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CONTRIBUTORS

Maciej Banach
WAM University Hospital in Lodz, Medical University of Lodz, Lodz, Poland
Alexander E. Berezin
State Medical University of Zaporozhye, Zaporozhye, Ukraine
Giorgio Brocco
Research Institute of the Hospital 12 de Octubre (i + 12), Madrid, Spain
Filippo Cellai
Cancer Risk Factor Branch, Regional Cancer Prevention Laboratory, ISPO-Cancer
Prevention and Research Institute, Florence, Italy
Stephen A.P. Chubb
PathWest Laboratory Medicine WA, Fiona Stanley Hospital, Murdoch; School of Pathology
and Laboratory Medicine, School of Medicine and Pharmacology, University of Western
Australia, Nedlands, WA, Australia
Elisa Danese
Section of Clinical Biochemistry, University of Verona, Verona, Italy
Michelle R. Davis
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States
Sarah Feldman
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States
Andrea Galli
Department of Experimental and Clinical Biomedical Sciences, University of Florence,
Florence, Italy
Roger W. Giese
Bouve College of Health Sciences, Barnett Institute, Northeastern University, Boston, MA,
United States
Julie Hirschhorn
Medical University of South Carolina, Charleston, SC, United States
Gunnar Houen
Statens Serum Institut, Copenhagen, Denmark
Giuseppe Lippi
Section of Clinical Biochemistry, University of Verona, Verona, Italy
Martina Montagnana
Section of Clinical Biochemistry, University of Verona, Verona, Italy
Armelle Munnia
Cancer Risk Factor Branch, Regional Cancer Prevention Laboratory, ISPO-Cancer
Prevention and Research Institute, Florence, Italy

vii
viii Contributors

Marco E.M. Peluso

Cancer Risk Factor Branch, Regional Cancer Prevention Laboratory, ISPO-Cancer
Prevention and Research Institute, Florence, Italy
Simone Polvani
Department of Experimental and Clinical Biomedical Sciences, University of Florence,
Florence, Italy
Manfredi Rizzo
University of Palermo, Palermo, Italy
Fabian Sanchis-Gomar
Research Institute of the Hospital 12 de Octubre (i + 12), Madrid, Spain
Nicole H. Trier
Statens Serum Institut, Copenhagen, Denmark
Samuel D. Vasikaran
PathWest Laboratory Medicine WA, Fiona Stanley Hospital, Murdoch, WA, Australia
Yun Wang
Medical University of South Carolina, Charleston, SC, United States
Kerry J. Welsh
National Institute of Health, Bethesda, MD, United States
Zhen Zhao
National Institute of Health, Bethesda, MD, United States
Yusheng Zhu
Pennsylvania State University Hershey Medical Center, Hershey, PA, United States
PREFACE

The fourth volume of the Advances in Clinical Chemistry series for 2017 is
presented.
In Chapter 1, the role of circulating microparticles in the diagnosis and
prognosis of heart failure is reviewed. Microparticles, a heterogeneous sub-
population of extracellular vesicles containing markers derived from their
cell of origin, are continuing to stimulate considerable study and research
as diagnostic and potentially therapeutic tools in many disease processes
including heart failure. In Chapter 2, the importance of peptide antibodies
in clinical laboratory diagnostics is explored. Due to their high specificity
and sensitivity, these multifunctional molecules are indispensable in the
generation of novel clinical assays for the identification and quantification
of disease markers. In Chapter 3, biomarkers of bone turnover are
highlighted with emphasis on the c-terminal cross-linked telopeptide for
type I collagen. Accurate assessment of bone status remains a continuing
clinical problem due to the potential for catastrophic nontraumatic fracture
in the growing elderly population. In Chapter 4, testing for human papil-
loma virus, the causative agent in cervical cancer, is reviewed. The molec-
ular basis of this virus, its pathogenesis, and the epidemiology of infection
will be discussed. Guidelines for cervical cancer screening and treatment
will also be considered. In Chapter 5, the role of exercise in physiology
and pathophysiology will be explored. Although low-intensity physical
activity is considered beneficial, strenuous exercise may enhance inflam-
mation and trigger the generation of free radicals, thus mediating damage
to intracellular targets including DNA. In Chapter 6, damage to nucleic
acid via the generation of bulky chemical complexes, i.e., adducts, is
reviewed with emphasis on smoking and lung cancer. The generation of
large reactive electrophiles during detoxification increases adduct risk and
can lead to mutations in oncogenes/tumor suppressor genes, thus promoting
carcinogenesis.
I thank Volume 81 contributors and colleagues for their peer review.
I extend thanks to Shellie Bryant and Vignesh Tamilselvvan for expert
editorial support.

ix
x Preface

I hope the fourth volume for 2017 will be enjoyed. Comments and
feedback from the readership are always appreciated.
I would like to dedicate Volume 81 to Chris on the occasion of his 40th
birthday.
GREGORY S. MAKOWSKI
 CHAPTER ONE

Microparticles in Chronic Heart

Failure
Alexander E. Berezin1
State Medical University of Zaporozhye, Zaporozhye, Ukraine
1
Corresponding author: e-mail addresses: dr_berezin@mail.ru; aeberezin@gmail.com

Contents
1. Introduction 2
2. Definition, Classification, Structure, and Regulation of MPs 4
3. Biological Role and Function of MPs 8
4. Measurement of MP 14
4.1 Flow Cytometry 15
4.2 Nanoparticle Tracking Analysis 16
4.3 Western Blot Analysis 17
4.4 Nanoparticles—Surface Plasmon Resonance-Based Imaging Microscopy 17
4.5 Highly Sensitive Fluorescent Microscopy 18
4.6 Surface-Assisted Laser Desorption/Ionization Mass Spectrometry 18
4.7 Micronuclear Magnetic Resonance Technique 18
4.8 Raman Microspectroscopy 18
4.9 Small-Angle X-Ray Scattering 19
4.10 Future Perspectives for MP Detection 19
5. MPs in Cardiovascular Disease 20
5.1 Erythrocytes-Derived MPs 20
5.2 Leukocyte-Derived MPs 21
5.3 Platelet-Derived MPs 22
5.4 Endothelial Cells-Derived MPs 23
6. Diagnostic and Predictive Value of Circulating MPs in HF 26
7. MPs and Therapeutic Aspects 28
8. Conclusion 30
Acknowledgments 31
References 31

Abstract
Heart failure (HF) continues to have a sufficient impact on morbidity, mortality, and dis-
ability in developed countries. Growing evidence supports the hypothesis that micro-
particles (MPs) might contribute to the pathogenesis of the HF development playing a
pivotal role in the regulation of the endogenous repair system, thrombosis, coagulation,
inflammation, immunity, and metabolic memory phenomenon. Therefore, there is a

Advances in Clinical Chemistry, Volume 81 # 2017 Elsevier Inc. 1

ISSN 0065-2423 All rights reserved.
http://dx.doi.org/10.1016/bs.acc.2017.01.001
2 Alexander E. Berezin

large body of data clarifying the predictive value of MP numerous in circulation among
subjects with HF. Although the determination of MP signature is better than measure-
ment of single MP circulating level, there is not yet close confirmation that immune
phenotype of cells produced MPs are important for HF prediction and development.
The aim of the chapter is to summarize knowledge regarding the role of various
MPs in diagnosis and prognosis of HF. The role of MPs as a delivery vehicle for drugs
attenuated cardiac remodeling is considered.

ABBREVIATIONS
BNP brain natriuretic peptide
CV cardiovascular
EVs extracellular vesicles
HF heart failure
HFpEF chronic HF with preserved ejection fraction
HFrEF chronic HF with reduced ejection fraction
HSP heart shock protein
ICAM intracellular adhesion molecule
MI myocardial infarction
MPs microparticles
PGF placental growth factor
PLGA poly(lactic-co-glycolic acid)
STEMI ST-segment elevation myocardial infarction
VCAM vascular cell adhesion molecule
VEGF vascular endothelial growth factor

1. INTRODUCTION
Heart failure (HF) continues to have a sufficient impact on morbi-
dity, mortality, and disability in developed countries [1]. However, within
last decades, the prevalence of HF have been progressively decreased pre-
dominantly HF with reduced left ventricular ejection fraction (HFrEF)
[2]. In contrast, frequency of novel cases of HF with preserved left ven-
tricular ejection fraction (HFpEF) appears to be raised [3]. These changes
in epidemiology of HF depend in particularly on the implementation of
contemporary strategy regarding early diagnosis, prevention, treatment
of HF [4], as well as resulting in effect of aging, sex, socioeconomic status,
and comorbidities [5–8].
Cardiac dysfunction that accompanies various types of HF development
is a complex and rather controversial issue and results from the trophic effects
Microparticles in Chronic Heart Failure 3

of pure mechanical overload, and susceptibility factors (i.e., ischemia,

inflammation, overload, dysmetabolic reasons) and the neurohormonal
reaction [9]. There are current available data regarding the role of cardiac
remodeling, worsening of adrenergic signaling mechanisms in the cardiac
response, catecholamines toxicity, inflammation, thrombosis, worsening
of endothelial integrity, and endothelium injuries are common for HF onset
and development beyond etiology [9–12]. Indeed, there are evidence
regarding the important role of dysregulation of sympathetic nervous system
and renin–angiotensin–aldosterone system (RAAS) in the HF [9,13]. To our
knowledge, adrenal signaling and RAAS overdrive accompany non-
cardiovascular (non-CV) pathologies (i.e., hyperglycemia and diabetes
mellitus, obesity and metabolic syndrome, obstructive sleep apnea, and renal
disease) with cardiac impairment [12,13]. Moreover, dysregulation of intra-
cellular signaling mechanisms in HF is considered a determined higher risk
of arrhythmias and cardiac remodeling contributing to worsen the prognosis
of this disease [13]. In this context, the investigations regarding the under-
lying molecular mechanisms of failing heart could be promised in the dis-
covery of novel diagnostic tools and predictive biomarkers in several
phenotypes of HF.
Nevertheless, male gender, current smoker status, increased highly sen-
sitive troponin T, and previous myocardial infarction (MI) were associated
with new onset HFrEF, whereas female gender, history of atrial fibrillation,
increased urinary albumin excretion, and cystatin C were conferred new
onset HFpEF [14]. However, higher age, obesity, and increased
N-terminal pro-B-type natriuretic peptide (NT-proBNP) increased the risk
for both HFpEF and HFrEF [14].
Although improving the management of HF remains a priority for health
care services, the outcome of HF patients remains poor despite modern
pharmacological and none-pharmacological therapies including established
devices, i.e., cardiac resynchronization therapy devices and implantable defi-
brillator/cardioverters [8,15]. Furthermore, the clinical outcomes of both
phenotypes of HF have been occurred similar or at least not sufficiently dis-
tinguished [16] that is important challenge for contemporary medical care
service.
There is growing awareness of the role of several predictive tools
reflecting various pathophysiological stages of cardiac dysfunction develop-
ment for risk stratification of the patients with various of HF. Most studies
have described the utility of biological markers in HF for diagnosis,
4 Alexander E. Berezin

prediction, and even biomarker-guided therapy, but by now natriuretic

peptides, soluble ST2, galectin-3, and high-sensitive cardiac specific tropo-
nins were validated only [4,17]. As expected, the routine use of biomarkers
on diagnosis of HF might help to stratify the patients at higher risk of death
and clinical outcomes. In fact, both 2012 European Society of Cardiology
(ESC) Guidelines for the Diagnosis and Treatment of Acute and Chronic
Heart Failure and 2013 American College of Cardiology Foundation/
American Heart Association (ACCF/AHA) Guideline for the Management
of Heart Failure are well accepted by many clinicians regarding diagnosis and
prognosis of HFrEF. In contrast, diagnosis and prediction of HFpEF with
biomarkers is still challenging for practitioners [18]. However, there was
not a large body of evidence regarding perspectives to may provide clinically
useful prognostic information both concerning the future risk of HFpEF/
HFrEF manifestation in asymptomatic subjects, the risk of fatal events,
and primary/readmissions in the hospital in individuals for those have
already established symptomatic acute, acutely decompensated/advanced,
and chronic stable HF related to ischemic and nonischemic causes [19]. It
is suggested that multimorbidity in HF may limit the diagnostic and predic-
tive utility of biomarkers [20–22]. HF may closely associate with release of
newly detectable circulating biomarkers currently called microparticles
(MPs) [23,24]. The aim of the chapter is to summarize knowledge regarding
the role of various MPs in diagnosis and prognosis of HF.

2. DEFINITION, CLASSIFICATION, STRUCTURE, AND

REGULATION OF MPs
MPs are defined a heterogeneous subpopulation of extracellular ves-
icles (EVs) with diameter average from 100 to 1000 nm originated from
plasma membranes of mother’ cells (Table 1). EVs are phospholipid-based
endogenously produced particles (30–1000 nm in diameter), which contain
cell-specific collections of proteins, glycoproteins, lipids, nucleic acids, and
other molecules. Abundant cells including cardiomyocites, blood cells,
endothelial cells, immune cells, and even tumor cells are capable to secrete
MPs of different size and compositions [25].
Depending on their origin EVs are graduated to follow subsets, i.e., the
exosomes (30–100 nm in diameter), the microvesicles (50–1000 nm in
diameter), ectosomes (100–350 nm in diameter), small-size MPs (<50 nm
Table 1 Classification and Key Features of Extracellular Vesicles
Population Diameter Best-Characterized
of Vesicles (nm) Origin Main Contained Components Cellular Sources Markers
EV 30–1000 nm Cell Regulatory proteins (i.e., All cell types Annexin V binding, tissue
membranes heat-shock proteins, tetraspanin), factor, and cell-specific
lipids, active molecules, nucleic markers
MPs 100–1000 nm Plasma Platelets, RBC, and
acids (mRNA, miRNA), cytokines,
membranes endothelial cells
growth factors, hormones,
MV 50–1000 nm Plasma procoagulant phosphatidylserine, Platelets, RBC, and
membranes likely complement endothelial cells
Small-size <50 nm Plasma Endothelial cells CD133 +, CD63
MPs membranes
Exosomes 30–100 nm Endosomal Immune cells and tumors CD63, CD61, CD63,
membranes CD81, CD9, LAMP1,
and TSG101
Ectosomes 100–350 nm Plasma Platelets, RBC, activated TyA, C1q
membranes neutrophils, and
endothelial cells
Late 50–1000 nm Endosomal Close-packed luminal vesicles Immune cells and tumors Annexin V binding, DNA
endosomes membranes content
Apoptotic 0.5–3.0 μm Plasma Proapoptotic molecules, oncogenic Cell lines
bodies membranes receptors
Abbreviations: EVs, extracellular vesicles; MPs, microparticles; MV, microvesicles; RBC, red blood cells.
6 Alexander E. Berezin

in diameter) known as membrane particles and apoptotic bodies (1–5 μm in

diameter).
The exosomes are formed by inward budding of the endosomal mem-
brane and are released on the exocytosis of multivesicular bodies known
as late endosomes, whereas the microvesicles are attributed via budding
from plasma membranes. However, the exosomes have been predomi-
nantly labeled in the case of immune cells (macrophages, T cells,
B cells, and dendritic cells) and tumor cells. Unlike the exosomes, the
ectosomes are ubiquitous microvesicles assembled at and released from
the plasma membrane [26].
MPs are released by cellular vesiculation and fission of the membrane
of cells [27]. Under normal physiological condition, a phospholipid bila-
yer of plasma membrane of cells represented phosphatidylserine and phos-
phatidylethanoalamine in inner leaflets, whereas phosphatidylcholine
and sphingomyelin represent in the external leaflets. The asymmetrical
distribution of phospholipids in the plasma membrane is supported by
activity of three major intracellular ATP-dependent enzyme systems, i.e.,
flippase, floppase, and scramblase. Because aminophospholipids are neg-
atively charged, but phospholipids exhibit neutral charge, the main role
of intracellular enzyme systems is supporting electrochemical gradient.
Both flippase and floppase belong to the family of ATP-dependent phos-
pholipid translocases. The flippase translocates phosphatidylserine and
phosphatidylethanoalamine from the external leaflets to the inner one.
The floppase transports phospholipids in the opposite direction. Finally,
scramblase being to Ca2+-dependent enzyme system exhibits unspe-
cific ability of moving of phospholipids between both leaflets of plasma
membrane.
Importantly, disappearing of the asymmetrical phospholipid distribution
in the bilayer of the cell membrane is considered a clue for vesiculation and
forming of MPs. Indeed, both processes of apoptosis or cell activation are
required asymmetry in phospholipid distribution that leads to cytoskeleton
modifications, membrane budding, and MPs release. The mechanisms of
vesiculation directly affect genome and may mediate by some triggers
including inflammation [28], while in some cases, there is a spontaneous
release of MPs from stable cells or due to injury from necrotic cells or from
mechanically damaged cells. Particularly, the MPs are released in both con-
stitutive and controlled manners, regulated by intercellular Ca2+ and
Rab-GTP-ases and activation of μ-calpain. μ-Calpain is a Ca2+-dependent
Microparticles in Chronic Heart Failure 7

cytosolic enzyme belong to protease, which cleaves talin and α-actin, lead-
ing to decreased binding of integrins to the cytoskeleton and a reduction in
cell adhesion and integrity. Finally, interaction of the actin and myosin is a
main component for cytoskeleton modification that creates a contractile
force and drives the formation of membrane MPs.
Recently MPs are considered a cargo for various molecules. Indeed, MPs
carry proteins, RNA, micro-RNA, and DNA fragments from their cells of
origin to other parts of the body via blood and other body fluids. Within last
decade, it has become to know that MPs would act as information transfer
for target cells. However, the difference between innate mechanisms
affected the release of MPs from stable cells, activated cells, or apoptotic cells
is yet not fully investigated and requires more studies.
The majority (more than 90%) of MPs in healthy controls are of platelet
origin, whereas less than 10% originate from granulocytes and less than 5%
from endothelial cells, red blood cells, and monocytes [29]. Since all types of
particles contain surface proteins derived from their cell of origin (including
antigen-presenting cells), while there are additional biomarkers confirming
origin of the MPs (Fig. 1). The key features of several MP populations are

Tissue injury, inflammation and chemotaxis,

plaque instability, coagulation, stimulating
cytokine release, and tissue factor induction

Immunity,
atherogenesis, and tissue
injury
Coagulation,
atherogenesis, and
inflammation

Vascular tone,
permeability,
hemostasis, tissue
repair, modulating nitric
oxide, and prostacyclin
production Platelets
Endothelial cells

Abbreviation: APCs, antigen-presenting cells

Fig. 1 Origin and main biological function of several MPs.
8 Alexander E. Berezin

Table 2 Key Features of MP Populations

Types of MPs Markers Detection
Derived from resting or activated cells
Granulocytes CD24 + CD11c CD66b/CD66acde Flow cytometry
western blotting,
Monocytes CD14
mass spectrometry,
Microphages CD11b+ CD64  Ly6Clo electron microscopic
technique, SPRi
Endothelial cells CD144, CD62E microscopy
T cells CD4 or CD8
B cells CD20
Dendritic cells CD1a, CD14, CD141, CD80,
CD85, CD86
ICAM(+) cells CD54
VCAM(+) cells CD106
Platelets CD41 and/or CD61
Erythrocytes CD235a, CD44, CD47, CD55,
CFSE, annexin V, and
antiglycophorin A
Derived from Annexin V binding, CD63, CD81, Flow cytometry,
activated or CD9, LAMP1, and TSG101 capture-based assays
tumor cells
Derived from Annexin V, DNA content, histones Flow cytometry
apoptotic cells
Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; ICAM, intracellular adhesion
molecule; SPRi microscopy, nanoparticles—surface plasmon resonance-based imaging microscopy;
VCAM, vascular cell adhesion molecule.

reported in Table 2. Taking into consideration the difference between con-

tents and number of MPs various origin, it has been suggested that signature
of MPs might be used as potential biomarker of several disease, i.e., meta-
bolic and CV disease including HF.

3. BIOLOGICAL ROLE AND FUNCTION OF MPs

MPs have great potentiality in material science-based applications [30],
while initially they were recognized as cell debris beyond any biological
Microparticles in Chronic Heart Failure 9

function. Developments of technologies that attenuate recognize, determi-

nation, and measurements of MPs obtained from various cells appear to be
indispensable tool to clinical medicine [26].
Recent investigations have been shown that MPs as derivate of cellular
membrane are discussed powerful paracrine regulators of target cell func-
tions [31–33]. Indeed, MPs possess a wide spectrum of biological effects
on intercellular communication by transferring different molecules (auto-
antigens, cytokines, mRNA, iRNA, hormones, tissue coagulation factors,
and surface receptors) able to modulate other cells affected growth of tissue,
reparation, vasculogenesis, inflammation, apoptosis, infection, and malig-
nancy. However, MPs are not only cargo for biological active substances.
Growing evidence supports the idea that regarding association between
immune pattern of MPs originated from different cells (endothelial cells,
mononuclears, dendritic cells, platelets) and nature evolution of various dis-
eases including CV diseases, cancer, sepsis, eclampsia, autoimmune, and
metabolic states [34–37].
Mononuclear cell-derived MPs are involved in inflammation, blood
coagulation, and thrombosis [38,39]. Mononuclears may generate proinfla-
mmatory MPs upon activation and apoptosis with a calcium-dependent
and p38 mitogen-activated protein kinase-dependent mechanisms resulting
in impact of cytokines, bacterial products, P-selectin, histamine, catecho-
lamines, angiotensin-II, and cigarette smoking [40–44]. Furthermore,
mononuclear cell-derived MPs may appear spontaneously beyond obvious
cause in physiological state [43,44].
Circulating mononuclear cell-derived MP-like RBCs-derived MPs
may provide an additional procoagulant phospholipid surface enabling
the assembly of the clotting enzymes complexes and thrombin generation
[45,46]. It has noted the release or recruitment of procoagulant MPs at
sites of endothelium injury or worsening of integrity through P-selectin
pathway could be enhanced or triggered by tissue factor (TF) activity [47].
Converging evidences from experimental or clinical data highlight a role
for MP harboring TF in the initiation of disseminated intravascular
coagulopathy.
Additionally, their role in the regulation of lipid metabolism through
peroxisome proliferator-activated receptor-γ (PPAR-γ) is exerted. More-
over, some PPAR-γ agonists have been linked to an increased risk of
thrombotic diseases [48]. Interestingly, angiotensin-II may upregulate
the generation of procoagulant MPs by human mononuclear cells that
confirms a hypothesis about linking between the renin-angiotensin
10 Alexander E. Berezin

systems to thrombosis [49]. Therefore, there is evidence regarding that the

older persons compared with young people may exhibit different patterns
of expression of MPs with procoagulant activity derived from mononu-
clear cells [50].
It is well known that MPs appear to be found into circulation in
response to many situational changes (physiological conditions, stress, lam-
inar shear stress on endothelium) microenvironmental stimulation, coag-
ulation/thrombosis, endotoxinemia, activated cells or those undergoing
apoptosis, ischemic injury, hypoxia, and malignancy [51–53]. Optionally,
it is well known that several hemodynamic conditions via laminar shear
stress may stimulate a secretion of MPs from endothelial cells. There are
some controversial in understanding of regulation in MPs’ secretion.
There are data that confirm a close link between high endothelial shear
stress and release of MPs from endothelial cells [54]. In opposite, an inverse
association between number of endothelial cell-derived MPs in circulation
and shear stress values was found [55]. Authors have suggested that
increased release MPs following apoptosis of endothelial cells may be trig-
ger of low laminar stress.
Thus, MPs depending on their origin, structure, and inducers secretion
might possess both physiological (cell-to-cell cooperation, regulation of
endogenous reparation, angiogenesis) and pathological effects (promoting
oxidative stress, vascular inflammation, coagulation, neovascularization).
Key mechanisms by which MPs may exert their biological functions on
cells are shown in Fig. 2.
Currently, the role of MPs in pathogenesis of several diseases is eluci-
dated and the numbers of studies devoted MPs-regulated processes in the
CV diseases, rheumatic diseases, infections, are dramatically raised [55–58]
(Table 3). However, MPs play critical roles in almost all physiological events
occurring in tissues and organs (Fig. 3).
Because MPs formation and shedding involve reconstitution of cell
membrane phospholipid structure, which contains procoagulant TFs, there
is suggesting that MPs especially originating from RBCs may act as indu-
cers and regulators of coagulation. Indeed, erythrocytes actively shed
phospholipid-bound MPs [58]. MPs originating from erythrocytes are nat-
urally produced in vivo during normal aging processes or they have associ-
ated with a variety of pathophysiological conditions including hematology
diseases (hemolysis, sickle cell disease, and thalassemia), chronic kidney
disease (IgA nephropathy), uremia, stroke, acute infections, sepsis, trauma,
Microparticles in Chronic Heart Failure 11

Moving across the cells

Endocytosis of
and fuse with the
microparticles by target
plasma membrane of
cells
the target cells

Recruiting of signal
Degradation with molecules from
release of signal Microparticles microparticles by
molecules target cells

Interacting with specific

Direct interacting with
molecules on the surface
the target cells
of the target cells (fusion)

Fig. 2 The key mechanisms of MP exertion on target cells.

thrombosis/embolia, and allograft dysfunction [59–64]. Therefore,

erythrocytes-derived MPs may secrete ex vivo during cold storage of
RBCs [65].
It has been defined that lipopolysaccharides, immune complexes,
complement components, abnormal hemoglobin variants might lead to
vesiculation, membrane instability, and loss of membrane asymmetry
of erythrocytes with exposal of phosphatidylserine [59,60]. This poten-
tiates thrombin generation resulting in activation of the coagulation cas-
cade via the tenase and prothrombinase complexes responsible for
subclinical phenotypes and increase of the atherothrombotic and CV
risk [66]. However, there is serious controversial in understanding an
ability of MPs derived from RBS, leukocytes, and endothelial cells reg-
ulate coagulations cascade through generation of plasmin formation.
Endothelial cell-derived and leukocyte-derived MPs provide the real
support to plasminogen activator activity, whereas platelets-derived
and RBCs-derived MPs do not contribute to the fibrinolytic activity
of MPs isolated from peripheral blood [66]. Therefore, circulating
erythrocyte-derived MPs exhibits procoagulant properties related to fac-
tor XI presentation on their surface [67]. Furthermore, complement acti-
vation on the RBCs leads to the shedding of erythrocytes-derived-MPs
12 Alexander E. Berezin

Table 3 The Role of MPs in Pathogenesis of Diseases

Relation to Pathogenic
Type of MPs Processes Relation to Diseases
Platelet-derived MPs Coagulation, inflammatory ACS, myocardial infarction,
processes, thrombosis, and HF, tumor progression,
malignancy systemic lupus
erythematosus, vasculitis
Leukocytes-derived Endothelial dysfunction and CV diseases, infections,
MPs vascular inflammation sepsis, autoimmune disease
Endothelial Endothelial dysfunction, Hypertension, HF, CV
cell-derived MPs angiogenesis, tumor growth, diseases, diabetes, metabolic
and increased oxidative stress syndrome, obesity,
vasculitis, sepsis,
inflammation, thrombosis,
pulmonary hypertension,
acute kidney injury, chronic
kidney disease,
antiphospholipid syndrome,
preeclampsia
Erythrocytes-derived Immunomodulation, Thrombosis, vasculitis,
MPs coagulation, inflammation sepsis, ACS, myocardial
infarction, autoimmune
disease, chronic kidney
disease, antiphospholipid
syndrome, preeclampsia,
graft rejection
Immune cells-derived Cargo of tumor rejection Tumor progression
MPs antigens
Abbreviations: ACS, acute coronary syndrome; CV, cardiovascular disease; MPs, microparticles.

that may express complement and TF thus promoting inflammation and

thrombosis [68]. On the other hand, erythrocytes-derived MPs present
fibrinolytic activity mainly due to the presence of plasminogen on
them [69].
Koshiar et al. [70] concluded that the erythrocyte-derived MP surface is
suitable for the anticoagulant reactions of the protein C system, which is
important to balance the initiation and propagation of coagulation in vivo.
Therefore, extracellular protein-bound RNAs (such as micro-RNA)
derived from RBCs-MPs may play a role in transfusion-related
immunomodulation [58].
24 Alexander E. Berezin

was lower than in healthy volunteers. Among T2DM patients, an increased

level of CD31+/annexin V+ MPs and a decreased level of CD62E + MPs
were significantly associated with asymptomatic atherosclerosis. Recent
clinical studies have shown that numerous of CD31+/annexin V+ endo-
thelial cells-derived MPs strongly correlate with endothelial function and
CV outcomes in stable CAD patients [133,134]. Moreover, Huang et al.
[135] reported that increased circulating CD31+/annexin V+ EMPs and
decreased circulating EPCs predict target organ damage in hypertensive
patients. The higher concentrations of endothelial-derived MPs have been
found in patients with acute coronary syndrome, sudden cardiac death due
to acute coronary occlusion and stable angina [114]. Moreover, number of
endothelial-derived MP (CD42-CD31+) closely and inversely relate to
indexes of microvascular obstruction in acute MI patients [136]. Yet,
it has reported that patients with metabolic (obesity, metabolic syndrome,
and T2DM) and CV (stable coronary artery disease, asymptomatic athe-
rosclerosis, acute coronary syndrome, MI, hypertension, HF) may have
impaired ratio between number of apoptotic endothelial cells-derived
MPs and MPs derived from activated endothelial cells [126,137].
This imbalance was predominantly associated with increased number of
MPs derived from apoptotic endothelial cells and labeled as CD31+/
annexin V+, whereas number of activated endothelial cells-derived MPs
with the phenotype CD62E + did not change or appeared to be tendency
to decrease [137]. Indeed, elevated CD31+/annexin V+ to CD62E + ratio
was found as indicator of impaired immune phenotype of endothelial
cells-derived MPs, which allows determining pattern of MPs in CV disease
patients [138]. This phenomenon was recognized as “impaired phenotype”
of endothelial cells-derived MPs and it has related to cellular injury,
inflammation, coagulation/thrombosis leading to vascular dysfunction
and contributing to CV risk [137–139]. Finally, “impaired phenotype”
of endothelial cells-derived MPs appearing as epigenetic reprogram-
ming of mother’ cells play a pivotal role in the development of CV
complications [139–141].
Fig. 4 is reported a principal mechanism of shaping of “impaired imm-
une phenotype” of cells secreting MPs. It has been suggested that not
only endothelial cells that epigenetically transformed into “functionally
incompetence cells” might produce wide spectrum of MPs, which may
directly worse target cells. However, in HF patients, the role of the endo-
thelial cells-derived MPs is more profoundly investigated than MPs origi-
nated from other types of cells.
Another random document with
no related content on Scribd:
para condenarte que tu inocencia
para hazerte salua. Beuire en
soledad de ti y en conpañia de los
dolores que en tu lugar me dexas
los quales de conpasion
viendome quedar sola por
acompañadores me diste. Tu fin
acabará dos vidas; la tuya sin
causa y la mia por derecho, y lo
que biuiere despues de tí me será
mayor muerte que la que tú
recibirás, porque muy mas
atormenta deseada que
padecella. Pluguiera á Dios que
fueras llamada hija de la madre
que muryo y no de la que te vido
morir. De las gentes serás llorada
en quanto el mundo durare. Todos
los que de tí tenian noticia auian
por pequeña cosa este reyno que
auies de eredar, segund lo que
merecias. Podiste caber en la yra
de tu padre y dizen los que te
conoscen que no cupiera en toda
la tierra tu merecer. Los ciegos
deseauan vista para verte y los
mudos habla por alabarte y los
pobres riqueza para seruirte; á
todos eras agradable y á Persio
fuiste odiosa. Si algund tiempo
biuo, él recebirá de sus obras
galardon iusto, y avnque no me
queden fuerças para otra cosa
sino para desear morir para
vengarme dél, tomallas he
prestadas de la enemistad que le
tengo, puesto que esto no me
satisfaga, porque no podra sanar
el dolor de la manzilla la secucion
de la vengança. ¡O hija mia! ¿por
qué si la onestad es prueua de la
virtud no dió el rey mas crédito á
tu presencia que al testimonio?
En la habla, en las obras, en los
pensamientos siempre mostraste
coraçon virtuoso, ¿pues por qué
consiente Dios que mueras? No
hallo por cierto otra causa sino
que puede mas la muchedumbre
de mis pecados que el
merecimiento de tu iustedad y
quiso[274] que mis errores
comprehendiesen tu innocencia.
Pon, hija mia, el coraçon en el
cielo; no te duela dexar lo que se
acaba por lo que permanece.
Quiere el señor que padezcas
como martyr porque gozes como
bienauenturada. De mi no leues
deseo, que si fuere dina de yr do
fueres, sin tardança te sacaré dél.
¡Qué lastyma tan cruel para mi
que suplicaron tantos al rey por tu
vida y no pudieron todos
defendella y podrá vn cuchillo
acaballa el qual dexará el padre
culpado y la madre con dolor y la
hija sin salud y el reyno sin
eredera! Detengo me tanto
contigo, luz mia, y digote palabras
tan lastimeras que te quiebren el
coraçon porque deseo que
mueras en mi poder de dolor por
no verte morir en el del verdugo
por iusticia, el qual avnque
derrame tu sangre no terna tan
crueles las manos como el rey la
condicion. Pero pues no se
cumple mi deseo, antes que me
yaya recibe los postrimeros besos
de mí, tu piadosa madre; y assi
me despido de tu vista y de mas
querer la mia.

EL AUCTOR
Como la reyna acabó su habla, no
quise esperar la respuesta de la
innocente por no recebir doblada
manzilla, y assi ella y las señoras
de quien fue aconpañada se
despidieron della con el mayor
llanto de todos los que en el
mundo son hechos. Y despues
que fue yda enbié á Laureola vn
mensaiero suplicandole
escriuiese al rey, creyendo que
auria más fuerça en sus piadosas
palabras que en las peticiones de
quien auia trabaiado su libertad.
Lo qual luego puso en obra con
mayor turbacion que esperança.
La carta dezia en esta manera:

CARTA DE LAUREOLA AL REY

Padre, he sabido que me
sentencias á muerte y que se
cumple de aquí á tres dias el
termino de mi vida, por donde
conozco que no menos deuen
temer los inocentes la ventura
que los culpados la ley, pues me
tiene mi fortuna en el estrecho
que me podiera tener la culpa que
no tengo, lo qual conocerias si la
saña te dexase ver la verdad.
Bien sabes la virtud que las
coronicas pasadas publican de
los reyes y reynas donde yo
procedo; pues ¿por qué nacida yo
de tal sangre creyste mas la
informacion falsa que la bondad
natural? Si te plaze matarme, por
voluntad obralo, que por iusticia
no tienes porqué; la muerte que tú
me dieres, avnque por causa de
temor la rehuse, por razon de
obedecer la consiento, auiendo
por meior morir en tu obediencia
que beuir en tu desamor. Pero
todavia te suplico que primero
acuerdes que determines, porque,
como Dios es verdad, nunca hize
cosa porque mereciese pena.
Mas digo, señor, que la hiziera,
tan conuenible te es la piedad de
padre como el rigor de iusto. Sin
dubda yo deseo tanto mi vida por
lo que á ti toca como por lo que á
mi cunple, que al cabo so hija.
Cata, señor, que quien crueza
haze su peligro busca. Mas
seguro de caer estaras siendo
amado por clemencia que temido
por crueldad. Quien quiere ser
temido forçado es que tema. Los
reyes crueles de todos los onbres
son desamados y estos á las
vezes buscando cómo se
venguen hallan cómo se pierdan.
Los suditos de los tales mas
desean la rebuelta del tienpo que
la conseruacion de su estado; los
saluos temen su condicion y los
malos su iusticia. Sus mismos
familiares les tratan y buscan la
muerte vsando con ellos lo que
dellos aprendieren. Digote, señor,
todo esto porque deseo que se
sostente tu onrra y tu vida. Mal
esperança teman los tuyos en ti
viendote cruel contra mi;
temiendo otro tanto les darés
en[275] exemplo de qualquier
osadia, que quien no está seguro
nunca asegura. ¡O quanto estan
libres de semeiantes ocasiones
los principes en cuyo coraçon
está la clemencia; si por ellos
conuiene que mueran sus
naturales, con voluntad se ponen
por su saluacion al peligro,
velanlos de noche, guardanlos de
dia; más esperança tienen los
beninos y piadosos reyes en el
amor de las gentes que en la
fuerça de los muros de sus
fortalezas; quando salen á las
plaças el que más tarde los
bendice y alaba más temprano
piensa que yerra. Pues mira,
señor, el daño que la crueldad
causa y el prouecho que la
mansedumbre procura, y si
todavia te pareciere meior seguir
antes la opinion de tu saña que el
conseio propio, malauenturada
sea hija que nacio para poner en
condicion la vida de su padre, que
por el escandalo que pornas con
tan cruel obra nadie se fiará de ti
ni tú de nadie te deues fiar porque
con tu muerte no procure algund
su seguridad. Y lo que más siento
sobre todo es que daras contra mi
la sentencia y harás de tu
memoria la iusticia la qual será
siempre acordada mas por la
causa della que por ella misma.
Mi sangre ocupará poco lugar y tu
crueza toda la tierra. Tú serás
llamado padre cruel y yo sere
dicha hija innocente, que pues
Dios es iusto él aclarará mi
verdad. Assi quedaré libre de
culpa quando aya recebido la
pena.

EL AUCTOR
Despues que Laureola acabó de
escreuir, enbió la carta al rey con
vno de aquellos que la
guardavan, y tan amada era de
aquel y todos los otros
guardadores que le dieran libertad
si fueran tan obligados á ser
piadosos como leales. Pues como
el rey recibio la carta, despues de
avella leydo mandó muy
enoiadamente que al leuador
della le tirasen delante, lo qual yo
viendo començe de nueuo a
maldezir mi ventura y puesto que
mi tormento fuese grande
ocupaua el coraçon de dolor mas
no la memoria de oluido para lo
que hazer conuenia, y a la ora
porque auia mas espacio para la
pena que para el remedio hablé
con Gaulo tio de Laureola, como
es contado, y dixele como Leriano
queria sacalla por fuerça de la
prision, para lo quél le suplicaua
mandase iuntar alguna gente para
que sacada de la carcel la tomase
en su poder y la pusiese en saluo,
porque si el consigo la leuase
podria dar lugar al testimonio de
los malos onbres y a la acusacion
de Persio. Y como no le fuese
menos cara que a la reyna la
muerte de Laureola, respondiome
que aceutaua lo que dezia, y
como su voluntad y mi deseo
fueron conformes dió priesa en mi
partida porque antes quel hecho
se supiese se despachase. La
qual puse luego en obra, y
llegado donde Leriano estaua dile
cuenta de lo que hize y de lo poco
que acabé, y hecha mi habla dile
la carta de Laureola, y con la
compasion de las palabras della y
con pensamiento de lo que
esperaua hazer traya tantas
rebueltas en el coraçon que no
sabia qué responderme. Lloraua
de lastyma, no sosegaua de
sañudo, desconfiaua segund su
fortuna, esperaua segund su
iusticia. Quando pensaua que
sacaríe á Laureola alegrauase,
quando dudaua si lo podrie hazer
enmudecia. Finalmente dexadas
las dubdas, sabida la respuesta
que Galio me dió, començo a
proueer lo que para el negocio
conplia, y como onbre proueydo,
en tanto que yo estaua en la
corte, iuntó quinientos onbres
darmas suyos, sin que pariente ni
persona del mundo lo supiese. Lo
qual acordó con discreta
consideracion, porque si con sus
deudos lo comunicara, vnos por
no deseruir al rey dixieran que era
mal hecho y otros por asegurar su
hazienda que lo deuia dexar y
otros por ser al caso peligroso
que no lo deuia enprender; assi
que por estos inconuenientes y
porque por alli pudiera saberse el
hecho quiso con sus gentes solas
acometello; y no quedando sino
vn dia para sentenciar á Laureola,
la noche antes iuntó sus
caualleros y dixoles quanto eran
mas obligados los buenos á temer
la verguença que el peligro. Alli
les acordo como por las obras
que hizieron avn biuia la fama de
los pasados; rogoles que por
cobdicia de la gloria de buenos no
curasen de la de biuos, traxoles a
la memoria el premio de bien
morir y mostroles quanto era
locura temello no podiendo
escusallo.
Prometioles muchas mercedes y
despues que les hizo vn largo
razonamiento dixoles para qué los
auia llamado, los quales a vna
boz iuntos se profirieron a morir
con el. Pues conociendo Leriano
la lealtad de los suyos tuuose por
bien aconpañado y dispuso su
partida en anocheciendo, y
llegado a vn valle cerca de la
cibdad estuuo alli en celada toda
la noche, donde dió forma en lo
que auia de hazer. Mandó a vn
capitan suyo con cient onbres
darmas que fuese a la posada de
Persio y que matase a él y a
quantos en defensa se le
pusiesen. Ordenó que otros dos
capitanes estuviesen con cada
cinquenta caualleros a pie en dos
calles principales que salian a la
prision, a los quales mandó que
tuviesen el rostro contra la cibdad
y que á quantos viniesen
defendiesen la entrada de la
carcel entre tanto que él con los
trezientos que le quedauan
trabaiaua por sacar á Laureola. Y
al que dió cargo de matar á
Persio díxole que en
despachando se fuese á ayuntar
con él y creyendo que a la buelta
si acabase el hecho auia de salir
peleando, porque al sobir en los
cauallos no recibiese daño,
mandó aquel mismo caudillo quél
y los que con el fuesen se
adelantasen a la celada a
caualgar para que hiziesen rostro
a los enemigos en tanto quél y los
otros tomauan los cauallos, con
los quales dexó cinquenta onbres
de pie para que los guardasen. Y
como acordado todo esto
començase amanecer, en
abriendo las puertas mouio con
su gente, y entrados todos dentro
en la cibdad cada vno tuuo a
cargo lo que auia de hazer. El
capitan que fué a Persio dando la
muerte a quantos topaua no paró
hasta el que se comenzaua a
armar, donde muy cruelmente sus
maldades y su vida acabaron.
Leriano que fue á la prision,
acrecentando con la saña la virtud
del esfuerço tan duramente peleó
con las guardas que no podia
pasar adelante sino por encima
de los muertos quél y los suyos
derribauan, y como en los
peligros mas la bondad se
acrecienta, por fuerça de armas
llegó hasta donde estaua
Laureola a la qual sacó con tanto
acatamiento y cerimonia como en
tienpo seguro lo podiera hazer, y
puesta la rodilla en el suelo
besole las manos como a hija de
su rey. Estaua ella con la
turbacion presente tan sin fuerça
que apenas podia mouerse,
desmayauale el coraçon,
falleciale la color, ninguna parte
de biua tenia. Pues como Leriano
la sacaua dela dichosa carcel que
tanto bien merecio guardar, halló
á Galio con vna batalla de gente
que la estaua esperando y en
presencia de todos gela entregó,
y como quiera que sus caualleros
peleauan con los que al rebato
venian, púsola en una hacanea
que Galio tenia adereçada, y
despues de besalle las manos
otra vez fue á ayudar y fauorecer
su gente boluiendo siempre a ella
los oios hasta que de vista la
perdio. La qual sin ningun
contraste leuó su tyo a Dala, la
fortaleza dicha. Pues tornando á
Leriano, como ya ell alboroto llegó
a oydos del rey, pidio las armas y
tocadas las tronpetas y atabales
armose toda la gente cortesana y
de la cibdad; y como el tienpo le
ponia necesidad para que Leriano
saliese al canpo començolo á
hazer esforçando los suyos con
animosas palabras, quedando
siempre en la reçaga, sufriendo la
multitud delos enemigos con
mucha firmeza de coraçon. Y por
guardar la manera onesta que
requiere el rretraer, yva ordenado
con menos priesa que el caso
pedia, y assi perdiendo algunos
delos suyos y matando a muchos
de los contrarios llegó a donde
dexó los cauallos, y guardada la
orden que para aquello auie dado,
sin recebir reues ni peligro
caualgaron él y todos sus
caualleros, lo que por ventura no
hiziera si antes no proueyera el
remedio. Puestos todos como es
dicho a cauallo, tomó delante los
peones y siguio la via de Susa
donde auie partido, y como se le
acercauan tres batallas del rey,
salido de paso apresuró algo ell
andar con tal concierto y orden
que ganaua tanta onrra en el
retraer como en el pelear. Yva
siempre en los postreros
haziendo algunas bueltas quando
el tiempo las pedia, por entretener
los contrarios, para leuar su
batalla mas sin congoxa. En el fin,
no auiendo sino dos leguas como
es dicho hasta Susa, pudo llegar
sin que ningund suyo perdiese,
cosa de gran marauilla, porque
con cinco mill onbres darmas
venia ya el rey enbuelto con él.
El qual muy encendido de coraie
puso a la ora cerco sobre el lugar
con proposito de no leuantarse de
allí hasta que dél tomase
vengança. Y viendo Leriano que
el rey asentaua real repartio su
gente por estancias segund sabio
guerrero. Donde estaua el muro
mas flaco ponia los mas rezios
caualleros; donde auia apareio
para dar en el real ponia los mas
sueltos; donde veya mas
dispusicion para entralle por
traycion ó engaño ponia los mas
fieles. En todo proueya como
sabido y en todo osaua como
varon. El rey como aquel que
pensaua leuar el hecho a fin,
mandó fortalecer el real, y proueó
en las prouisiones; y ordenadas
todas las cosas que a la hueste
cumplia, mandó llegar las
estancias cerca de la cerca de la
villa, las quales guarnecio de muy
bona gente, y pareciendole
segund le acuciaua la saña gran
tardança esperar á tomar á
Leriano por hanbre, puesto que la
villa fuese muy fuerte, acordo de
conbatilla lo qual prouo con tan
brauo coraçon que vuo el cercado
bien menester el esfuerço y la
diligencia. Andaua sobre saliente
con cient caualleros que para
aquello tenia diputados; donde
veya flaqueza se esforçaua,
donde veya coraçon alabaua,
donde veya mal recaudo proueya.
Concluyendo, porque me alargo,
el rey mandó apartar el combate
con perdida de mucha parte de
sus caualleros, en especial de los
mancebos cortesanos que
sienpre buscan el peligro por
gloria. Leriano fue herido en el
rostro y no menos perdió muchos
onbres principales. Pasado assi
este conbate diole el rey otros
cinco en espacio de tres meses,
de manera que le fallecian ya las
dos partes de su gente, de cuya
razon hallaua dudoso su hecho,
como quiera que en el rostro, ni
palabras, ni obras nadie gelo
conosciese, porque en el coraçon
del caudillo se esfuerçan los
acaudillados. Finalmente como
supo que otra vez ordenauan dele
conbatir, por poner coraçon a los
que le quedauan hizoles una
habla en esta forma.

LERIANO Á SUS CAUALLEROS

Por cierto, caualleros, si como
soys pocos en número no
fuésedes muchos en fortaleza yo
ternia alguna duda en nuestro
hecho segun nuestra mala
fortuna, pero como sea mas
estimada la virtud que la
muchedumbre, vista la vuestra
antes temo necesidad de ventura
que de caualleros y con esta
consideracion en solos vosotros
tengo esperança. Pues es puesta
en nuestras manos nuestra salud,
tanto por sustentacion de vida
como por gloria de fama nos
conviene pelear. Agora se nos
ofrece causa para dexar la
bondad que eredamos á los que
nos han de eredar, que
malauenturados seriamos si por
flaqueza en nosotros se acabasse
la eredad. Assi pelead que libreys
de verguença vuestra sangre y mi
nombre. Oy se acaba ó se
confirma nuestra onrra;
sepamosnos defender y no
avergonçar, que muy mayores
son los galardones de las
victorias que las ocasiones de los
peligros. Esta vida penosa en que
bevimos no sé porqué se deua
mucho querer, que es breue en
los días y larga en los trabaios, la
qual ni por temor se acrecienta, ni
por osarse acorta, pues quando
nascemos se limita su tiempo, por
donde escusado es el miedo y
devida la osadía. No nos pudo
nuestra fortuna poner en meior
estado que en esperança de
onrrada muerte ó gloriosa fama.
Cudicia de alabança, auaricia de
onrra acaban otros hechos
mayores quel nuestro; no
temamos las grandes conpañas
llegadas al real, que en las
afrentas los menos pelean; á los
sinples espanta la multitud de los
muchos y á los sabios esfuerça la
virtud de los pocos. Grandes
apareios tenemos para osar; la
bondad nos obliga, la iusticia nos
esfuerça, la necesidad nos
apremia. No ay cosa porque
deuamos temer y ay mill para que
deuamos morir. Todas las
razones, caualleros leales, que os
he dicho eran escusadas para
creceros fortaleza pues con ella
nacistes, mas quíselas hablar
porque en todo tiempo el coraçon
se deue ocupar en nobleza, en el
hecho con las manos, en la
soledad con los pensamientos, en
conpañia con las palabras como
agora hazemos, y no menos
porque recibo ygual gloria con la
voluntad amorosa que mostrays
como con los hechos fuertes que
hazeys. Y porque me parece
segund se adereça el combate
que somos costreñidos á dexar
con las obras las hablas, cada
vno se vaya á su estancia.

EL AUCTOR
Con tanta constancia de animo
fue Leriano respondido de sus
caualleros que se llamó dichoso
por hallarse dino dellos; y porque
estaua ya ordenado el conbate
fuese cada vno á defender la
parte que le cabia; y poco
despues que fueron llegados
tocaron en el real los atauales y
tronpetas y en pequeño espacio
estauan iuntos al muro cincuenta
mill onbres los quales con mucho
vigor començaron el hecho,
donde Leriano tuuo lugar de
mostrar su virtud y segund los de
dentro defendian creya el rey que
ninguno dellos faltaua. Duró el
conbate desde medio dia hasta la
noche que los departio. Fueron
heridos y muertos tres mill de los
del real y tantos de los de
Leriano, que de todos los suyos
no le auian quedado sino ciento y
cincuenta, y en su rostro segund
esforçado no mostraua ayer
perdido ninguno, y en su
sentimiento segund amoroso
parecia que todos le auian salido
del anima. Estuuo toda aquella
noche enterrando los muertos y
loando los biuos, no dando menos
gloria á los que enterraua que á
los que veya. Y otro día en
amaneciendo, al tienpo que se
remudan las guardas acordo que
cincuenta de los suyos diesen en
vna estancia que vn pariente de
Persio tenía cercana al muro,
porque no pensase el rey que le
faltaua coraçon ni gente; lo qual
se hizo con tan firme osadia que
quemada la estancia mataron
muchos de los defensores della, y
como ya Dios tuviese por bien
que la verdad de aquella
pendencia se mostrase, fue preso
en aquella vuelta vno de los
damnados que condenaron á
Laureola, y puesto en poder de
Leriano mandó que todas las
maneras de tormento fuesen
obradas en él hasta que dixese
porqué leuantó el testimonio, el
qual sin premia ninguna confesó
todo el hecho como pasó. Y
despues que Leriano de la verdad
se informó, enbiole al rey
suplicandole que saluase á
Laureola de culpa y que mandase
iusticiar aquel y á los otros que de
tanto mal auien sido causa. Lo
qual el rey sabido lo cierto aceutó
con alegre voluntad por la iusta
razon que para ello le requeria. Y
por no detenerme en las
prolixidades que en este caso
pasaron, de los tres falsos onbres
se hizo tal la iusticia como fue la
maldad. El cerco fue luego alçado
y el rey tuuo á su hija por libre y á
Leriano por desculpado, y llegado
á Suria enbió por Laureola á
todos los grandes de su corte, la
qual vino con ygual onrra de su
merecimiento.
Fue recebida del rey y la reyna
con tanto amor y lagrimas de
gozo como se derramaran de
dolor; el rey se desculpaua, la
reyna la besaua, todos la seruian
y assi se entregauan con alegria
presente de la pena pasada. A
Leriano mandole el rey que no
entrase por estonces en la corte
hasta que pacificase a él y a los
parientes de Persio, lo que recibio
a graveça porque no podria ver á
Laureola, y no podiendo hazer
otra cosa sintiolo en estraña
manera. Y viendose apartado
della, dexadas las obras de
guerra, boluiose á las congoxas
enamoradas, y deseoso de saber
en lo que Laureola estaua rogome
que le fuese á suplicar que diese
alguna forma onesta para que la
pudiese ver y hablar, que tanto
deseaba Leriano guardar su
onestad que nunca penso hablalla
en parte donde sospecha en ella
se pudiese tomar, de cuya razon
él era merecedor de sus
mercedes. Yo que con plazer
aceutaua sus mandamientos,
partime para Suria, y llegado allá,
despues de besar las manos á
Laureola, supliquele lo que me
dixo, a lo quél me respondió: que
en ninguna manera lo haria por
muchas causas que me dió para
ello. Pero no contento con dezir
gelo aquella vez todas las que
veya gelo suplicaua; concluyendo
respondiome al cabo que si mas
en aquello le hablaua que
causaria que se desmesurase
contra mi. Pues visto su enoio y
responder fui á Leriano con graue
tristeza y quando le dixe que de
nueuo se comenzauan sus
desauenturas, sin duda estuuo en
condicion de desesperar. Lo qual
yo viendo, por entretenelle, dixele
que escriuiese á Laureola
acordandole lo que hizo por ella y
estrañandole su mudança en la
merced que en escriuille le
començo á hazer. Respondiome
que auia acordado bien, mas que
no tenia que acordalle lo que auia
hecho por ella pues no era nada
segund lo que merecia y tanbien
porque era de onbres baxos
repetir lo hecho; y no menos me
dixo que ninguna memoria le
haria del galardon recebido
porque se defiende en ley
enamorada escreuir que
satisfacen se recibe, por el peligro
que se puede recrecer si la carta
es vista, asi que sin tocar en esto
escriuio á Laureola las siguientes
razones:

CARTA DE LERIANO Á
LAUREOLA
Laureola, segund tu virtuosa
piedad, pues sabes mi pasion, no
puedo creer que sin alguna causa
la consientas, pues no te pido
cosa á tu onrra fea ni á ti graue.
Si quieres mi mal ¿por qué lo
dudas? á sin razon muero,
sabiendo tú que la pena grande
assi ocupa el coraçon que se
puede sentir y no mostrar. Si lo
has por bien pensado que me
satisfazes con la pasion que me
das porque dandola tú es el
mayor bien que puedo esperar,
iustamente lo harias si la dieses a
fin de galardon. Pero ¡desdichado
yo! que la causa tu hermosura y