Density Lipoprotein……………………….……………..………. (ox

LDL)

Pulmonary Arterial Hypertension………………………………………………… (PAH)

Peroxisome-Proliferator Activated Receptors-γ …………………………..…….

(PPARγ)

Reactive Oxygen Species………………………………………...…………………...

(ROS)

Soluble Suppression of Tumorigenesis-2 ……………………………………………(Sst2)

Triglyceride……………………………………..……………………..……………… (TG)

Type-2 Diabetes ……………………………………………………………………(T2DM)

T-helper 2 cytokines cells …………………………………………...…………...…...(Th2)

Transmembrane Receptor ST2 Ligand …………………….……...…...……. (TM-

ST2L)

Tumor Necrosis Factor-α …………………………………………………..……...(TNF-α)

Triglyceride Glucose …………………………………………………..………..…...(TYG)

Vascular Smooth Muscle Cells………………………...……………………...…...

(VSMC)

Waist Circumference ……………………………………………………………...….

(WC)

World Health Organization …………………………………………………...........

(WHO)

xii
 Evaluation of Soluble Suppression of Tumorigenesis-2 (Sst2) &

Endothelin -1 in Patients with Metabolic Syndrome

By

Shahed Munther Mahmoud

Supervisor

Prof. Dr. Khaled Abdul-Aziz Ahmed

Co. Supervisor

Dr. Husni Shukri Farah

ABSTRACT

Background: Metabolic syndrome is greatly associated with metabolic biomarkers yet, role

of (s ST2), (ET-1) and their contribution to complexity of metabolic biomarkers must be taken

into account.

Objectives: To explore the correlation relation of SSt2 and ET-1 (CVD parameters) with

metabolic indexes (lipid profile and FBS) in patients with hypertension and heart disease then

later to investigate the SST-2 and ET-1 value correlation with development of hypertension

and heart disease.

Methods: A total of 88 patients with medium age of 40, majority male participants chosen.

The multiple responses analysis used to identify the type of the disorder, the spearman test

correlation used to establish correlation between metabolic biomarkers with Sst2 and ET-1.

xiii
The point biserial correlation test employed to establish any correlation between experience of

chronic disease (hypertension & heart disease) and value of Sst2 and ET-1.

Results: 74.3% and 71.4% of patients suffered from high blood pressure and cardiac

disorder, followed by 40% thyroid disorder and 8.6% diabetes. A positive correlation was

between metabolic biomarkers (LDL, TG, FBS, TSH, FT4) with SSt2 and ET-1 however, the

HDL showed an inverse significant correlation. Moreover, a significant positive correlation

was detected between experience of hypertension and heart diseases with higher values of

SSt-2 and ET-1.

Conclusion: This study established positive correlative relation between metabolic

biomarkers with high value of Sst2 &ET-1. This study established positive relation between

experience of hypertension and heart diseases with higher values of SSt-2 and ET-1.

xiv
 ‫تقييم التثبيط القابل للذوبان في تكوين األورام ‪( 2-‬‬
‫‪ )Sst2‬والبطين الداخلي ‪ 1-‬في المرضى الذين يعانون‬
‫من متالزمة التمثيل الغذائي‬

‫‪ :‬إعداد‬
‫شهد منذر محمود‬

‫‪ :‬المشرف الرئيسي‬
‫د‪.‬خالد عبد العزيز‬

‫‪ :‬المشرف المشارك‬
‫د‪.‬حسني فرح‬

‫الخلفية‪ :‬ترتبط متالزمة التمثيل الغذائي ارتباًطا وثيًقا بالواصمات الحيوية األيضية ‪ ،‬ولكن يجب أن يؤخذ‬
‫دور (‪ )ET-1( ، )s ST2‬ومساهمتها في تعقيد المؤشرات الحيوية األيضية في االعتبار‪.‬‬

‫األهداف‪ :‬الستكشاف عالقة االرتباط بين ‪ SSt2‬و ‪( ET-1‬معلمات ‪ )CVD‬مع مؤشرات التمثيل الغذائي‬
‫(ملف تعريف الدهون و ‪ )FBS‬في المرضى الذين يعانون من ارتفاع ضغط الدم وأمراض القلب ‪ ،‬ثم في‬
‫وقت الحق للتحقيق في ارتباط قيمة ‪ SST-2‬و ‪ ET-1‬مع تطور ارتفاع ضغط الدم وأمراض القلب‪.‬‬

‫‪xv‬‬
‫الطريقة‪ :‬ما مجموعه ‪ 88‬مريضا بمتوسط عمر ‪ ، 40‬تم اختيار غالبية المشاركين الذكور‪ .‬يستخدم تحليل‬
‫االستجابات المتعددة لتحديد نوع االضطراب ‪ ،‬وهو ارتباط اختبار سبيرمان المستخدم إلنشاء ارتباط بين‬
‫المؤشرات الحيوية األيضية مع ‪ Sst2‬و ‪ .ET-1‬اختبار االرتباط الثنائي النقطي المستخدم لتحديد أي‬
‫ارتباط بين تجربة المرض المزمن (ارتفاع ضغط الدم وأمراض القلب) وقيمة ‪ Sst2‬و ‪.ET-1‬‬

‫النتائج‪ ٪74.3 :‬و ‪ ٪71.4‬من المرضى يعانون من ارتفاع ضغط الدم واضطراب القلب ‪ ،‬يليه ‪٪40‬‬
‫اضطراب الغدة الدرقية و ‪ ٪8.6‬السكري‪ .‬كان هناك ارتباط إيجابي بين المؤشرات الحيوية األيضية (‬
‫‪ )LDL ، TG ، FBS ، TSH ، FT4‬مع ‪ SSt2‬و ‪ ، ET-1‬ومع ذلك ‪ ،‬أظهر ‪ HDL‬ارتباًطا معنوًيا‬
‫عكسًيا‪ .‬عالوة على ذلك ‪ ،‬تم الكشف عن ارتباط إيجابي معنوي بين تجربة ارتفاع ضغط الدم وأمراض‬
‫القلب مع ارتفاع قيم ‪ SSt-2‬و ‪.ET-1‬‬

‫الخالصة‪ :‬أنشأت هذه الدراسة عالقة ارتباط موجبة بين المؤشرات الحيوية األيضية ذات القيمة العالية لـ‬
‫‪ .Sst2 & ET-1‬أسست هذه الدراسة عالقة إيجابية بين تجربة ارتفاع ضغط الدم وأمراض القلب مع‬
‫ارتفاع قيم ‪ SSt-2‬و ‪.ET-1‬‬

‫‪xvi‬‬
 PREFACE

The following thesis is written according to Al-Ahliyya Amman University

Guidelines outlining the thesis manual of the Deanship of Postgraduate and Scientific

Research, 2023.

xvii
CHAPTER ONE

INTRODUCTION

18
1.0 Historical Background of Metabolic Syndrome

The disorder of metabolic syndrome is dated back to 80 years. In 1920s, a Swedish

medics known as Kylin for the first time addressed the concept of metabolic syndrome,

depicted it as combination of couple of disorders including; hypertension, hyperglycemia and

gout disease, leading to an imminent threat of cardiovascular disorder (Samson, & Garber,

2014). Subsequently, in 1947, another physician known as Vague pointed at upper body

adipose mass known as android or male-phenotypic obesity with ability to trigger metabolic

complications, leading to type-2 diabetes and cardiovascular complications (Eckel et al.,

2010).

In 1988, insulin resistance was suspected to be the driver behind Cardio-Vascular

Disease (CVD), on top of type-2 diabetes (T2DM) and Reaven (1988) argued that insulin

resistance mostly would be in company with other metabolic abnormalities, hence hailed the

consolation as metabolic syndrome X and suggested that the syndrome would encompass,

abnormal parameters of hyperlipidemia, hyperinsulinemia, hyperglycemia, hypertension,

causing cardiac disorders even without co-occurrence of diabetes (Cheng, 2007).

In last decades, worldwide the numbers of people who suffer from symptoms of

metabolic syndrome have widely intensified. Such intensification is corresponded to global

outbreak of obesity and diabetes which also, have caused cardiovascular symptoms with high

rate of death toll (Samson, & Garber, 2014).

The complication of the syndrome is due to its asymptomatic nature as it can express

itself in assorted modes based on heterogeneity of parameters which generating the syndrome,

hence, there has always been a global campaign to bring awareness on the gravity and

sensitivity of this disguised disorder with the greatest rate of morbidity from cardiac point of

view (Swarup et al., 2022).

19
 A diverse combination of metabolic deviations entailing glucose intolerance, insulin

insensitivity, insulin resistance, impaired glucose tolerance, impaired fasting glycaemia,

hyperglycemia followed by abdominal/ android obesity, hyperlipidemia and high level of

blood pressure all have been introduced as the diagnostic risk factors behind cardiovascular

disorder (Lopes et al., 2016; Rondanelli et al., 2020).

Study by Després, (2012) also, accentuated on the significance of central obesity

known as abdominal fatness in conjunction with hypertriglyceridemia waist appearance as

underlying parameters of metabolic syndrome behind cardiovascular disorder. Considering

the fact that Metabolic syndrome is still treated as an enigmatic disorder with elaborate

mechanisms, justifies the debate that its inducing parameters whether or not forge

independently on their own or constitute under a greater pathogenic pathway (Dizaji, 2018).

Meanwhile, genetic and epigenetic interferences, life style factors entailing; poor diet,

sedentary and lack of mobility have also been attested to be as underlying triggers behind the

syndrome. In particular, dense-caloric food has specifically singled out as pivotal trigger

behind invocation of pathways, leading to metabolic syndrome due to accumulation of

visceral adiposity (Pekgor et al., 2019). Among broad diversity of etiologies behind the

metabolic syndrome, it seems, insulin resistance, chronic inflammation and neuro-hormonal

activation are perceived to have imperative roles in the proliferation of the syndrome and its

ensuing transition into diabetes and cardiac disorders (Fig1.1) (Fahed et al., 2022).

20
 Figure (1.1) Mechanisms Manifesting Metabolic Syndrome Pathophysiology; Source:
Fahed et al., (2022).

1.1 Concept of Metabolic Syndrome from Different Health-Related Bodies

Metabolic syndrome is a series of metabolic irregularities which lead to risk of

vascular failures causing cardia arrest or stroke. The syndrome itself has been addressed

through heterogeneous names entailing; insulin resistance syndrome, syndrome X, syndrome

X-plus, deadly quartet and others (Fahed et al., 2023).

Although, there are still controversies surrounding metabolic syndrome causes, yet in

1998 there was an international consensus on a globally-acknowledged definition proposed by

World Health Organization (WHO) as a set of introduced criteria to guide clinicians and

21
researchers to detect the syndrome better (Wang et al., 2020). In its following years, the

National Cholesterol Education Program’s Adult Treatment Panel III (NCEP: ATP III) as well

as European group for the study of insulin resistance came up with conceptualized definitions,

consenting on crucial parameters of hyperglycemia, hypertension, hyperlipidemia and obesity

as the leading cause of the syndrome (Rafey, 2021). However, the detailed of the standard was

differed between these entities as WHO and European group for the insulin resistance study

had mutual consent on calling insulin resistance or intolerance as underlying parameter

behind metabolic abnormality (Nwankwo et al., 2022). On the contrary, for NCEP-ATP III

such principles were not incorporated (Subramani et al., 2019).

Considering differences in definitions, the WHO definition was more suitable for

research purposes as opposed to NCEP-ATP III definition which was more tailored for

clinical applications, due to the fact that the latter one definition seemed to be more primitive

and easy to apply in clinical assessment of patients (Rivadeneyra-Domínguez et al.,2023).

There was also, a new definition by American association of endocrinology who

implied that to diagnose the metabolic syndrome leading to cardiac complication, all

collaborative parameters of heightened triglyceride, lowered high density lipoprotein,

amplified low-density lipoprotein, increased blood pressure, risen fasting blood sugar and

highly loaded blood sugar had to be taken into account. Clearly, in American definition, the

component of obesity was excluded despite, the rising empirical evidence on abdominal

obesity introduced concentrated centered body fat as the chief contributor behind risk of type-

2 diabetes and cardiac disorders (Dabke et al., 2019).

Such discrepancy in definitions has made it elusive to gauge the syndrome prevalence

its etiologies and its health outcomes among different countries (Wang et al., 2020). Later,

International Diabetic Federation (IDF) followed by National Heart, Lung and Blood Institute

(NHLBI) and the American Heart Association, introduced a novel definition, immersing

22
abdominal adipose and emphasizing on obesity as the critical contributor behind

hypertension, diabetes, cardiovascular disease, gout and gallstone complications as well as

some certain carcinomas, which later was attested that the degree of obesity leading to risk

factors behind metabolic syndrome would be varied among different ethnicities based on

geographic location and factor of age, gender and genome composition (Bovolini, 2019), such

discrepancy forced international association for the study of obesity in company with world

health organization to revise the set-up criteria for abdominal obesity and the body mass

index (BMI) addressing Asian over-weight and obese status versus Western population

( former: women 80>cm , men 90cm >, latter: BMI >23 over-weight, BMI>25 obesity for

Asians) ( Bovolini, 2019).

1.2 Metabolic Syndrome International Incidence and Prevalence

Although, a unanimous consensus has been reached on different definitions of

metabolic syndrome, yet still its comparison incidence at global level and its worldwide

prevalence based on published articles are a daunting task due to availability of diverse

criteria which each has defined the underlying risk factors behind the metabolic syndrome

differently and in accordance to diverse geographical locations, age, gender, genomic type and

life style factors (Reinehr et al., 2007). For example; Iranian national survey in 2020 revealed

prevalence of metabolic syndrome by 41% in accordance to National Cholesterol Education

Program’s Adult Treatment Panel III while, it seemed to be a little bit more up to 44% in

accordance with International Diabetic Federation criteria and 41% based on national heart,

lung and blood institute (Bahar et al., 2020).

However, in other Middle Eastern countries like Tunisia, the incidence of metabolic

syndrome between different age and gender were chronologically 23% and 95% in

accordance to National Cholesterol Education Program’s Adult Treatment Panel III while, it

was 36% and 95% in accordance to International Diabetic Federation criteria (Daouas et al.,

23
2022). Meanwhile, the only common sense was the fact that females more than males were

afflicted. The occurrence of metabolic syndrome is often in association with occurrences of

obesity and diabetes (Fahed et al., 2022). However, obesity is not always leading to metabolic

syndrome. Indeed, there are metabolically healthy obese people who have none of metabolic

syndrome –induced parameters of hypertension, hyperglycemia, insulin resistance,

hyperlipidemia and others. Indeed, studies implied that substantial portion of obese population

are metabolically healthy without signs of metabolic syndrome (Stefan et al.,2013; Engin,

2017).

According to the Center of Disease Control and Prevention (CDC), USA encountered

three-folds higher increment in prevalence of metabolic syndrome and the prevalence of 50%

has been associated with obesity and diabetes which both are highly related to the high

occurrence of cardio-vascular disease in particular in severely obese younger generation

(Moore et al., 2017; Saklayen, 2018). In accordance with CDC- published statistics in 2017

close to 30 million of American young adults (12%) were diagnosed for type-2 diabetes which

surprisingly, ¼ of them had no clue of their situation and its prevalence intensified by age

(>65), concerning 25% of American senior population. Furthermore, incidence of pre-diabetes

or signs of metabolic syndrome was reported 3 folds higher among American adult

population.1

Furthermore, in accordance to American National Health and Nutrition Examination

Survey (NHANES), from the birth of metabolic syndrome around 1988 till 2010, body mass

index of Americans on average heightened by 0.37% annually in both gender while Waist

Circumference (WC) rose up to 0.37 % in men as compared with women which was estimated

at 0.27% per year (Yi et al., 2017).

Addressing parameter of ethnic origin on the metabolic syndrome studies by ford et

al., (2002); O'Bryant et al., (2022); Zolfagharian et al., (2020) demonstrated lower percentage
1
National Center for Health Statistics (US), & National Center for Health Services Research. (2010).

24
in non-Hispanic white population in contrast to Mexican-Americans as well as African-

Americans. There is also, the parameter of age which influences metabolic syndrome as in

countries like Iran and France in younger population the prevalence is less than 10% as

compared with elderly (>60) which is 70% (Ebrahimi et al.,2011; Eckel et al.,2005).

However, despite metabolic syndrome has been addressed as disease of adults and

senior population, yet, due to parameter of obesity and its rising pace in younger population, it

is evident that the syndrome can move into a much younger age group regardless of ethnicity,

hence the diagnosis of cardiac arrest and diabetes will inevitably occur in the younger

population (Cook et al., 2008).

Concerning parameter of ethnicity, the incidence of diabetes seemed to be greater

among some certain groups as for instance, it was reported as high as 15% among American-

Indians yet, as low as 4.3% among Chinese- Americans. In addition, south Asian- Americans

were diagnosed for a higher possibility of metabolic syndrome incidence followed by greater

incidence of central obesity (Palaniappan et al., 2010).

Meanwhile, study by Swarup et al (2020) demonstrated a newly established data

suggesting that in accordance to (NHANES), the cases of metabolic syndrome have been

falling down by 24% in USA males followed by 22% in females.

Studies on Chinese cases concerning prevalence of obesity as the most concerning

risk factor of metabolic syndrome argued that between (1992-2002) obesity occurrence

intensified from 15 to 22% in accordance to WHO criteria however, applying Chinese obesity

definition standard which generally, stands for lower BMI, the increase in rate of obesity was

up to 30%. Such high rate of obesity resulted in up to 10% increase of metabolic syndrome

cases in urban areas versus 5% increase in countryside and it continued with prevalence of

15% up to 2017 (Wang et al., 2007).

25
 Study on Tai adolescent students as well as Chinese students (6-18 ages) demonstrated

that students who had diabetes, concurrently suffered all metabolic syndrome parameters of

hypertension, higher BMI, higher rate of cholesterol and hyperglycemia as opposed to those

with normal fasting sugar. The same study also, confirmed higher diagnosis of metabolic

syndrome in the school students (Wei et al., 2003; Pan et al., 2008).

1.3 Prevalence of Metabolic syndrome in Middle East & Kingdom of

Jordan

Middle East region is well-renowned for its high prevalence in metabolic syndrome. In

accordance to meta-analysis of 59 reviewed cross sectional studies by Ansarimoghaddam et

al., (2018) it was demonstrated that metabolic syndrome percentage was up to 4% in Turkey,

up to 40% in Saudi Arabia, up to 63% in Pakistan, in Qatar was up to 33%, in Kuwait up to

36% in Emirate up to 55%, in Iran up to 42% and up to 23% in Yemen which such high

occurrence of metabolic syndrome inevitably perceived to create vascular failures leading to

cardiovascular disease, coronary heart disease and stroke with high prevalence of 16%, 12 and

16% respectively.

Another study on non-western population, based- Iran argued that low level of

physical activity, in addition to, poor level of education and family history of chronic diseases

resulted in high risk of metabolic syndrome in 14 percent of participants (Kelishadi et al.,

2008).

Another case study in Saudi Arabia focused on the relation of diet and metabolic

syndrome and established a high prevalence of metabolic syndrome in particular greater in

women than men population due to deficiency of calcium, zinc, magnesium followed by A, C,

E and K (Al-Daghri et al., 2013).

Another study by Al Suwaidi et al., (2010) focused on prevalence of metabolic

syndrome in patients with acute heart coronary failure, concerning some middle east countries

26
which results demonstrated that metabolic syndrome is highly widespread among women who

suffering acute coronary syndrome.

In regard to prevalence of metabolic syndrome in Jordan, study by Obeidat et al.,

(2015) reported 51% of adults in both gender suffered from the disorder. The

featuring risk factors were estimated as follow, 71% for waist circumference, 46% for

hypertension, 42% for high FBS, 43% for low High Density Lipoprotein (HDL), followed by

50% for total TG. Indeed, the study confirmed that the prevalence of metabolic syndrome was

absolutely high in the kingdom thus, national screening was recommended in order to

decrease the incidence of type-2 diabetes as well as, CVD.

Another study by Ajlouni et al., (2020) concentrated on metabolic syndrome and

percentage of its key contributing risk factors through two different guidelines of adult

treatment panel as well as, international diabetes federation criteria. Compiled data from 2017

CVD risk factor survey patients established that in accordance to IDF guideline, both gender

with prevalence of 48% suffered from metabolic syndrome, as compared with ATP guideline

which just established the prevalence of 44%. This study established that socio-demographic

factors of age, gender, profession, geographical region and marital status are substantially

associated with metabolic syndrome. This study also, concluded that the prevalence of

metabolic syndrome is absolutely high in kingdom of Jordan and it is on the rise, thus,

changes in life style is essential such as; targeting dietary patterns of Jordanian citizens with

high quality foods followed by physical activity.

Another study by Al-Shami et al., (2022) through anthropometric measures of BMI,

WC, Conicity Index (CI), BSI, Body Roundness Index (BRI) determined the prevalence of

metabolic syndrome among Jordanians between the year 2018-19 period. The study

confirmed that obese men and women both demonstrated higher percentage in metabolic

syndrome, which confirmed at 42%. This study argued that among all employed

27
anthropometric measures, the best trustworthy measures would be waist to height ratio as well

as body roundness index to anticipate the early detection of metabolic syndrome among

Jordanian adults, in particular, due to the fact that these indexes are affordable, thus, can be

relevant in detecting and deterring metabolic syndrome.

1.4 Problem Statement

Nowadays, metabolic syndrome is multiple risk factors, leading to chronic diseases

such as; Atherosclerotic cardiovascular disorder, followed by type 2 diabetes. Indeed,

metabolic syndrome covers all conditions of insulin resistance, heightened glucose, high

blood pressure, pro-inflammatory condition, pro-thrombotic condition, atherogenic

dyslipidemia. However, the primary environmental and genetic antecedents behind metabolic

syndrome are excess energy consumption followed by inherited obesity and bad life style

which all can be altered.

Long has been perceived that insulin resistance is the imperative pathophysiology in

metabolic syndrome, since all the diagnoses under name of metabolic syndrome are all

associated with insulin resistance. Nevertheless, despite, insulin resistance is the onset of type

2 diabetes or it is aggressively related to atherogenic dyslipidemia alongside, pro-

inflammatory disorders, still there is less related to hypertension, however, studies on cardio

vascular disorders suggested that hyperinsulinemia or insulin resistance are independent risk

factors for CVD, yet, such relation requires further clinical investigations (Silveira Rossi et

al., 2022).

Obesity is also, entertained as key contributor in metabolic syndrome since it is

responsible for hyperglycemia, hypertension, high level of cholesterol, low level of HDL,

insulin resistance which all are greater risks for CVD (Silveira Rossi et al., 2022). The

contribution of obesity type and fat composition in particular, fat distribution inside body for

28
instance, central fat as compared with intra-abdominal fat or subcutaneous can have higher

risk of metabolic syndrome (Silveira Rossi et al., 2022).

When it comes to risk factor of obesity, application of drug therapy as well as,

bariatric gastric surgery can all be helpful in reversing the obesity risk factor behind

metabolic syndrome (Swarup et al., 2022).

In clinical and epidemiological studies, obesity is the major risk factor for CVD.

However, the mechanism which corresponds the obesity with metabolic syndrome is not fully

comprehended, yet, studies suggest that endothelial dysfunctions, life style changes and

particular gene composition all can contribute to obesity and later CVD (Kaur, 2014).

Most patients who are diagnosed for metabolic syndrome are subjected to an obese or

sedentary life due to the fact that city life reinforced by processed food easy access are major

drivers behind such metabolic disorder, and can be amplified by growing in age or genetic

complications. Thus, it makes it quite justifiable that the primary intervention target should

focus on persons’ life style, dealing with calorie counts, calorie restriction, dietary

improvement such as; food quality side by side increment in physical activity (Lim et al.,

2021), in order to reverse the metabolic indexes as the initiator of hypertension and CVD.

1.5 Significance of the Study

Most patients who are diagnosed for high metabolic indexes are subjected to an

obese or sedentary life, thereby this study signifies the fact that primary intervention should

focus on persons’ life style, dealing with calorie counts, calorie restriction, dietary

improvement such as; food quality side by side increment in physical activity.

1.6 Study Aim

In this thesis, it is aimed to explore the correlation relation of SSt2 and ET-1 (CVD

parameters) with metabolic indexes (lipid profile and FBS) in patients who suffer

29
hypertension and heart disease then later to investigate the SST-2 and ET-1 value correlation

with development of hypertension and heart disease.

1.7 Study Objectives

To investigate that how high Soluble Suppression of Tumorigenesis-2 (Sst2) would

lead to higher possibility of CVD [ heart failure, hypertension].

To investigate that how higher level of Endothelin-1 (ET-1) would lead to

hypertension followed by heart and vascular failure.

1.8 Hypothesis/ Research Question

Do higher level of SSt2 and ET-1 will correlatively associated with metabolic

syndrome indexes, leading to development of hypertension and CVD?

30
 CHAPTER TWO

LITRATURE REVIEW

31
2.0 Over view on Cardio-Vascular Disease (CVD)

CVD is the major induction of morbidity around the globe and their occurrence is on

the rise in both developed and developing societies (Fauci et al., 2005; Ahern et al., 2011)

which substantially has attenuated the individual quality of life and overall life expectancy.

In order to avert the negative outcomes of CVD both primary and secondary

preventive measures should be taken into account which have pushed physicians, seeking for

tools in order to inspect the advent of CVD progress to diminish the negative ramifications.

Among reliable biomarkers these two following known as; “Carotid intima-media thickness”

and “brachial artery flow-mediated dilatation” are well-meticulous biomarkers which can be

relied on in early detection of atherosclerosis and Chronic Heart Failure (CHF) (Ciccone et

al., 2011).

In addition, in order to identify the onset of any change and alteration in Cardio

vascular system (CVS), application of bio-chemical markers accompanied by ultra -

sonographic devices are very much helpful (Ciccone et al., 2011). In another study by Ciccone

et al., (2013), he argued that in order to identify the development of acute/chronic heart

coronary disease, the biomarker of osteoprotegerin and its role is instrumental.

Furthermore, it has proven by Thygesen et al., (2012) that biomarkers of Brain

Natriuretic Peptide (BNP) and (NT-pro-BNP) known as; Natriuretic Peptides are the most

reliable biomarkers of Heart Failure condition (HF). Meanwhile, all above-mentioned

biomarkers suffer one common ground which is limitation in their application or accurate

detection which have coerced the researchers to seek for more precise biomarkers addressing

both CVD and HF conditions (Ciccone et al., 2013).

More studies have proven that in cardiac remodeling, which is the initiation of CVD,

some certain heterogeneous biochemical pathways get invoked, which result in secretion of

some certain chemicals into circulation. The release of these chemicals are underlying since

32
their early catch can diagnose the beginning of any Coronary Artery Disease (CAD) and HF

(Baba et al., 2012). In identification of such chemical compounds inside serum, genomic

science has contributed substantially through discovery of related genes behind appearance of

biochemical pathways and chemical release into bloodstream.

Considering such breakthrough, a novel gene which is called Suppression of Tumor

tumorigenicity (ST2) and encodes the STD protein receptor located at epithelial membranes

of cardiac myocytes has been discovered. It is intriguing to know that there is a mutual

influence between immune system cells, their cytokines type production and ultimate

expression of ST2. During T-helper2 detection, the antigen targeting cells, through secretion

of IL-6 start to amplify the ST2 gene expression and its protein receptor encoding which is

situated at membrane of epithelial myocytes (Meisel et al., 2001).

In addition to, IL-6 other cytokines including; IL-1, IL-5 and TNF-Alfa have the same

effect as IL-6 but in a more temperate way. Th-type-2 gets activated through their STD

receptors binding with IL-33, leading to regulation of inflammation and at the same time, they

control and rectify the gene expression of STD, clearly, the mutual effect will lead to a

harmonized inflammation (Ciccone et al., 2013).

The STD location implies that during cardiac myocytes exposure to mechanical stress,

pressure, trauma and others, they get encoded, leading to CVD, HF and Ischemic Heart

Disease (IHD) (Schmitz et al., 2005). Indeed, in this following literatures, the researcher aims

to unveil the possible significance of STD protein as an early biomarker in detection of heart

complications as the major risk factor behind metabolic syndrome.

33
2.1 Biology of Suppression of Tumorigenicity (ST2) & its Relationship with

Heart

ST2 is the member of the toll-like interleukin (IL-1) receptor family which was

discovered in 1989, detected by two separate lab experts who are investigating the Growth

Stimulated Fibroblasts (GSF) (Konukoglu, 2018). Due to its huge cell signaling capacity, IL-

1 casts a substantial role in modulation of immunity, infection and inflammations which are

key responses of immune system. ST2 gene is situated on human’s chromosome- 2q12 and it

encodes three different isomer receptors including; a Transmembrane Receptor ST2 Ligand

(ST2L), a secreted soluble or Serum Circulating Receptor ST2 (sSt2), followed by a variant

form STV receptor which abundantly is expressed and dominated inside Gastrointestinal Tract

(GIT) (Pascual-Figal, & Januzzi, 2015).

ST2L is composed of an extra-cellular immunoglobulin domain, followed by

transmembrane domain and intracellular cytoplasmic domain in contrast to sST2 which

deficits the two domains of transmembrane and cytoplasm. The extracellular domain of ST2

matches homogeneity with the analogous region on IL-1 receptor ((Konukoglu, 2018).

While the origins of circulating Sst2 is matter of debate, its predominant expression is

observed ubiquitously in all living cells in particular, Cardiac fibroblasts, cardio myocytes,

heart macro vascular including arteries, coronaries and heart microvascular including heart

endothelial cells in reaction to diastolic load. Release of Sst2 is profoundly observed from

Cardiac fibroblasts and cardio myocytes due to their exposure to mechanical stress, pressure,

injury and similar (Ghashghaei et al., 2016; Kuroiwa et al., 2001) Meanwhile, it is known that

ST2L primary expression happened inside hematopoietic cells (Konukoglu, 2018).

Before 2005, it was largely accepted that ST2 has an instrumental role in mediating

inflammatory reactions, resulted in its involvement in assorted immune-based diseases

34
entailing; Asthma, pulmonary fibrosis, rheumatoid arthritis, vascular diseases, and septic

shock (Hur et al., 2015).

However, the signaling mechanism of ST2 was reviewed in year 2005 due to a new

discovery on IL-33 which is produced by a broad variety of cell types encompassing;

fibroblasts, mast cells, dendritic cells, macrophages, osteoblasts, endothelial cells, and

epithelial cells, prompting mast cells, Helper T cells, eosinophils, and basophils to discharge

type-2 cytokines which are pro-inflammatory agents inside body (Seyfizadeh et al., 2015).

IL-33 is a cytokine belongs to the member of IL-1 ligand family which is involved

intracellularly and extracellularly in both adaptive and innate immune system. IL-33

intracellularly is the independent active performing ligand of ST2L transmembrane receptor

and in case it binds with the receptor it regulates and modifies immune reactions and

functions while, extracellularly, it acts as pro-inflammatory cytokines (Schmitz et al., 2005;

Konukoglu, 2018; Lecart et al., 2002; Homsak, & Gruson, 2020).

As the cytokine IL-33 binds with transmembrane receptor of STL2, results in

regulation and modification of mitogen-activated protein kinases and several biochemical

pathways, entailing; invocation of NF-KB kinases signaling pathway which is concerned with

cellular regulations including; growth, survivals, proliferation, immune and inflammatory

responses, hypertrophy and other cellular behaviors. Indeed, IL-33/ST2L gets engaged in

cellular immune responses via activation of T helper -2 and their associated cytokines. IL-33

interaction with its ST2L receptor has also a role in some immune inflammation- induced

diseases (Takezako et al., 2006; Konukoglu, 2018) (Fig 2.1).

35
 Figure (2.1): Interaction of IL-33 with the Transmembrane Receptor of ST2L, leads to
regulation and modification of protein kinases and several biochemical pathways, leading to
inflammatory cytokines and immune reactions. Meanwhile, the soluble ST2 receptor acts as a
decoy receptor and in case of increase in circulation binds with IL-33, and diminishes the
systematic biological effects of IL-33 inflammatory processes; source: Konukoglu, (2018).

Clearly, as Weinberg et al., (2002); Sanada et al., (2007) implied mechanical stretch of

living cells could intensify the secretion of IL-33 from cytoplasmic vesicles furthermore, both

cardiac fibroblasts and cardiac myocytes could demonstrate an even increase in activation of

ST2L and sST2 due to exposure to biomechanical stress including volume/pressure overload.

Such activation due to mechanical overload has substantial impact on cardiac remodeling and

CVS alteration.

During exposure to biomechanical stress, naturally biomarkers of angiotensin II or

phenylephrine will be released into circulation, resulting in cardiac hypertrophy through

activation of NF-KB pathway. However, IL-33 ligand due to its anti-hypertrophic effect can

36
clog the activity of angiotensin II or phenylephrine on myocardium cells via interaction with

ligand of ST2, thus alleviating the NF-KB hypertrophic pathway alongside deterring the

phosphorylation of IKK (Sanada et al., 2007). Moreover, Sanada et al., (2007) argued that

biomarkers of angiotensin II or phenylephrine which promote hypertrophy as physiological

response to any stress can activate MAPK pathway, leading to further hypertrophic growth

and proliferation, yet, IL-33 binding with ST2L can deactivate such pathways, in order to

shield CVS which makes sense why presence of ST2L can decline cardiac fibrosis in mice

models.

In vivo-vitro experiments, IL-33 application could drop the activity of p38 MAPK

followed by Jun N-terminal kinase phosphorylation in MAPK pathway which both have risen

up by biomarker of angiotensin II. The knock-out of Angiotensin II is instrumental since it has

a noticeable role in formation of Reactive Oxygen Species (ROS) responsible for cardia

destruction. Yet, IL-33 injection curbed ROS manufacturing caused by angiotensin II

biomarker in vitro-vivo models. Additionally, use of IL-33 purified extract /gram in mice

models for consecutive of 4 weeks could trim down the impediment of hypertrophy and

fibrosis possibility of cardio myocytes and their altered structure (Sanada et al., 2007).

Indeed, role of IL-33 proven to rectify the ST2L activation, leading to improvement of

its own intracellular biochemical pathway. Such behavior on IL-33 self-improvement

vindicated to be crucial in ceasing or reining in the aggravation of heart functional and

structural condition (Ho et al., 2013). Such results were reinforced by Sanada (2007) mice-

model as the model manifested echocardiographic enhancement in mice- heart hypertrophy

and systolic behavior due to application of IL-33 ligand which led to invocation of ST2L.

such results consolidated the hypothesis of engineering drugs to hone ST2 activation in

human cardiac cells as one part of clinical improvement.

37
 Meanwhile, binding of IL-33 with Sst2 (due to its accumulation in circulation as a

result of mechanical stress), will deter any linkage between IL-33 and ST2L, thus, it will

hinder the good biological effects of IL-33 on T- helper2 inflammatory process and regulation

of (CVS) (Tajima et al., 2007). Indeed, as Weinberg et al., (2002) implied, intensification in

level of s ST2 plasma in myocardial infraction subject will be entertained as a negative

prognostic element for overall CV risk profiling.

Clearly, the sST2 operates as a decoy receptor (tricking IL-33) in such situational

interactions, thereby, it is fair to conclude that ST2 system is composed of two isomer

receptors which one (ST2L) has stimulating effect on IL-33, guaranteeing cardiac health

while, the second (sSt2) has antagonistic effect on IL-33 mechanism and determining cardiac

failure (Konukoglu, 2018; Dominguez et al., 2017; Doganyigit et al., 2022; Michailidou et al.,

2022) (Fig2.1).

Although, biological and biochemical analysis has revealed on IL-33/ST2 interaction

and how its different isomers operate contradictory generating positive and negative

consequences for CVS health, yet still more remains enigmatic in regard to such interaction,

thus more studies need to be conducted addressing therapeutic action of IL-33/ST2 on human

organs in particular CVS.

2.2 sST2 and its Biomarker Relationship with Cardiovascular Disorders

As it was aforementioned, IL-33 has an anti-hypertrophic action inside heart via

establishing a bind with ST2L receptors located at membrane cells of cardio myocytes.

Meanwhile, Sst2 is a decoy receptor which in case of binding with IL-33, declines IL-33

beneficial effects on the heart health (Sanada et al., 2007).

Weinberg et al., (2002) argued that in accordance to results of vitro-studies as cardio

myocytes get exposed to mechanical stress, level of Sst2 will amplify, indeed Weinberg et al.,

(2002); Sinning et al., (2017); Dieplinger et al., (2014) argued that in assorted types of (CVD)

38
including; HF, CAD and in case of artery implantation the level of Sst2 will increase

substantially which has brought the attention on its possible role as a potential prognostic

biomarker for diagnosing cardiovascular disorders.

Experimentally, it is demonstrated that myocardial expression of sST2 gene intensifies

in rats with myocardial infraction problem, led to increment in level of inflammatory

cytokines and fibrosis biomarkers which all contribute to HF (Sánchez‐Más et al.,2014).

Despite Sanchez in his study did not establish any correlation between IL-33 and

Cardiac biomarkers nor correlation between sST2 and cardiac apoptosis, yet in other studies

were demonstrated that for instance; interaction between IL-33 and ST2L could decline the

numbers of myocardial fibroblasts, deter cardio myocytes hypertrophy, diminish apoptosis

while, could enhance myocardial cell functionality (Seki et al., 2009).

Additionally, in vitro studies with Atherosclerosis, IL-33injection could diminish the

size of atherosclerotic plaque size, level of Oxidized Low Density Lipoprotein (ox LDL)

alongside decrease in level of pro-inflammatory macrophages and T-cell numbers inside

aortic sinus while, could promote T-helper 2 cytokines cells (Th2) which are in charge of

secreting Interleukins-4,5 and 13 (IL-4, IL-5 and IL-13) during type-2 immune responses ,

indeed, these ILs are instrumental in regular activation of airway inflammation, IGE reactions

and regulation of a balanced inflammatory function inside CVS, furthermore, Th2 cytokines

could stimulate IL-10 which has anti-inflammatory response thus, helping to create that

inflammation balance to protect cardio myocytes and fibroblasts of CVS (Miller et al., 2008).

Meanwhile, Miller argued that injection of sST2 could lead to further growth of fatter

atherosclerotic plague followed by intensification in numbers of T helper-1 responses which

are in charge of pro-inflammatory reactions including; Interferon Gamma (IF-Nγ) and IL-12.

In support of above literature, De la Fuente et al., (2015) demonstrated that induction

and accumulation in serum level of ST2 (s ST2) would cause atherosclerosis as well as HF.

39
The serum concentration of Sst2 will amplify in inflammatory driven conditions such as;

“rheumatoid arthritis, type 2 diabetes, sepsis, autoimmune diseases, liver failure, cancers,

fibro proliferative diseases, and ulcerative colitis”.

Increase in level of sST2 due to mechanical stress, pressure or similar risk factors, will

lead to wrong bonding between sST2 and IL-33, thus antagonizing the anti-hypertrophic and

anti-apoptotic effects of IL-33 inside cardio myocytes (Miller, & Liew, 2011), similarly,

blocking the SLT2 receptor will have the same inhibitory effect on functionality of IL-33, on

the contrary, binding between IL-33 and ST2L receptor allows IL-33 demonstrates its cardio-

protective effects on cardio myocytes and brings over all protection on the CVS (Konukoglu,

2018).

Dyspnea is the condition of breathlessness or heavy hard-taking breaths which stems

from different pathological diagnosis including; “heart failure, chronic obstructive pulmonary

disease, bronchitis/asthma, pneumonia, malignancy, pulmonary embolism and plenty more”

(Zwaan et al., 2013). Finding a biomarker which can relate to dyspnea different pathological

outcomes can hone the diagnosis alongside the fact that it can also, distinguish the diverse

pathological conditions of dyspnea from one another to emanate better treatment method

(Socrates et al., 2010).

In dysphonic patients with or without Acute Heart failure (AHF), the concentration of

C-Reactive protein (CRP), the level of sST2 and another biomarker known as “amino-terminal

pro-brain natriuretic peptide” (NT-pro- BNP) were measured (Januzzi et al., 2007) and

results revealed a higher level of s ST2 biomarker in patients with acute HF as compared with

none HF however, the landmark biomarker as the diagnostic determinant for acute HF was

affirmed to be (NT-pro- BNP) rather than sST2, nevertheless, sST2 was still reliable as it

appeared with prognostic predictions . Meanwhile, a conjunction of high (NT-pro- BNP) and

low sST2 was detected in dyspnea patients who their condition was not associated with HF

40
(Januzzi et al., 2007). On the contrary, patients with acute HF, their sST2 level was higher

than (NT-pro- BNP) which determined further the reliance on sST2 biomarker when it comes

to CVS health condition (Januzzi et al., 2006).

Meanwhile, sST2 biomarker could not distinguish between dyspneic patients due to

HF versus dyspneic patients due to chronic obstructive pulmonary disease, thereby, sST2

versatility in differentiating dyspneic pathological causes were debunked (Dieplinger et al.,

2010).

In another study, the evaluation of s ST2 as a strong biomarker in myocardial function

considering patients with HF or without HF was strongly approved, as a matter of fact, s ST2

biomarker was associated with both “Left Ventricular (LV) and Right (RV) ventricular

function” and further was treated as the prognostic biomarker concerning morbidity (Shah et

al., 2009).

McLaughlin et al., (2015) in his research indicated that Pulmonary Arterial

Hypertension (PAH) is a lung condition, diagnosed by obstruction of lung’s small arteries,

leading to hypertension inside lung tissues. This condition is detected through progressive

remodeling in the pulmonary arteries, and enforces mechanical stress on heart, led to

intensification in arterial withstanding and increase in pulmonary artery pressure.

These intensifications will amplify the workload of the right side of the heart, creating

heart failure (Fig 2.2). Under such circumstance, the biomarker of sST2 which has detrimental

effects on heart will elevate (Chida et al., 2014).

41
 Figure (2.2): Pulmonary Arterial Hypertension (PAH); Source: Orriols et al., (2017).

Zheng et al., (2014) pointed at direct association between high level of sST2 in blood

and high pulmonary vascular resistance condition, cardiac index and clinical deterioration.

In accordance with Shao et al., (2014), in vitro studies, suppression of IL-33 elevated the

discharge of sST2 from human endothelial cells in PAH patients.

There has been varied studies, highlighted the correlation between heightened level of

sST2 and PAH conditions in heart patients, referring to sST2 as a determining biomarker for

purpose of vascular remodeling and for anticipating the outcomes and intensity of PAH

(Zheng et al., 2014).

In a systematic review focusing on sST2 and PAH, was indicated that high rate of

sST2 prompted high morbidity and poor clinical outcomes in PAH patients. Indeed, was

proven that in PAH non-survivors as compared with survivors, the level of sST2 was

measured higher (Aimo et al., 2017). Chida et al., (2014) argued that high level of sST2 could

prompt an increase in ventricular dilatation followed by systolic dysfunctionality.

The significance and role of ST2L/IL-33 axis and sST2 in circulation have been

affirmed in multiple inflammatory based conditions including cancer, cardiac disorders and

42
any inflammation –based condition. sST2 meddles with homeostasis and pathogenesis of

abovementioned diseases to counteract the interaction between ST2L/IL-33, generating the

onset of fibrosis, tissue destruction and inflammation followed by remodeling CVS. In clinical

studies, s ST2 is considered as an imperative marker for patients with cardiac disease in

addition to renal failure and dialysis, furthermore, sST2 is entertained as a monitoring

biomarker for patients with Heart Failure (HF) who are under treatment (Homsak, & Gruson,

2020).

HF is a biological and clinical complication which stems from failure of ventricular

filling or lack of ability of pumping blood out. The major diagnosis of HF is heart fatigue and

difficulty in breathing which gets accompanied by pain and shortened breaths. There are no

single diagnostic tests available which can determine HF clinical diagnosis since, the whole

diagnostic scenario relies on historical and physical examination (Yancy et al., 2013).

At the contemporary time, the best guideline to detect HF prognosis is to measure the

dosage of natriuretic peptides followed by cardiac troponin (Yancy et al., 2013).

Furthermore, the biomarker of Sst2 which is responsible for inflammation, fibrosis and

cardiac arrest has been incorporated in 2013-HF guidelines.

Blood concentration of sST2 intensifies in inflammatory condition and heart disorders

which has proven to be a reliable prognostic biomarker (Breidthardt et al., 2013; Vorovich et

al., 2014). Sst2 also, has been a valuable biomarker in prognosis and diagnosis of CVD

and myocardial infarction and furthermore, can predict heart failure prognosis in low-risk

based communities (Wang et al., 2020; Muntner, & Whelton, 2017).

In diagnosis of Chronic Heart Failure (CHF) sST2 is reliable since the biomarker is

the determinant of cardio myocytes stress level and fibrocystic condition thus, such biomarker

helps to identify patients with broad spectrum of CVD.

43
SsT2 based on Bayes-Genis et al., (2015) study has valuable prognostic credit which makes it

reliable to be incorporated into the clinical practice to anticipate any subsequent cardiac risk.

Indeed, the number of evidence, reinforcing application of sST2 for prognosis and diagnosis

purposes of chronic heart failure perpetuates to grow.

Conversely, concentration on level of s ST2 and its measurement in blood sample could be a

relevant clinical prognostic biomarker to divide and differentiate the risk morbidity of patients

who sustain from myocardial infarction, HF and dyspnea (Dattagupta, & Immaneni, 2018).

2.3 Biomarker of ST2 in Other Clinical Settings

ST2 in anticipating mortality is the best biomarker, in particular in accompany with

beta-type natriuretic peptide can even have more assuring effect on increment in risk of death

(Daniels et al., 2010). In study by Coglianese et al., (2012) high concentration of sST2 was

attributed to systolic blood pressure, consumption of anti-hypertension drugs followed by

diabetes and the biomarker concentration of sST2 raised even higher in men and elderly.

Brown et al., (2007) also, argued that in patients with chest pain compliant due to

pulmonary diseases of obstructive sleep apnea, chronic obstructive pulmonary disease,

asthma, pulmonary embolism, and pulmonary hypertension in addition to, alcoholism,

systematic inflammation or infection, a greater concentration of sST2 in their serum was

observed.

2.4 Biology of Endothelin-1 (ET-1) and its Relationship with its Receptors

Endothelin is the peptide which is composed of 21-amino acids with three

recognizable isomers of ET-1, ET-2, ET-3 .ET-1 is the most prevalent and well-investigated

isomer, yet two more ET-2 and ET-3 are not well known (Motte et al., 2006).

ET-1 is generated by great deal of different organs (Table.1), yet it is believed that it

is mostly released by endothelial cells of Vascular Smooth Muscle Cells (VSMC) where they

44
are in charge of vascular contraction, cell proliferation and mitoses as well as, pro-

inflammatory reactions (Wort et al., 2009; Alves Lopes et al., 2019).

Table.1: Cells-Releasing ET-1 and ETA, ETB Receptors Expression; Source:

Kowalczyk etal., (2015).
Different CVS Urinary Immune Nerve system Other tissues
Organs system system & skin
Cells-releasing Endothelium, Renal Neurons Macrophages, Fibroblasts,
ET-1 VSMCs, cardio medulla mast cells, sinusoids…...
myocytes leukocytes ,Ku
epfer cells
Cells- VSMCs, cardio Glomerular Melanocytes, Neurons Adipocytes ,
,
expressing myocytes nuclear capillaries , keratinocytes reproduction
receptor ETA membranes in human cell cells, liver
aortic VSMC collecting cells….
ducts
Cells- Endothelium, Renal ----------- Vagus nerves Different
expressing VSMCs, coronary tubes …. and other endocrine
receptor ETB vascular, neurons tissues,
atrioventricular osteoblasts,
conducting tissue, hepatocytes
atrial and ventricular
myocardium, nuclear
membranes in human
aortic VSMC

Endothelin-1 (ET-1) is an active endogenous vasoconstrictor and apart from vaso-

constrictive role, it can instigate fibrosis of the vascular cells while, induces production of

ROS (Guionaud, 2015). The consequences of ET-1 activation will be vasoconstriction,

hypoxemia, hyperplasia, hypertrophy, fibrosis and increment in vascular permeability

(Makino et al., 2011; Balistrieri et al., 2023).

45
 It is argued that ET-1 provokes pro-inflammatory pathways such as; NF-KB,

intensifies production of superoxide anion radical of O2. (-) as well as pro-inflammatory

cytokine secretion of TNF-α, IL-1, and IL-6 (Poli, 2000; Ammar et al., 2021).

There are two types of endothelin receptors in mammals, ETA, ETB receptors, and ET-

1function is regulated by both of these ETA and ETB receptors which are classified under

family of G- protein –coupled receptors family (Khalil, 2011; Kubale et al., 2020).

ETA and ETB receptors have been identified in many cell types on top of blood vessels,

but mainly are situated in Cardiovascular tissues (Hynynen, & Khalil, 2006). The regulatory

receptors of ETs can influence performance of variety of organs (heart, liver, kidney, lungs)

(Rodriguez et al., 2013) and in addition to, they are involved in modulation of vascular tone,

in re-designing of vascular, myocardial and bone structure, followed by suppression of

apoptosis and salt-water maintenance. On top of that, ETs can stimulate and support

bronchoconstriction, angiogenesis and neuropathic pain (Rodriguez et al. 2013; Thakkar et

al. 2006).

ETA receptors (Fig 2.3) are situated on the membrane of (VSMC) where they are in

charge of vascular contraction, cell proliferation, mitoses as well as, pro-inflammatory

reactions while, ETB receptors are composed of two sub categories of ET B1 and ETB2. ETB1 are

situated on endothelial cells and invokes Nitric Oxide (NO) which is a pulmonary vasodilator

mediator (in the walls of arteries and veins) and antagonistic of fibrosis, in addition to, ETB1

stimulates release of prostacyclin (Prostaglandin I2) which is a vasodilator and it induces

secretion of endothelium-derived hyperpolarizing biomarker, ETB1 also, curbs proliferation of

vessels (angiogenesis) and suppresses ET-1 releases by endothelial cells (Marzoog, 2023). On

the contrary, ETB2 are situated on VSMC and induces contraction (Hynynen, & Khalil, 2006;

Khalil, 2011).

46
 It is posited that ETB1 receptors of endothelial cells, have a substantial role in sweeping

away ET-1 from plasma, yet findings are not well-established on this mechanism (Kawanabe,

& Nauli, 2011). The pulmonary endothelial ETB1 receptors are in charge of wiping off up to

50% of released pulmonary ET-1 from the blood circulation (Schneider et al., 2007).

Figure (2.3): ETA Receptor Located on VSMC, it activates IP3 -signaling pathway, MAPK
signaling cascade leading to contraction and pro-inflammatory responses, in addition to cell
mitosis and growth; Source: Andersson et al., (2008); Khalil, (2011).

47
There so many different regulators which can trigger or suppress ET-1 secretion (Table.2)

Table.2: Stimuli and Suppressors behind ET-1 Release; Source: Kowalczyk etal., (2015).
Triggers behind ET-1 Release Suppressors of ET-1

Inflammatory cytokines, growth factors, High sheer stress

thrombin, neuro-hormones, any vascular

stretch.

Low sheer stress, adrenaline, angiotensin II, Nitric oxide (NO)

Hypoxemia, Insulin, free radicals, IL-1, Prostacyclin , prostaglandin (PG-I2)

Endotoxin (LPS)

Vasopressin, cardiotrophin-1,homocysteine, Heparin, Atrial natriuretic peptide

IL-6, Calcium-ions, TNF-A

In general physical and chemical stimulants of activator protein-1, GATA-2, smad,

hypoxia inducible factor-1 and NF-KB pathway invoke ET-1 gene expression on endothelial

cells (Rodríguez-Pascual et al., 2003).

2.5 ET-1 Relationship with Heart Diseases

Reis et al., (2013) argued that patients with PAH suffer a higher level ET-1 and

accumulation in expression of ET-1 receptors are observed in PAH patients with condition of

plexiform lesion in their lung system (Price et al., 2012). In addition, higher level of ET-1 in

plasma will alert on intensity of the disease and also, it is an applicable parameter for adverse

prognosis (Tsutamoto et al., 1993).

48
 In patients with Chronic Obstructive Pulmonary Disease (COPD), also, there is a high

level of ET-1 and as COPD advances, ET-1 concentration inside urine will get heightened

(Biernacki et al., 2003). Study by Schommer et al., (2012) implied high level of ET-1 in

subjects who their level of O2 saturation gets exacerbated due to exercise or at night. In study

by Barberà, & Blanco, (2009) patients with PH-COPD, the level of ET-1 trans pulmonary

heightened as compared with normal subjects.

Study by Vancheri et al., (2010) also, demonstrated accumulation in expression of

lung tissues ET-1 receptors followed by plasma level of ET-1 in subjects with Idiopathic

Pulmonary Fibrosis (IPF) with or without PAH.

Study by Khalid et al., (2016) argued that during endo-toxemia which is presence of

Lipopolysaccharide (LPS) of dead bacteria, the plasma level of ET-1 elevated. Some studies

established articulation between ET-1 in plasma and morbidity rate in sepsis patients

(Piechota et al., (2007).

Meanwhile, the antagonist receptor of ET-1 known as ET B1, believe to be helpful in

obstructing ET-1 activation thus, ablating its pathological effects, protecting against assorted

vascular diseases (Ariyarajah, 2008).

Study by Rivera et a., (2005) demonstrated the association between high concentration

of ET-1 in plasma with intensity and prognosis of heart failure, furthermore, high ET-1

positively was anchored to NT-pro-BNP levels and conversely was correlated to Ejection

Fraction (EF) which shed a light on the fact that high level of ET-1 could be connected to

ventricular malfunction or remodeling and activation of neuro-hormonal pathways.

In relationship between an induced- ET-1 endothelium atherosclerosis scholars argue

that a healthy endothelium can modulate the biological activities of vessels thus shield them

against atherosclerosis. Endothelium is a single cell monolayer which covers the entire

vasculature. Endothelium has a barrier line which can segregate blood from organs and tissues

49
and has a mechanical and biochemical role in anti-coagulation, vascular sensation, hormone

secretions, regulation of inflammation and others. Endothelial cells are the home to

endothelin receptors, vaso regulatory biomarkers, endocrines, dissolve lipophilic gas with

vasodilator effects, NO, and vascular constricting G protein receptor agonists. It has been

proven that ET-1 and angiotensin (Ang)-II are involved in fostering hypertension and

atherosclerosis (Ibañez, & Fuster, 2017).

Endothelial dysfunction is underlying in development of obstructive or non-

obstructive forms of coronary artery disease. Coronary heart dysfunctionality occurs, due to

asymmetric secretion between vaso constrictive (ET-1, superoxide, hydrogen peroxide and

thromboxane) vaso protective and vaso relaxer biomarkers including (NO, prostacyclin

(PGI2), endothelium-derived hyper polarizing biomarkers) (Taqueti, & Di Carli, 2018).

In endothelial injuries-induced atherosclerosis the pro-inflammatory cytokine of IL-1,

IL-6 and TNF-α get activated through discharge of ET-1, as indeed, Kagami et al., (2010)

argued that ET-1 in cooperation with TH-17 produces the major pro-inflammatory cytokine of

IL-17 and similarly generates, other pro-inflammatory cytokines of IL-6, IL-21, IL-22 and

TNF-α which all have a substantial role in atherosclerosis. However, the concentration of ET-

1 and Interleukin-17 (IL-17) get more amplified in CVD as compared with atherosclerosis.

Rizzoni et al., (2019) argued that in inflammation-induced atherosclerosis the

accumulated level of TNF- α and IL-17 will reinforce the increment of other pro-

inflammatory cytokines like IL-6, leading to formation of an inflammatory cascades which

will promote oxidative stress which makes the formed plagues to be volatile in blood

circulation., leading to atherosclerosis and ischemic heart as well.

2.6 Endothelin- 1 and Hypertension

In genetically modified mice has been proven that ET-1 has been engaged with

normalization of blood pressure as well as proliferation of vascular diseases (Lund et al.,

50
2003). ET-1 by inducing the rise of [Ca2+] entry gets involved in the pathway of stromal-

interaction molecule-1, leading to induction of vascular smooth muscles contractions. Such

mechanism also, needs the involvement of activated p63Rho guanine nucleotide exchange

factor and intensification in O-GlcNAcylation which is a kind of post-translational modifier.

Endothelial overexpression of ET-1 genes in mice samples manifested increase in

genes expression of lipid metabolism, as well as pro-inflammatory signal transduction. Such

events led to expedition of atherosclerosis in case, the samples were on a high fat diet which

resulted in hypertension (Kudryavtseva et al., 2013).

However, in study by Rautureau, & Schiffrin, (2012), ET-1 receptor antagonist of

ETB1 as well as antagonistic gene of calcitonin gene-peptide, both showed capability to

dissolve ET-1 contraction effect.

Study by Montani et al., (2007) on ratio of ET1/ET3 as an underlying prognostic

factor of PAH proved that high rate of ET-1, high ratio of ET1/ET3 and low level of ET3 in

plasma could be relied on as severity of the heart condition and be assessed as prognostic

factors in PAH condition.

In salt-diet hypertensive induced subjects, the plasma level of ET-1was intensified

through endothelium secretion. ET-1 which is released from endothelial cells, ignited

oxidative stress on the walls of vascular vessels, tapping into redesigning vasculatures’

structures and performance followed by their endothelial dysfunctionality, very symptoms

which are diagnosed in hypertensive samples who demonstrate a high rate of ET- mediating

biomarker. The antagonistic receptor of ET-1 could drop hypertension alongside vascular

hypertensive reshaping structure (Schiffrin, 2005).

As it was aforementioned, ET-1 is an instrumental modulator in vascular tone and

pro-inflammatory reactions which will lead to a vascular damage diagnosed in hypertensive

cases. In addition, in hypertensive cases, high ET-1 would curb the biomarker of Peroxisome-

51
Proliferator Activated Receptors-γ (PPARγ) which exhibits pro-cardio properties through off-

setting (COX-2) which is a pro-inflammatory enzyme as well as ROS production. Thus,

proven that (PPARγ) has a regulatory antagonistic effect on hypertensive induced ET-1,

through deactivation of ETA receptor and increment in activity of ET B1 receptor. Indeed, ET-1

through ETA receptors can amplify the production of ROS as well as phenylephrine-induced

contraction under a mechanism which relies on activation of TP receptors in connection with

COX-2 enzyme. Cox2 enzyme is in charge of accumulation of prostacyclin and alleviation of

NO, leading to hypertension and heart failure (Palacios-Ramírez et al., 2019).

Study by Touyz, & Schiffrin,E. L. (2003) argued that despite the precise role of ET-1

in human’s hypertensive condition is enigmatic, yet some certain groups of hypertensive

patients suffer ET-1 dependent hypertension including black Africans, patients with salt-diet,

or low level of renin control hypertension, in addition to, obese and diabetic people. ET-1 has

been detected in intensive hypertension, heart failure, atherosclerosis, and pulmonary

hypertension, thus clearly, ET-1 is the inducer of CVD and symptoms of metabolic syndrome.

Study by Kaoukis et al., (2013) demonstrated that harmonized equation between ETA/ETB

agonists and antagonists’ receptors will have noticeable effects on attenuating the blood

pressure which can enhance protection against hypertension target-organ damage thus, better

management of CVD.

Although, there are myriad options with a good endurance capable of treating

hypertension, yet, still there are so many focuses on novel anti-hypertensive drugs which can

identify and nullify novel pathways of hypertension ignition, thus helping to modulate blood

pressure. Among all different medications, the drug of Aprocitentan is an oral non-selective

ET-1 receptor antagonist which can control arterial hypertension (Hocht et al., 2022).

52
2.7 Association between Hypertension and Metabolic Syndrome

Hypertension has posed a substantial threat in the general population, causing high rate

of morbidity due to CVD. Furthermore, the correlation between hypertension and

dyslipidemia, obesity, diabetes (hyperglycemia/ insulin resistance) also, identified as

symptoms of metabolic syndrome, will elevate the risk of CVD. The elaborate

pathophysiological overlap between triggers of metabolic syndrome has proven that to what

extent antidiabetic and antihypertensive drugs can be responsible in controlling the syndrome,

not leading to CVD (Behi et al., 2022).

As these studies Da silva et al., (2020); Minh et al., (2021) suggested, there is a

connection between hyperglycemia, hyperinsulinemia, insulin resistance and hypertension.

Hypertension can be generated though different genetic or epigenetic ways, as for instance it

can occur due to retention of water and sodium via proximal renal tube reabsorption supported

by insulin (Kawarazaki, & Fujita, 2021).

Study by Reaven, (1988) argued that based on empirical evidence high level of

fructose feeding in animal models could trigger hypertension. Furthermore, Hwang et al.,

(1987) argued that hypertension could also, be seen in sucrose-fed mice, which furthermore

Reaven, (1988) determined that changes in diet and life style could result in insulin resistance

and hyperinsulinemia and these collectively could promote hypertension.

Meanwhile, as Stetic et al (2021) implied exercise can diminish hypertension in people

with early hyperinsulinemia. Resistance to insulin, glucose intolerance, high triglycerides, low

HDL, and hypertension are all parameters of metabolic syndrome, influencing health of

(CAD). Indeed, link between epigenetic, and genetic can tamper with expressions of orphan

G Protein-Coupled Receptors (GPCR) in metabolic syndrome.

Small RNA (mi RNA) are in charge of gene expression and its hindrance can hone the

performance of metabolic syndrome patients, due to the fact that mi RNA activation will

53
promote metabolism of glucose and cholesterol, resulting in atherosclerosis (Skuratovskaia et

al., 2020).

Indeed, insulin resistance and low HDL could trigger the expression of SREBP 1 and

2 genes, followed by overexpression of miRNA –mi-R33a-b, yielding to the risk of metabolic

syndrome.

The biomarkers of miR-221 and let-7 are also, both high in patients suffering

metabolic syndrome as indeed, let-7 was correlated with high blood pressure and low HDL

(Wang et al., 2013). Study by Natali, & Nesti, (2021) implied that Hyperinsulinemia creates

hypertension due to lack of insulin ability to have its vasodilatory effects on walls of blood

vessels, followed by increment in vasoconstriction due to high level of free fatty acids which

are formed under reactive oxygen species (ROS).

In metabolic syndrome, insulin resistance occurs as the main component or rather

predominately precursor behind the syndrome (Lonardo et al., 2015). Empirical evidence has

demonstrated that insulin by provoking sodium reabsorption generates anti-natriuretic

properties which in one hand, enhance insulin resistance while, in another hand, it will

amplify its escalation (Yani et al., 2008), thereby can generate hypertension in metabolic

syndrome.

There are other attributes of insulin which can lead to hypertension in metabolic

syndrome, including, absence of insulin vasodilation effect, which trigger generation of free

fatty acid and their vasoconstriction functionality (Mendizábal et al., 2013). Clearly, drugs

which attenuates hyperinsulinemia and hones insulin resistance are also, effective on

hypertension due to proven links between hypertension and insulin resistance.

Simultaneously, some anti-hypertensive medications can rise the insulin sensitivity like

antagonists of angiotensin II receptors (da Silve et al., 2020).

54
 Apple shape obesity known as central Obesity which is behind metabolic syndrome,

can also, trigger hypertension due to the fact that adipose cells can produce adipocytokines

entailing; leptin, Tumor Necrosis Factor-α (TNF-α), interleukin-6 (IL-6), angiotensinogen,

and non-Esterified Fatty Acids (NEFA) which all have bioactive capability of triggering

arterial hypertension and ultimately, metabolic syndrome (Van de Voord et al., 2013).

Furthermore, the overreaction of sympathetic nerve system in obese patients followed

by activated renin-angiotensin system and its enhancing feedback on sympathetic nerve

system contributes to higher level of blood pressure alongside insulin resistance (Lent-

Schochet et al., 2019).

There is also, the elevation in level of leptin hormone which due to resistance in

insulin will increment. The leptin function positively affects the activity of sympathetic nerve

system, resulting in hypertension induced obesity and insulin resistance (Valensi, 2021;

Alvarez et al., 2002).

On top of that, as above literature argued, endothelial mal-functionality will contribute

to hypertension followed by insulin resistance (De Jongh et al., 2004; Lent-Schochet et al.,

2019).

Another factor which contributes to hypertension as the trigger behind metabolic

syndrome is pro-inflammatory cytokines of necrosis factor-α (TNF-α) and interleukin-6 (IL-

6) Which both cast a role in pathophysiology of vascular hypertension. High level of TNF-α in

serums could indicate the symptom of insulin resistance as well as systolic blood pressure.

Additionally, IL-6 through stimulation of sympathetic nerves will accumulate the plasmatic

dosage of angiotensinogen and angiotensin II, producing hypertension (Yanai et al., 2008).

There is also, the condition of sleep apnea which is caused by over activity of

sympathetic nerves and dysfunctionality of baroreflex (body homeostatic mechanism in

55
sustaining blood pressure) thus, causes hypertension, indeed patients with sleep apnea

condition mostly are diagnosed for metabolic syndrome (Venkataraman et al., 2020).

There are other triggers which obstructive sleep apnea used in accumulation of blood

pressure, encompassing; “heightened angiotensin II, high dosage of aldosterone levels,

oxidative stress, hyper-leptinemia, and inflammation” (Noce et al., 2019).

To wrap up this literature review, these above-mentioned factors have

vasoconstriction properties, they can indeed, curb vasodilation, prompt sympathetic over

activation, elevate intravascular fluids, resulting in cultivation of hypertension in metabolic

syndrome patients (Fig. 5) (Kirpichnikov, & Sowers, 2001; Hengel et al., 2022; Oduro-

Donkor, & Barber, 2023; Bovolini et al., 2021).

56
Figure (2.4): The Chief Mechanism Which Connects Insulin Resistance to Hypertension
and Hypertension to Metabolic Syndrome; Source: (Kirpichnikov, & Sowers, 2001).

CHAPTER THREE

MATERIALS AND METHODS

57
3.1 Purpose of this Research

The human serum Mid-Regional pro-Adreno-Medellin (MR-pro-ADM) Elisa kit is to

assess the MR -PRO-ADM levels inside any biological fluid encompassing; human serum,

plasma, culture media.

3.2Methods of Application

The Elisa-Kit employed method of Sandwich-Elisa. The Micro Elisa strip plate inside

the kit was pre-coated with an anti-body which was particularized for MR-pro-ADM. Chosen

samples were added to each Micro Elisa strip plate well, in combination with specific

antibody. In the next phase, Horseradish Peroxidase conjugated antibody specifically designed

for (MR-PRO-ADM) inserted to each strip plate well and went for incubation. Free

components were rinsed away and later the TMB substrate solution injected into each well.

Those wells contained MR-pro-ADM as well as HRP-Conjugated MR-pro-ADM

Antibody turned blue in color and appeared yellow after injection of stop-solution. The

Optimal Density known as (OD) was measured at wavelength of 450nm by device of

spectrophotometric. The OD value is proportional to concentration of MR-PRO-ADM in each

sample, by comparing the OD of the sample to the standard curve, the concentration of MR-

PRO-ADM was quantified.

58
 Table.3 Equipment Provided Alongside the Kit
Other Equipment 96 Storage
Determinati
ons
1. User manual 1 R-T
2. Closure plate 2 R-T
membrane
3. Sealed bags 1 R-T
4. Micro Elisa strip plate 1 2-8 C
5. Standard 27nm/L 0.5 ml * one 2-8 C
bottle
6. Standard diluent 1.5 ml * 2-8 C
one bottle
7. HRP-Conjugation 6ml * 1 2-8 C
Reagent bottle
8. Sample diluent 6ml * 1 2-8 C
bottle
9. Chromogen Solution A 6ml * 1 2-8 C
bottle
10. Chromogen Solution B 6ml * 1 2-8 C
bottle
11. Stop Solution 6ml * 1 2-8 C
bottle

59
 12. Wash Solution 20 ml (30x) 2-8 C
* 1 bottle

3.3 Sample Preparation

3.3.1 Serum Preparation

After blood was collected, went on clotting under room temperature (10-20 minutes)

due to undistributed status. The formed clot was centrifuged at 2000-3000 RPM for 20

minutes.

3.3.2 Plasma Preparation

The collected blood was transferred into tube containing anti-coagulant EDTA or

citrate. After incubation for 10-20 minutes, centrifuging occurred for another 20 minutes at

2000-3000RPM, then, supernatant was skimmed away carefully as plasma-sample.

3.3.3 Urine Samples

Urine was collected by an aseptic tube, then 20 minutes was centrifuged and the

supernatant was skimmed away into another tube. The preparation of cerebrospinal and

pleuroperitoneal fluid is identical to urine sampling procedure.

3.3.4 Cell Sampling

To detect the secretion of cells, cell culture supernatant was collected into a

disinfected tube, and went under centrifuging for 20 minutes at 2000-3000 RPM.

To detect intracellular compounds, the culture must be dissolved in 1*100ML with

PBS (PH: 7.2,7.4) and then the cells through constant freezing and thawing were shattered in

order to discharge their intracellular compounds.

60
3.3.5 Tissue Sampling

Tissue samples were cut, weighed, then frozen in liquid nitrogen and stored in -80 C

for future use. The tissue samples were homogenized after inclusion of PBS at PH=7.4 and

later centrifuged for 20 minutes at the same PRM and the supernatant was skimmed away.

This supernatant known as; Aliquot was used later for ELISA assay.

3.4 Sample Distribution Procedures

1. sample/ standard was dissolved inside tube and later 50 microliters were pipped out into a

microplate well (1Tube goes for 2WELLS=10 wells).

Table.4: Different Standards

18 ng/L Standard.NO.1 300µl original standard
+150 µl standard diluent
12 ng/L Standard.NO.2 300 µl STno.1+150 µl
standard diluent
6 ng/L Standard.NO.3 150 µl ST no.2+150 µl
standard diluent
3 ng/L Standard.NO.4 150 µl ST no.3+150 µl
standard diluent
1.5 ng/L Standard.NO.5 150 µl ST no.4+150 µl
standard diluent

61
 Figure (3.1): Standard Diluent Distribution; Sample Distribution Procedures; Source:
lab- Source
2. In the micro Elisa strip plate, a well was left empty as blank control.

3. Incubation at 30 minutes, 37C was conducted.

4. Dilution: The concentrated washing buffer was diluted with distilled water (30 times for 96

tubes and 20 times for 48 tubes).

5. Rinsing: using wash solution for 30 seconds and then discarding it. Repeat the procedure

for 5 times.

6. Inserting 50 microliters HRP-conjugate reagent to each well, except the blank control well

7. Followed by incubation in step.3 and rinsing step.5

8. Coloring in darkness: inserting 50 microliter Chromogen Solution A, followed by 50

microliters Chromogen Solution B to each well, shaking gently to get mixed. Later incubating

under 37C, 15 minutes

9. Termination phase: Inserting end-solution 50 microliters to each well to cease the progress

of reactions, thus turning color from blue to yellow.

10. Read absorbance of optimal density at 450 nm, employing Microliter plate reader. The OD

value control of blank sample (control) is set at zero. Experiment should be conducted after 15

minutes adding termination solution.

3.5 Criteria and Lab Principles

1. The kit at arrival at once must be stored at temperature of 4 C in the fridge but upon

trial experiment the temperature must be harmonized to the normal room temperature.

2. In case of precipitates appearance in concentrated rinsing buffer, the buffer must

get heated to have all these precipitates diluted in order to avoid skewed results.

3. Accurate pipetting is underlying to refrain from any experimental error, followed

by standard curve inclusion in every experiment. In case optimal density > than the standard

62
inside well, the sample must be dissolved before testing. At the time of calculating MR-PRO-

ADM original, the total dilution factor must get multiplied (XnX5).

4. Miro-plate sealers are one-time use to deter any cross contamination

5. Substrate must be avoided from light exposure

6. All lab operations must be determined by manufacturers’ instructions and the results

must get determined by micro titer plate reader.

7. All the samples, rinsing buffers, and wastes must be treated as potential infectious

agents.

3.6 How to Calculate Results

The concentration of human MR-pro-ADM standard and the optimal density is plotted

on the log scale (x-axis) and (y-axis) respectively. The original concentration is quantified by

multiplication of dilution factor.

3.7 Precision Involvement

- Three samples with low, medium and high level human ET-1, tested for 20 times on

one plate. This process is known as low-assay precision.

- Three samples with low, medium and high level human ET-1, tested on 3 different

plates, 8 replicates in each plate. This assay called, intra-assay precision.

- CV%= SD/mean X100

-Intra-Assay: CV<10%

-Inter- Assay: CV/12%

Assay Range: 0.8 to 20 ng/L

Sensitivity: 0.1 ng/L

Storage: 2-8 C at 6 months

63
CHAPTER FOUR

RESULTS

64
4.1 Participants’ Socio-Clinical Characteristics

A total of 88 patients, their health records were examined. More than half of samples

were male patients 47(53.4%), with medium age of 40 years old. The multiple responses

analysis demonstrated that chronologically, 74.3% and 71.4% of patients suffered from high

blood pressure and cardiac disorder, followed by 40% thyroid disorder and 8.6% diabetes.

In this study, the medium was employed as Central Tendency Index (CTI), to depict

the sample critical parameters level. In (Table.5) the medium level of TSH and FT4

chronologically, were 3.05 ml/ul and 1.36 ng/dl, followed by cholesterol level at borderline of

Md=200.0mg/dl, Triglyceride (TG) on desirable level of 138 mg/dl, HDL was within optimal

level of 42mg/dl while LDL was out of desirable =102 mg/dl. Additionally, the medium of

fast blood sugar followed by endothelin-1 and suppression of tumorigenesis-2 were

chronologically were at 98mg/dl, 4.17 Mdn, and 2.51 Mdn.

65
 Table 5. Participants’ Socio and Clinical Characteristics.
Variables Category Frequency Percentage

Gender Male 47 53.4

Female 41 46.6

*Chronic diseases Hypertension 26 74.3

Heart diseases 25 71.4

Thyroid 14 40.0

Diabetes mellitus 3 8.6

Age in years Mean SD Median

38.71 12.87 40.0

Thyroid Stimulating Hormone 3.48 2.37 3.05

(TSH) mIU/L

Free Thyroxin -FT4 ng/dl 1.45 0.4 1.36

Cholesterol (Chol) mg/dl 194.42 63.83 200.0

Triglyceride (TG) mg/dl 155.08 75.47 138.0

High Density Lipoprotein 48.63 15.30 42.0

(HDL) mg/dl

Low Density Lipoprotein 117.35 41.38 102.0

(LDL) mg/dl

Fast Blood Sugar (FBS) mg/dl 101.24 34.70 98.0

Endothelin-1 (ET-1) mol/ml 5.74 3.07 4.17

Suppression of 2.65 1.21 2.51

Tumorigenesis-2 (SSt2)

66
 ng/ml
* Multiple response analysis

4.2 Correlation Analysis of Thyroid Function Tests, Lipids Profile, Fast

Blood Sugar and Chronic Diseases with ET-1 and Suppression of

Tumorigenesis-2

The nonparametric Spearman rho correlation was used to correlate the SSt-2, ET-1 with

thyroid function test and metabolic indices (lipids profile and fast blood sugar) while the point

biserial correlation utilized to correlate the hypertension, heart diseases with SSt-2 and ET-1.

The results in (Table.6) revealed that the TSH and FT4 were significantly positively

correlated with SSt2 and ET-1, similarly the cholesterol, triglycerides, LDL and fast blood

sugar demonstrated a significant positive correlation with SSt2 and ET-1, however the HDL

showed an inverse significant correlation since lower value of HDL correlated with higher

values of SSt-2 and ET-1.

Regarding the chronic disease which coded as zero for no and one for yes, the point biserial

correlation exhibited a significant positive correlation indicating those experienced

hypertension and heart diseases are correlated with higher values of SSt-2 and ET-1.

67
 Table 6. Correlation Analysis of Thyroid Function Tests, Lipids Profile, FBS and
Chronic Diseases with ET--1 and sST-2.
Parameters SSt-2 ET-1

TSH Correlation Coefficient .225* .258*

Sig. (2-tailed) .035 .015

FT4 Correlation Coefficient .306** .221*

Sig. (2-tailed) .004 .038

CHOL Correlation Coefficient .226* .290**

Sig. (2-tailed) .034 .006

TG Correlation Coefficient .227* .317**

Sig. (2-tailed) .033 .003

LDL Correlation Coefficient .257* .246*

Sig. (2-tailed) .016 .021

HDL Correlation Coefficient -.239* -.244*

Sig. (2-tailed) .025 .022

FBS Correlation Coefficient .235* .244*

Sig. (2-tailed) .027 .022

Hypertension Correlation Coefficient .232* .234*

Sig. (2-tailed) .030 .028

Heart diseases Correlation Coefficient .218* 0.207*

Sig. (2-tailed) .036 .039

** Correlation is significant at <0.01, * significant at <0.05

68
4.3 Clinical Parameters Mean Difference Comparison between Healthy and

Metabolic Participants

Using independent sample t-test to investigate clinical parameters between those

healthy participants’ and those experienced metabolic disorders, the results in (Table.7)

revealed that the metabolic participants significantly had higher TSH level (5.63±1.36

vs1.43±0.78) and FT4 (1.75±0.32 vs1.17±0.21) p<.001 respectively. Likewise, the metabolic

participants had significantly higher reported Cholesterol (248.6±33.58 vs. 142.64±37.02),

Triglyceride (219.14 ± 54.83 vs. 93.87±23.47) and Low-density lipoprotein level

(149.02±29.22 vs.87.09±25.6) than healthy participants’. p<0.001 respectively. However, the

high-density lipoprotein was significantly lower among metabolic than healthy participants’

(41.03 ± 6.9 3vs. 55.90 ± 17.51) p<.001.

In the same context, the Fast blood sugar, Tumorigenesis-2 and Endothelin-1 were

significantly higher among metabolic than healthy participants’

(122.35±37.13vs.81.07±14.61), (2.95±1.62vs.2.36±0.48) and (6.88±4.34vs.4.65±3.52)

p<0.001 respectively. And finally those having metabolic disorders were older in age than

healthy participants (43.78±12.02vs.34.0±11.93) p<.001

69
 Table7. mean differences of clinical parameters between healthy and metabolic
participants
Parameters Metabolic disorders Healthy people t-value p-value
Mean SD Mean SD
TSH. mIU/L 5.63 1.36 1.43 0.78 17.824 <.001
FT4. ng/dl 1.75 0.32 1.17 0.21 10.073 <.001
CHOL mg/dl 248.60 33.58 142.64 37.02 14.044 <.001
TG. mg/dl 219.14 54.83 93.87 23.47 14.044 <.001
HDL. mg/dl 41.03 6.93 55.90 17.51 5.194 <.001
LDL. mg/dl 149.02 29.22 87.09 25.60 10.589 <.001
FBS. mg/dl 122.35 37.13 81.07 14.61 6.920 <.001
Tumorigenesis-2 2.95 1.62 2.36 0.48 2.367 <.001
ng/ml
Endothelin-1 6.88 4.34 4.65 3.52 2.655 <.001
fmol/ml
Age/years 43.78 12.02 34.00 11.93 3.763 <.001

70
CHAPTER FIVE

DISCUSSION

71
 Substantial evidence has proven the role of metabolic indexes, as determinants behind

development of chronic disease such as; Cardiac disorder (Abdul Ameer, 2017; Nyman et al.,

2013). Epidemiological evidence has frequently argued that genetics and epigenetics both

influence diet and life-style choices of individuals, altering their lipid profiles, blood sugar,

leading to condition of distorted metabolic indexes and onset of developing chronic

pestilences (Mishra et al., 2022; Tzika et al., 2018; Park et al., 2021).

Studies by Isomaa et al., (2001); Alexander et al., (2003); Lakka et al., (2001);

Galanopoulou et al., (2021); Zwack et al., (2021) argued that the diagnosis of metabolic

syndrome indexes are associated with the high risk for CVD and type -2 diabetes.

As Clark, & El‐Atat, (2007) implied, metabolic syndrome embodies an ensemble of

risk factors including; hypertension, pro-inflammatory status, pro-thrombotic status, high

FBS, insulin resistance, abdominal central obesity, and dyslipidemia (high LDL, low HDL

and high total cholesterol) which induce chronic disease such as; CVD.

The prevalence of distorted metabolic indexes is disproportionately higher in African-

American population as compared with other ethnicities, followed by higher percentage in

women than men of the same race (Summer et al., 2010). However, Leiter et al., (2011)

argued that the correlation between metabolic indexes and Coronary Artery disease (CAD)

seems to be heterogeneous among differed ethnicities due to different set of benchmarks as

for instance between two ethnicities of European-American and African-American, the lipid

profile in the serum of the latter one surprisingly, seemed to be less chronically inductive

which can lead to a lower frequency of CAD as compared with the European ethnicity

(Anuurad et al., 2007).

In support of metabolic indexes Su et al., (2022) discovered that the new biomarker of

Triglyceride Glucose (TYG) is anchored to intensity of CAD and such intensity would get

72
altered based on heterogeneity in patient’s glucose metabolic status, leading to high risk of

multi-vessel CAD.

In this thesis research, it was aimed to investigate the correlation relation of SSt2 and

ET-1 (known as CVD parameters) with metabolic indexes (lipid profile and FBS) in patients

who were suffering high metabolic indexes and later to assimilate the correlation value of

these indexes and their role on development of hypertension and heart disease.

The produced results of this thesis through application of spearman test

demonstrated that the majority out of 88 participated patients who suffered hypertension

(74%) followed by cardiac disorder (71%) demonstrated higher lipid profile in their serums (

cholesterol, triglycerides, LDL), followed by lower lipid profile of HDL and also, high value

of FBS which would be the indication of possible insulin resistance and pathophysiology

diagnosis of subsequent chronic diseases in accordance to following studies (Gallagher et al.,

2010; Zitzmann, 2009; Smith, & Ryckman, 2015).

This thesis findings also, demonstrated that ET-1 and SST-2 were highly expressed in

patients’ serum. The results argued that through adoption of spearman correlation test there

was a significantly positive correlation between metabolic indexes and value of ET-1 and

SST-2 as cardiac parameters.

Evidently, in cardiology field and among heart diseases, chronic heart failure is one of

the most common one with prognosis or diagnosis of preliminary hypertension (Qin, X., & Li,

2020). Chronic heart failure as Zoanni et al., (2023) implied is the end-phase results of

numerous cardiac disorders, yet its main distinguishing diagnosis are changes in ventricular

structure, cardiac pump function, neuro-humoral malfunction and other pathological

diagnoses including chronic hypertension (Bosch et al., 2017).

73
 Aronow, (2017) in his study argued that as cardiac disorder develops into chronic

stages, the cardiac heart function gradually drops due to increment in blood flow resistance

which occurs because of damaged endothelial vessels, bracing the status of cardiac failure.

In this thesis research the established results through adoption of the point biserial

correlation demonstrated that participants with diagnosis of hypertension and heart disease

were positively correlated with higher index of SST-2 and ET-1in their serums. Clearly, in

support of this thesis, current studies indicate that amplified levels of SST-2 are the predictor

of all different types of cardiac disorders including Acute Coronary Syndrome (ACS) (Jha et

al., 2018) or Pulmonary arterial hypertension (PAH) (McLaughlin et al., 2015), which both

can lead to heart failure (Chida et al., 2014).

Zheng et al., (2014) pointed at direct relation between high level of sST2 in blood

circulation and high pulmonary vascular resistance condition, high cardiac failure index and

high clinical deterioration. In accordance to study by Shao et al., (2014), vitro results asserted

elevated release of Sst2 from human endothelial cells in patients with PAH condition. In a

systematic review addressing the relation between sST2 and PAH- caused heart failure

condition, was proven that high rate of sST2 triggered high cardiac failure death toll and poor

clinical survival rate or recovery outcomes in PAH patients (Zheng et al., 2014).

Indeed, Aimo et al., (2017), in his research displayed that in PAH non-survivors as

compared with survivors, Sst2 had higher concentration level. Chida et al., (2014), also

argued that high level of sST2 could ignite spike in ventricular dilatation followed by systolic

dysfunctionality.

In accordance to results of vitro-studies Weinberg et al., (2002) argued that as cardio

myocytes get constantly prone to mechanical stress, level of Sst2 will gradually amplify

which in support of this argument, indeed Sinning et al., (2017); Dieplinger et al., (2014) also

intimated that in heterogeneous types of Cardiovascular disease (CVD) including; HF, CAD

74
or Artery Implantation (AI) the level of Sst2 substantially will intensify which has determined

its potency as a prognostic or diagnostic biomarker for cardiovascular disorders. In case of HF

under clinically verified status of myocarditis, Coronado et al., (2019) argued that the elevated

level of Sst-2 increasingly guarantees the risk of cardia arrest known as heart failure in

accordance to NEWYORK heart association class. Also, Sánchez ‐Más et al., (2014) asserted

that myocardial expression of sST2 gene intensifies in rat models suffering from myocardial

infraction problem, such intensification resulted in increased numbers of inflammatory

cytokines alongside fibrosis biomarkers, all are known as contributors behind HF.

Weinberg et al., (2002) also in support of this thesis results, implied that

intensification in level of s-ST2 plasma in subjects with myocardial infraction condition will

be entertained as a negative prognostic biomarker for overall CV risk profiling. Miftode et al.,

(2021) also, argued that Sst2 has some certain features which make it reliable to be used even

better than conventional biomarker of NT-pro-BNP for prognostic diagnosis since it possesses

more accuracy in anticipating any fatal possibility in case of HF.

In addition, as reviewed literature suggested in dysphonic patients with acute Heart

failure the level of sST2 and another biomarker known as “N-Terminal pro-Brain Natriuretic

Peptide” (NT-pro- BNP) were measured high and Sst2 was trusted even better than NT-pro-

BNP (Januzzi et al., 2007) as it appeared with prognostic predictions. (Januzzi et al., 2006)

suggested that patients with acute HF, their sST2 level was higher than (NT-pro- BNP) which

determined further the reliance on sST2 biomarker as CVD identifier. On the contrary, Jin et

al., (2018) argued that in chronic heart failure (CHF) the s ST2 and NT-Pro-BNP both can

work as prognostic and diagnostic indicators for CHF, since their serum concentrations were

risen substantially in the ventricular activities. Shah et al., (2009), also highlighted that

significance of sST2 as a strong biomarker to gauge the patients’ heart myocardial function

with HF or without HF and results verified that s ST2 biomarker was related to both “left (LV)

75
and right (RV) ventricular function” and further entertained as the prognostic biomarker

concerning fatality.

In addition, as the reviewed literature underpinned sST2 biomarker could not

differentiate between dyspneic patients caused- HF versus dyspneic patients caused- chronic

obstructive pulmonary disease, thereby, sST2 versatility in detecting and determining the

dyspneic pathological etiologies were debunked (Dieplinger et al., 2010).

In support of this Januzzi et al., (2007) pointed at the fact that dyspnea patients who

their condition was not associated with HF, subjected to a conjunction of high (NT-pro- BNP)

and low sST2 while Januzzi et al., (2006) supported the fact that patients with acute HF, their

sST2 level was higher than (NT-pro- BNP) which determined further the reliance on sST2

biomarker in CVD predictions.

In another study by miller and his colleagues (2008) which came in support of this

thesis results were also highlighted that injection of sST2 into patients’ leading to further

proliferation of fatter atherosclerotic plague followed by accumulation in numbers of T

helper-1 responses which are responsible for pro-inflammatory reactions of (IFNγ) and IL-12,

which all are prognostic elements in heart failure.

In support of above mentioned reviews as well as this thesis results, De la Fuente et

al., (2015) also, verified that climb in serum level of ST2 (Sst2) would yield to HF-induced

atherosclerosis. this thesis participants’ results demonstrated that high fasting blood sugar was

positively correlated with high SST-2, a perfect recipe for cardiac disorder. To elucidate such

outcomes, study by De la Fuente et al., (2015) posited that the serum concentration of Sst2

will ratchet up in any inflammatory driven conditions such as; type-2 diabetes which occur

due to glucose intolerance (high FBS) because of insulin resistance or insensitivity.

Altara et al., (2018) also, argued that SST2 in diabetic patients would jump up as a

result of dyslipidemia and higher FBS, hence Sst2 could be a reliable prognostic biomarker in

76
diagnoses of acute coronary syndrome in patients with diabetes and the anticipatory factor of

future re-hospitalization, relapse of myocardial infarction (MI) or mortality events.

As this thesis reviewed literature cited the sST2 operates as a decoy receptor for IL-33

such manipulative interactions will change the cardiac remodeling and health profiling.

Indeed, as literature of this thesis confirmed ST2 system is composed of two isomer receptors

which one (ST2L) has inductive effect on IL-33, guaranteeing cardiac health while, the

second (sSt2) has antagonistic effect on IL-33 mechanism and determining cardiac failure

(Konukoglu, 2018 Dominguez et al., 2017; Doganyigit et al., 2022; Michailidou et al., 2022).

Addressing the second aim which was to assimilate whether or not there would be any

positive correlation between metabolic indexes and value of ET-1, and later their aftermath

outcomes on hypertension and heart failure, in this regard the thesis results established

positive correlation.

As a matter of fact, the produced results of this thesis through application of spearman

test correlation followed by point biserial test demonstrated that 74% of patients with

hypertension and 71% with cardiac disorder had higher lipid profile in their serums followed

by lower lipid profile of HDL and also, high value of FBS which had a significant positive

correlation with high value of ET-1 and high possibility of vascular or cardiac failure.

Reviewed literatures Makino et al., (2011); Balistrieri et al., (2023) argued that the

consequences of ET-1 activation will be vasoconstriction, hypoxemia, hyperplasia,

hypertrophy, fibrosis and increment in vascular permeability. In addition, it is argued that ET-

1 is regulated by ETA and ETB receptors, mostly located on endothelium of blood vessels or

CV tissues (Hynynen, & Khalil, 2006), which makes sense why its activation will lead to

vasocontraction.

ET-1 ignites pro-inflammatory pathways of NF-KB, and pro-inflammatory secretion

of TNF-α, IL-1, and IL-6 (Poli, 2000; Ammar et al., 2021) which are all involved in

77
vasoconstriction, fibrosis, hypertrophy, hyperplasia, leading to hypertension and vascular or

heart failure.

Since the literatures Rodriguez et al. (2013); Thakkar et al. (2006) suggested that ET-1

is involved in modulation of vascular tone, in re-designing of vascular, suppression of

apoptosis, bronchoconstriction, angiogenesis, thereby such results support this thesis

argument that higher metabolic indexes will lead to higher value of ET-1 and its

vasoconstriction, vascular remodeling activity, hypertrophy and fibrosis activity.

Literatures in support of this thesis results arguing positive correlation between lipid

indexes and higher value of ET-1, leading to higher risk of heart or vascular failure. Reis et

al., (2013) argued that patients with PAH and condition of plexiform lesion in their lung

system sustain a concentrated ET-1 in their serum followed by accumulation in expression of

ET-1 receptors (Price et al., 2012).

In addition, higher level of ET-1 is an applicable parameter for cardiac adverse

prognosis which can also inform on the intensity of the cardiac or vascular disease condition

(Tsutamoto et al., 1993). Zhao et al., (2022) in his study argued that genetic and epigenetic

factors including obesity is more underlying in regulation of ET-1 and in deterring the events

of CVD-morbidity based obesity as compared with other metabolic indexes such as; glycemic

control.

In support of this thesis study, Sari et al., (2009) argued that in patients with

endothelial dysfunction, there would be a high level of LDL and abnormalities in NO level,

which could lead to high ET-1.

In support of this thesis results, studies which have displayed ET-1 role in homeostasis

and function of endothelium have also, agreed on ET-1 role in CHD and how high level of

total cholesterol and TG can elevate ET-1 concentration thus severity of cardiac or vascular

failure (Isola et al., 2020).

78
 Study by Rivera et a., (2005) demonstrated positive correlation between high

concentration of ET-1 in plasma with intensity and prognosis of heart failure, furthermore,

high ET-1 positively was attributed to NT-proBNP levels and conversely was connected to

ejection fraction (EF) which proves that high level of ET-1 could be connected to ventricular

malfunction or vascular remodeling.

To cap it all, the covered literatures in unanimous consent with this thesis results

argued on the positive correlation between high value of ET-1 biomarker in blood circulation

and cardia failure

79
CHAPTER SIX

CONCLUSION

80
 The significance rivalry role of ST2L and sST2 in circulation have been affirmed in

multiple inflammatory conditions including cancers, diabetes and cardiac disorders. sST2

indeed interferes with homeostasis and pathogenesis of abovementioned diseases through

suppression of any interaction between ST2L/IL-33 (the regulatory and modifying cytokine

ligand behind immune reaction) thus, supporting the onset of tissue fibrosis, tissue destruction

and inflammation followed by remodeling CVS functionality.

In clinical studies, sST2 is contemplated as an imperative marker for patients with

cardiac disease furthermore, sST2 is entertained as a screening biomarker for patients with

heart failure (HF).

HF is a biological and clinical complication which stems from failure of ventricular

filling or paucity of ability of blood ejection. The chief diagnosis of HF is heart fatigue and

hard breathing allied with pain and shortened breaths. There are no single diagnostic tests

available which can detect and confirm HF clinical diagnosis since, the whole diagnostic

scenario rests on historical and physical examination. currently, the optimal guideline to

characterize HF prognosis is to gauge the concentration of natriuretic peptides alongside

cardiac troponin.

Intriguingly, since 2013 the biomarker of Sst2 which ignites inflammation, fibrosis

and cardiac arrest has been incorporated into 2013-HF guidelines.

At the moment, the best guideline to identify HF prognosis is to measure the dosage of

natriuretic peptides as well as cardiac troponin.

sST2 blood concentration climbs up in inflammatory condition and heart disorders.

As the reviewed literature in this thesis postulated, Sst2 has been a valuable biomarker in

prognosis and diagnosis of CVD and myocardial infarction and furthermore, has managed to

predict heart failure prognosis in low-risk based communities.

81
 In diagnosis of Chronic Heart Failure (CHF) sST2 is very much reliable since Sst2 is

the determinant of cardio myocytes stress level and fibrocystic condition thus, such biomarker

assists to single out patients with broad spectrum of CVD. Concerning covered literatures in

this thesis SsT2 it reliable to be embedded into the clinical practice for prediction of any

possible subsequent cardiac arrest. Indeed, recently, the number of evidence, backing up

adoption of sST2 for prognosis and diagnosis purposes of chronic heart failure has been on

the rise.

Conversely, measuring s ST2 in blood sample could be a pertinent clinical prognostic

biomarker which can decide and differentiate the life expectancy of those patients who bear

condition of myocardial infarction, HF and dyspnea.

In addition to Sst2, this thesis also focused on Endothelin-1 (ET-1) which is a peptide

released by endothelial cells of vascular smooth muscle cells (VSMC) instigate vascular

contraction, as well as, pro-inflammatory reactions.

In endothelial injuries-induced atherosclerosis, the pro-inflammatory cytokine of IL-1,

IL-6 and TNF-α get activated through discharge of ET-1, as indeed, ET-1 in cooperation with

TH-17 produces the major pro-inflammatory cytokine of IL-17 and similarly generates, other

pro-inflammatory cytokines of IL-6, IL-21, IL-22 and TNF-α which all have a substantial role

in vasoconstriction, vascular contraction, fibrosis, vascular and heart failure.

As reviewed literatures suggested Endothelial overexpression of ET-1 genes in

laboratory models exhibited increment in genes expression of lipid metabolism, as well as

pro-inflammatory signal transduction. Such events led to acceleration of atherosclerosis and

vascular failure. Literatures in this thesis in support of this thesis results verified that ET-1 has

been detected in intensive hypertension, heart failure, atherosclerosis, and pulmonary

hypertension, thus clearly, ET-1 is the inducer of CVD and symptoms of metabolic syndrome.

Indeed, the harmony between performance of ETA/ETB receptor- agonists and antagonists

82
perceived to have noticeable effects on ablating the blood pressure which can bring protection

against hypertension target-organ damage thus, better management of CVD.

CHAPTER SEVEN

LIMITATIONS & FUTURE STUDIES

83
 As it was aforementioned, IL-33 has an anti-hypertrophic action inside heart via

establishing a bind with ST2L receptors located at membrane cells of cardio myocytes.

Meanwhile, as literatures argued Sst2 is a decoy receptor which in case of binding with IL-33,

attenuates IL-33 beneficial effects on the heart health. Increment in level of sST2 due to

mechanical pressure, stress or physical stretch will lead to wrong bonding between sST2 and

IL-33, thus ceasing the anti-hypertrophic and anti-apoptotic effects of IL-33 inside cardio

myocytes. In contrast, binding between IL-33 and ST2L receptor allows IL-33 manifests its

cardio-protective effects on cardio myocytes resulting in generic protection of CVS.

Despite all, this thesis just focused on the correlative relationship between Sst2

biomarker and metabolic indexes (lipid profile+ FBS) and turned away from investigating any

impact of IL-33 on ST2L and Sst2 and their aftermath outcomes on hypertension and overall

heart disease.

In addition, this thesis did not entertain potential supplementation use of IL-33 in

deterring cardiac-induced metabolic syndrome. Conversely, as one part of future plan, this

gap should be addressed by including greater number of patients with metabolic syndrome-

induced heart condition.

Meanwhile, as the literature Kawanabe, & Nauli, (2011) argued ET B1 receptors are on

endothelial cells, have a substantial role in sweeping away ET-1 from plasma, yet findings are

not well-established on this mechanism, thereby such gap could be explored further as the

subsequent research agenda.

84
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APPENDEXES

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