ELSEVIER Contents lists available at ScienceDirect journal homepage: www.olsevier.com/locate/dsx Diabetes & Metabolic Syndrome: Clinical Research & Reviews Ai Review The role of collagen homeostasis in the pathogenesis of vascular disease associated to insulin resistance 2 Maria M, Adeva-Andany’, Elvira Castro-Quintela, Carlos Fernandez-Fernandez, Natalia Carneiro-Frei , Matilde Vila-Altesor mveral Meicine Depremen, Hosp General ua Cardona, Fado bazin sn, 1546, Fer pain ARTICLE INFO ‘Aeceped 1 Api 2019, 1. Introduction Insulin resistance is associated with devastating vascular dam- age that is not fully explained by traditional carciovascular tsk factors ar hypergiycemia, as the risk of cardiovascular disease re- mains elevated despite adequate glycemic control []. Patients with Insulin resistance experience a systemic disorder of collagen ho- ‘meostasis that may contribute to eause vascular injury among ather complications. Collagen abundance, structural properties and fibrillar arrangement are abnormal on the extracellular matrix of every tissue examined, including skin, tendons, bone, skeletal "muscle, eye, peripheral nerves, kidney, lung, liver. and blood vessels of any size [2]. Excessive collagen deposition occurs in the inter- stitial space of tissues and contributes to cause clinical manifesta- tions of diabetes such as fibrotic skin, predisposition to bone fractures, impaired lung function, diastolic dysfunction, and liver disease. Heavy deposits of collagen are present in the capillary basement membrane and the vascular wall of arteries af any size. In capillaries, the ensuing thickening of the capillary basement ‘membrane typifies microangiopathy associated to insulin resis- ‘ance. In the arterial wall, the striking accumulation of collagen on the tunica media causes intima-media thickening, Histological ex- aminations reveal similar findings (accumulation of disordered collagen) in blood vessel of any size from capillaries to large ar- teries, suggesting a common pathogenic mechanism to microvas- cular and macrovascular disease [3.i]. Histological ‘microangiopathy (thickening ofthe capillary basement membrane) Js present in normogiycemic subjects with high risk of developing, * Corresponding autor neds sso ns ML Ado Anda 1871-4021je 2019 Published by Elsevier Lid on behalf of Diabetes India diabetes but is very rare in hyperglycemic patients with diabetes secondary to pancreatitis. Glucagon deficiency in patients with global pancreas damage prevents insulin resistance from devel- ping and patients with pancreatitis tend to experience exquisite insulin sensitivity. These findings indicate that insulin resistance rather than hyperglycemia is the major factor responsible for vascular injury [5], Vascular smooth muscle cells synthesize extracellular matrix in the tunica media of the arterial wall, The interaction between smooth muscle cells and the surrounding extracellular matrix is essential ta normal structure and function of the blood vessel, Any modification in the composition of the extracellular matrix is sensed by smooth musele cells via plasma 'membrane receptors and elicits appropriate cellular adjustment, so ‘hat the extracellular matrix regulates cellular function. Collagen is 4 major component of the extracellular matsix throughout the human body. Alteration of collagen homeostasis in the arterial wall ‘may play a crucial role in the pathogenesis of vascular disease associated to insulin resistance, The cause of the profound disruption of collagen metabolism associated with insulin resis- tance is unknown [5] 2. Insulin resistance is associated with a systemic alteration of collagen homeostasis, Collagen is a crucial component of the extracellular matrix in human tissues. Three polypeptide chains (named a chains) twisted around each other into a triple helix compose one collagen mole- cle. numberof distinet2-chains have been identified in humans, Collagen molecules may assemble as homotrimers or heterotrimers of a chains. Mutations in the different genes coding each a-chain Jead to a variety of human disorders, including the syndrome of Ehlers-Danlos, characterized by fragility of the blood vessel wallwre ene Ate-dany ea Babes Mtoe Syarome: Cn Reser & Redes 15 (2019) 1877-186 ‘Amino acids in = chains are arranged ina specific repeating pattern, Gly-X-¥, Every third amino acid in any collagen chain is glycine. The presence of glycine at this positon is essential to bring the {three 2 chains together and form the triple helix. Proline and 4- hydroxyproline occupy frequently the X and ¥ positions, respec- tively, and contribute to the stability ofthe triple helix. Hydroxyl- ation of profine is @ key step to collagen formation, as a critical ‘number of prolyl residues must be hydroxylated to form a stable triple helix [7]. Hydroxyproline concentration in a tissue has been used as an index of the amount of collagen present in that tissue. Human collagen contains about 12-14% 4-hydroxyproline while clastin contains about 1.5% [8]. A number of collagen types achieve specific functions in every tissue, but structural and functional ifferences berween them are not well defined. Some types of collagen assemble into fibcis by formation of covalent cross-links between neighboring collagen molecules. Cross-linking of ‘collagen into fibrils occurs in several enzymatic steps. Collagen f= brils may represent the base for hyciroxyapatite crystals to deposit physiologically in bone tissue and pathologically in sot tissues such as the arterial wall [7]. Properties of human collagen undergo a marked deterioration with age that may be related with insulin resistance, as decline of insulin sensitivity is a physiological feature of aging. Consistently, ‘age-related collagen modifications mimic those observed in Younger patients with insulin resistance. Skin collagen undergoes ‘age-related changes characterized by increased stiffness and! frag- ‘mentation of collagen fibrils (9. In human bone, there is an age- related change in collagen content and cross-links from 3 to 89 years of age [10], Excessive collagen deposition resulting in thick- ‘ening of the basement membrane of capillaries associated with age has been documented in the retina the skeletal muscle, and the liver [1113]. A prominent feature of the aging process in the arterial wall is the progressive accumulation of collagen in the tunica media, In human aortas obtained at autopsy, mechanical ‘measurements suggest that collagen accumulation reduces the clastic response ofthe arterial wall [81,15], thas been long known that insulin resistance is associated with ‘marked alterations in the abundance, structure, and layout of collagen throughout the fnuman body. Collagen is less soluble by ‘acid and pepsin and less digestible by collagenases and cyanogen, bromide in patients with diabetes compared to controls. Defective collagen metabolism in the interstitial space contributes ta cause diabetic complications, such as skin changes, restricted joint mobility, tendon diseases, predisposition to bone fractures, and ‘layed wound healing [3,16], Alteration of collagen homeostasis, precedes the clinical onset of diabetes, having been detected in patients with insulin resistance. In 1975, Hamlin etal. stated: “The possibility must be considered that a connective ussue defect may precede the diabetes and play a sole in its pathogenesis" [17] Ia review of the literature, patients with diabetes have more than three times the odds of tendinopathy compared fo controls. Diabetes predisposes to a wide range of tendon diseases, including ‘contracture, rupture, impaired tendon healing, and asymptomatic ‘Achilles tendinopathy [18]. Abnormal collagen structure in tendons may contribute to cause tendinopathy in patients with diabetes. Properties of collagen (evaluated by enzymatic digestion) from the central tendon ofthe diaphragma obtained at autopsy ae altered in patients with diabetes. The collagen fibrils become stiff and insol- tuble compared to control subjects [17]. The ultrastructure of collagen in extensor tendons (evaluated by X-ray diffraction) shows ‘major changes in patients with diabetes compared to normal ten- dons [19]. In a cross-sectional study, Achilles tendon stiffness was higher and tendon fibril density was greater in patients wit di betes compared to control subjects [20] Limited joint mobility occurs commonly among, patients with diabetes and may affect any joint, including wrist, elbow, ankle, and spine, Joint stifiness is associated with sclerotic and microvascular ‘complications. Knee osteoarthritis is mae frequent inpatients with diabetes and the complication rate of total knee arthroplasty is higher compared to controls. Aseptic loosening and need for revi- sion surgery alter total knee arthroplasty occur more frequently in patients with diabetes, reducing functional outcome [21] Patients with diabetes have an increased incidence of interver- tebral disc herniation compared to nondiabetic individuals. Collagen isan important component ofthe intervertebral disc. Type 1 collagen accounts for 1~2% of all disc collagen, but its binding to type ll fbrils enhances the resistance of the disc, The evelof type IX collagen in the intervertebral disc is reduced in patients with dia- betes compared to control subjects. This reduction may facilitate the development of disc herniation [22] ‘A meta-analysis of the literature concluded that the risk of bone fracture is more elevated in patients with diabetes compared to ‘control subjects. Glycated hemoglobin (HDAIC) was not linked to bone mineral density [23]. Bone extracellular matrix is composed of collagen fibrils and mineral. Type I collagen is the most abundant collagen in bone, although types ll and V are also present. The elevated risk of bone fracture associated with diabetes is not ‘explained by 2 reduction in bone mineral density, suggesting that the poor bone quality is related to defective bone collagen [10] Dura mater is a dense collagen structure, The properties of dura ‘mater collagen obtained at autopsy are markedly altered in patients ‘with diabetes. Compared to normal specimens, dura mater collagen from diabeti patients is much more resistant co solubilization [24]. Ina biracial study with Chinese and Caucasian participants, collagen content in adipose tissue (assessed by the level of hy- droxyproline) increased with the degree of insulin resistance, evaluated by the homeostasis model assessment of insulin resis- tance (HOMAAIR) index, in both population groups. The level of hydroxyproline (ancl the extent of fibrosis) in adipose tissue was higher among more insulin-resistant patients [25]. Analogous re- sults were obtained among obese subjects that underwent hyper- insulinemic euglycemic clamp to evaluate insulin sensitivity. The ‘extent of fibrosis in adipose tissue was higher in more insulin resistant subjects compared to those more insulin sensitive. Fibrosis of adipose tissue in obese subjects is associated with in- sulin resistance [26] ina study that reeruited healthy males, the synthesis of type I and type Ill collagens in the adipose tissue increased in response to insulin resistance. Insulin resistance was induced by overfeeding-related weight gain and assessed by ‘euglycemic hyperinsulinemic clamps, Collagen content was ‘measured in biopsy samples, Insulin resistance was followed by elevated content of type! and type Il collagens in the adipose tissue ba 3. Collagen homeosta ‘with insulin resistance and microangiopathy associated ‘Numerous investigations have long documented the presence of systemic capillary Involvement in patients with insulin resistance, ‘characterized by collagen accumulation and subsequent thickening, ‘of the capillary basement membrane, the histopathological ball- mark of diabetic microangiopathy [3.5,28- 20]. This alteration precedes the clinical diagnosis of type 2 diabetes, so that capillary basement membrane thickening can be demonstrated in predia- betie subjects prior to detectable hyperglycemia. In contrast, the ‘width ofthe capillary basement membrane in patients with type 1 diabetes is normal at the time of diagnosis and thickens with increasing duration of the disease. These findings suggest that in- sulin resistance is a major factor causing microvascular damage [29-31], Histological examinations reveal that thickening of theLAL Ade nny Dees & Metab: yatome inc Research Reviews 12 (2013) 177-1883 oo basement membrane occurs in the capillaries of every tissue examined, including retina, kidney, skeletal muscle, skin, liver, Jung, and peripheral nerves. Microangiopathy may contribute ta cause complications of diabetes such as retinopathy, nephropathy and peripietal neuropathy [3], 31. Gingival tissue In patients with diabetes, basement membrane thickening is ‘dentified in terminal arterioles and capillaries from gingival tissue, compared to control subjects. In prediabetic patients, approxl- ‘mately 503 of the terminal arteriales and capillaries show base- ‘ment membrane thickening, In obese subjects, this alteration Is cccasionally present while no change is observed in control sub- jects [1 32. Kidney Diabetic nephropathy is characterized by thickening of the basement membrane ofthe glomerular capillaries and expansion of ‘mesangial matrix (Kimmelstiel and Wilson intercapillary sclerosis). ‘Thickening of the basement membrane of kidney tubules and capsular epithelivm is alsa present [1225]. Mesangial content of type VI collagen has been found 2.8-fold higher in patients with diabetes compared to control subjects [15] 33. Be Microangiopathy affecting retinal capillaries has been long documented in patients with diabetes. Diabetic retinopathy is characterized by capillary aneurysms and a cluster of exudates and hemorrhages in the retina surrounding the aneurysms. Thickening, ff the retinal capillary basement membrane is observed [22] Electron microscopy and histochemical studies performed on hu- ‘man eyes confirm that the retinal capillary wall is thickened in patients with diabetes campared to normal samples [11]. Micro- vascular abnormalities in the choroid have also been identified in patients with diabetes [36]. The prevalence of brain micro- angiopathy (evaluated as cerebral microbleces on magnetic reso- rnance imaging) is higher in diabetic patients with proliferative retinopathy, compared to nondiabetic subjects and diabetic pa- tients without retinopathy, suggesting that retinal changes may indicate a generalized microangiopathy [37] 3.4, Nervous system Brain small vessel disease expressed on magnetic resonance Simaging as lacunar Infarctons, white matter lesions and micro- bieedss common inpatients with diabetes compared with healthy Individuals. Cerebral microangiopathy is associated with abnormal sin capillary perfusion (assessed by capillary microscopy), Su- gesting that cerebral microangiopathy is 4 manifestation of gener- alized microvascular dysfunction [38,39}. ‘The capillary basement membrane of sural nervesis thicker (by 52.9%) in patients with diabetes compared to control nerves. Endoneuri microangiopathy is closely associated with clinical diabetic polyneuropathy {40}. Similarly to the capillary basement ‘membrane, the thickness ofthe perineural cll basement mem- brane (asessed by electron microscopy) is greater in sural nerves from patients with diabetes compared to nondiabetic subjects Thickening of perineural cel basement membrane may precede the recognition of hyperglycemia, n sciatic nerve samples from patients with diabetes, aggregation of type VI collagen fibrils i ‘dentified adjacent to te basement membranes of perineuril cells bs} 435, skin ‘Microangiopathy targets the skin of patients with diabetes. Histological examinations show thickening of the basement ‘membrane of dermal capillaries and deposition of hyaline periodic Aacid-Schiff (PAS)-positive material an the normpally fibrillar area external to the endothelial cell, compared to normal control bi- opsies [28,42]. Dermal collagen is also abnormal in the intetstitial space. Atomic force microscopy images of normal human skin reveal tightly packed and well-organized dermal collagen fibrils, In contrast, dermal collagen fibrils are fragmented and disorga- nized in patients with diabetes [9]. Patients with diabetes show increased cross-linking of the skin collagen fibrils, Skin collagen is stiffer and more insoluble than that of nondiabetic indivicuals 20} On skin biopsy samples, thickening of the epidermis and dermis with collagen accumulation is observed in patients with diabetes compared to controls. Type Ill collagen is the predomi- rant type deposited in diabetic skin, Fragmented and cross-linked collagen contributes to cause clinical abnormalities such as thick tight skin typical of diabetes [21]. Fibrocytes from diabetic pa- tients do not increase the expression of type | collagen on stim- ulation, unlike normal fibrocytes. This abnormality may Contribute to explain impaired wound healing [13], Diabetes is 2 frequent finding among patients with cutaneous collagenous vasculopathy, an idiopathic skin microangiopathy characterized by dilated dermal capillaries with walls thickened by hyaline ‘material containing collagen IV by immunohistochemisry. Simi- larly, collagenous colitis has been reported in patients with type 1 diabetes, Endoscopic biopsies showed marked thickening of the subepithelial collagen plate (Masson's trichrome positive, Congo red negative) in samples from stomach, duodenum, terminal ileum, and colon [44 36, Sketetat muscle ‘Thickening of the capillary basement membrane in the skeletal muscle is avery constant finding among patients with diabetes. The ‘width (measured by electron microscopy) ofthe capillay basement membrane is over twice compated to normal subjects. Hypersly= cemic patients with diabetes due to pancreatitis rarely experience this alteration while thickening of the capillary basement mem- brane is found in approximately 50% of normoglycemic subjects ‘with family history of diabetes, suggesting that insulin resistance rather than hyperglycemia is the major factor responsible for microvascular disease [5,23]. Follow-up skeletal muscle biopsy examinations on the subjects with family history of diabetes revealed that patients who developed diabetes (47%) during the {ollow-up period showed progressive thickening of their capillary basement membrane compared to the initial measurement [45]. Collagen synthesis increases inthe skeletal muscle of healthy males Jn response to insulin resistance. Participants underwent over- autttion to 10% weight gain, Skeletal muscle biopsies were ob- tained to measure collagen content and_hyperinsulinemic euglycemic clamps were performed to assess insulin sensitivity Overfeeding and weight gain worsened! insulin sensitivity. In response to insulin resistance, an increase in the mRNA level of type 1. 1, V, and VI collagens was observed in the skeletal muscle. Type Ill and type Vi collagen protein levels were unchanged [27] Comparable results were documented in healthy subjects who ‘underwent lipid infusion to induce insulin resistance, compared to saline infusion. Following insulin resistance, an overexpression of {ype I and type ll collagen genes with an increase of mRNA levels ‘was observed on biopsy samples of skeletal muscle [46], Consis- tently, the amount of fype | and type Il collagens in skeletal muscle (measured in biopsy samples) is increased in patients with insulinvss ene Ate-dany ea Babes Mtoe Syarome: Cn Reser & Redes 15 (2019) 1877-186 resistance (assessed by_hyperinsulinemic euglycemic clamp) compared to insulin-sensitive subject [47} 37. Heart “Myocardial microangiopathy is a complication of diabetes. His- tological examination of autopsy samples document intense PAS- positive deposits on the smallest branches of coronary arteries in patients with diabetes compared to control subjects. The presence (of PAS-positive material in the small vessels ofthe myocardium was closely related tothe presence of nodular glomeruloscleross in the kidney [8]. In addition to myocardial microangiopathy, interstitial fibrosis with accumulation of type Il collagen is typically present in the myocardium of patients with diabetes compared to controls [29,50]. Type I collagen mRNA level is elevated by twofold in car- diac fibroblasts (cells that synthesize extracellular matrix} derived from patients with type 2 diabetes compared t controls [51]. The association between insulin resistance and serum mazkers of collagen synthesis has been investigated in obese subjects. Serum procollagen type ll aminopeptide was measured and insulin sensitivity was evaluated by the HOMA-IR index. Regression ana- Iyses showed that serum procollagen type Ill aminopeptide level was independently correlated with HOMA.IR and HDL-c, suggest- Ing that markers of cardiac fibrosis are related to insulin resistance [50]. Myocardial interstitial fibrosis impairs diastolic function hin- ‘ering ventricular relaxation and contributes to increase the risk of heart failure in patients with insulin resistance. in addition, inter- stitial fibrosis may predispose (0 cardiac arthythmias, Impaired diastolic function has been reported in patients with insulin resis- tance and obesity [50] 38. Lung Diabetic microangiopathy occurs in the lung. Electron micro- scopy examination on human lung samples shows thickening of the alveolar capillary basement membrane. In addition, the thickness of the basement membrane of bronchial epithelial cells is greater in patients with diabetes compared to control subjects and interstitial fibrosis with collagen accumulation is observed. There is n0 close correlation between fasting blood glucose or HbAIc level and basement membrane thickness in lung tissue [35,52]. Patients with insulin resistance suffer from subclinical lung dysfunction long before the clinical onset of type 2 diabetes. Cross-sectional studies in different population groups reveal an independent association between insulin resistance and impaited ventilatory function, including reduced forced vital capacity (FVC) and forced expiratory volume in 1's (FEV1). I has been suggested that measurement of Jung function by spirometry may be useful to estimate the pro- _sression of systemic microvascular disease in patients with insulin resistance [53] In nondiabetic subjects the decline of FYC and FEV1 ‘was prospectively investigated during 5 years. After adjustment for confounders, subjects who developed diabetes during the follow- up period had greater deciine of ventilatory function compared to those who did not develop diabetes [=4]. The prospective associ tion between pulmonary function and incidence of diabetes was investigated by the National Health and Nutrition Examination Survey Epidemiologic Follow-Up Study, a cohort study of US adults. Individuals with restrictive lung disease are at risk for developing diabetes, but obstructive lung disease is not associated with di betes incidence [55] 39, Liver In the normal liver, the perisinusoidal space of Disse contains ‘occasional small deposits of collagen fibrils including types II, 1V, ‘and XIX, Patients with insulin resistance typically develop a non- Cirrhotic form of hepatic sinusoidal fibrosis (diabetic hepato- sclerosis) that is not associated with nonalcoholic steatohepatitis. ‘On histological examination, the most striking feature isthe pres cence of dense perisinusoldal fibrosis. On electron microscopy, ant increase in the amount of collagen fibrils within the space of Disse ‘with abundant collagen bundles and segments of perisinusoidal basement membrane-like deposition are observed among patients ‘with diabetes while collagen deposits are absent in control sub- jects, Perisinusoidal collagen accumulation is associated with hya- Tine thickening of small hepatic artery branches and perivascular fibrosis composed mainly of fibrillar collagen, Patients with dia- betes who develop hepatic sinusoidal fibrosis (collagenization of the space of Disse) usually have evidence of microvascular disease, ‘such a5 retinopathy, nephropathy, and peripheral and autonomic ‘neuropathy. By immunocytochemistry, an increase in type { and type Ill collagens is identified that corresponds to the collagen bundles seen under electron microscopy. Type IV collagen shows linear segments of perisinusoidal staining, imparting a basement ‘membrane-like appearance. Stellate cell activation is observed in patients with diabetic hepatosclerosis [90.57], Hepatic peri- sinusoidal fibrosis has been observed in obese patients as wel. Electron microscopy examination of liver biopsy samples from ‘obese patients shows deposition of abundant collagen fibrils and tlectron-dense material resembling basement membrane within the space of Disse [57] 4 Collagen homeostasis and macroanglopathy associated ‘with insulin resistance Patients with insulin resistance develop vascular damage char- acterized by atherosclerasis and medial calcification (Monckebere's ‘slerosis) that may be explained by defective extracellular matrix homeostasis on the arteriat wall Fatty streaks or intimal xanthomas are focal accumulations of fa-laden macrophages (foam cells) in the arterial intima, Micrascopically, the media is preserved and ‘extracellular matrix accumulation has not been found an these lesions. Intimal xanthomas are at lesions that usually egress, The ‘extent of fatty streaks in young petsons docs not predict the extent ‘of raised lesions (fibrous plaques) in later life. Potentially progres- ‘sive atherosclerotic lesions include intima-media thickening of the arterial wall and fibrous plaques. Vascular calcification is associated, With either of them. intima-media thickening is a difuse widening fof the intima-media of the arterial wall, Or histological examina- tion, the lesion is characterized by collagen accumulation, Micro ‘calcification foci are a very common finding. There are no foam cells, Fibrous plaques are focaly distributed in the same area that the intima-media thickening lesion. Fibrous plaques are raised le- sions that protrude into the lumen of the artery. Dense bundles of collagen are found in every fibrous plaque. The densely fibrotic ‘areas are fee from large accumulations of fat. Small lacunar spaces ‘re commonly seen in this fibrous structure. Calcification can be seen in the plaque and in the adjacent artery wall. In advanced fibrous plaques, the collagenous extracellular matrix forms a fibrous cap that encloses a necrotic core containing debris including lipids. The fibroatheroma plaque contains varying degrees of infil- tration by maeraphages and lymphocytes that represent a protec- tive response. Advanced fibrous plaques with a necrotic core (fibrous cap atheroma) may undergo complications such as rupture ‘or thrombosis that cause lie-threatening occlusive episodes [58-66], Inuima-media thickening and fibrous plaques are ‘commonly complicated by arterial wall calcification. Vascular ‘calcification is driven by smooth muscle cells in response to altered ‘extracellular matrix and involves deposition of carbonated hhy- droxyapatite (calcium phosphate) on the extracellular matrix.LAL Ade nny Dees & Metab: yatome inc Research Reviews 12 (2013) 177-1883 oo Arterial calcification is observed microscopically in the intima- ‘media thickening. lesion, indicating that microcacification foci develop at the beginning ofthe atherosclerosis process (56-69). In analyses of human coronary arteries conducted to quantify the calcium-to-phosphate ratio on the tssue, microcalifications composed of amorphous calcium phosphate can be demonstrated at a preatheroma stage, suggesting that the formation of calcium phosphate granules isan early event inthe atherosclerotic process [70}. Electron microscopy examination on human aorta samples dbtained at autopsy detects zones of microcaleiication on intima- ‘media thickening lesions, confirming that calcified deposits are formed at an early stage of atherosclerosis development [57] Autopsy studies reveal that atherosclerotic lesions begin to appear in young population groups indicating that preventive ac- tuons need to be implemented early in life. Among them, compel- ling evidence derived from prospective studies has demonstrated ‘hat animal protein ineveases the risk of both type 2 diabetes and cardiovascular disease by causing insulin resistance. Restriction or discontinuation of animal protein consumption is crucial to reduce vascular disease associated with insulin resistance [51/55] In a comprehensive histopathologic study of medium-to-large caliber blood vessels in 100 autopsy subjects, vascular tissue was analyzed to determine the percent fibrosis of over 700 vaseular segments, The percent fibrosis (% collagen) ofthe tunica media was strongly correlated within subjects across all blood vessels sug- gesting that fibrosis isa global process independent of the vessel im Investigation on human atherogenesis has been accelerated by the development of models of human atherosclerotic lesions and induced pluripotent stem cell technology that makes available human vascular cells generated from induced pluripotent stem cells [6,72 4.1. Role of collagen homeostasis in the pathogenesis of ‘macrovascular disease associated to insulia resistance In 1883, Thoma and Kaefer suggested that medial damage played a primary rolein the development of atherosclerosis. Medial lesions resulted in focal arcas of injury which became filled up by a fibrotic repair process leading to atherosclerotic plaques. Medial ‘Shinning was later confirmed to be an initial typical finding in fibrous plaques [4,58], Smooth muscle cells in the medial layer synthesize extracellular matrix. Abundant components of human arterial extracellular matrix are elastin, proteogiycans and collagen. ‘Additional constituents include enzymes that degrade extracellular ‘matrix (such as matrix metalloproteinases) and their inhibitors, and molecules that regulate the assembly of collagen into fib such as small leucine-rich repeat proteoglycans including decorin ‘The extracellular matrix interacts with smooth muscle cells via cell, membrane receptors such as integrins. Changes in extracellular ‘matrix composition are detected by smooth muscle cells and elicit pathophysiological_ responses. Molecular interactions between components of the extracellular matrix (such as collagen) and smooth muscle cells contribute to the normal function and adaptive responses to damaged arterial wall, Dysfunctional overproduction ‘of collagen in response toan unknown injury leads tothe structural changes that characterize vascular damage associated to insulin resistance [73,74], Mattix degrading proteins and theit inhibitors or other molecules that regulate collagen assembly into fbrilar structures may play a role modulating arterial damage, but their Impact has not been defined {75}. There is a pathological “diabetes phenotype" in vascular smooth muscle cells. Smooth muscle cells isolated from patients with diabetes exhibit abnormal morphology and greater adhesion and migration in cell cultures compared to those from nondiabetic subjects [76]. Collagen composition, distribution, and mechanisms of fibrillar assembly on the normal wall of human arteries are not well novi, Type [and type Ill collagens are the most abundant types in the medial layer. Type IV and type V collagens have been fouind around individual smooth muscle cell in the medial layer and surrounding endothelial cells in the intima forming a basement membrane. Unlike type IY, type V collagen is also distributed throughout the extracellular matrix in the aortic media [15]. Type It collagen has been detected in small amounts in the media of human aorta sec- tions. The average content of type Il collagen is approximately 1% of {otal arterial collagen while collagens types Il, and IV amount 10 {99% [77], Type XIX collagen has been identified on the endothelial basement membrane of human blood vessels [3] Patients with insulin resistance show abaormal collagen ho- ‘meostasis in the arterial wall including elevated collagen content, disordered collagen arrangement, and altered collagen composi- ‘ion, which contributes to cause vascular injury. Intima-media thickening and fibrous plaques are characterized by accumulation of collagen. The measurement of hydroxyproline reveals an in- crease ofthe collagen content in fibrous plaques, comprising 40% of the total protein versus 25% in normal human aorta. Collagen constitutes as much as 60% of the total protein in advanced lesions [811578] Fibrous plaques show disordered and shapeless collagen structures, in contrast to the organized assembly of fibrillar collagen in the normal arterial wall, Collagen fibrils degenerate in the dead tissue zones of atheromatous plaques [15]. Collagen dls- tribution is nor uniform within the flarous plaques. These lesions consist of regions with abundance of type Ian type II collagens and areas deficient in these collagen types [15,72,79]. The amount of type IV collagen increases with the advance of atherosclerosis. Heavily thickened type IV collagen structures surrounding incl- Vidual smooth muscle cells are found in fibrous plaques [15]. The abundance of type I collagen increases markedly in atherosclerotic lesions, particularly in samples with calcification. The amount of ‘ype Il collagen is higher in the tissue around the calcium deposit [77]. The relationship between collagen and arterial calification has been investigated én vitro in human carotid atherosclerotic plaques and three-dimensional collagen hydrogels (that mimic structural features of the atherosclerotic fibrous cap). There is ar Inverse relationship between collagen content and the size of microcaleifications in human fibrous plaques. The micro cakifications aggregate within fibrous plaques in gaps between collagen fibers. Similarly, increasing collagen content in the hydrogels reduces microcaleifcation size by 90% compared to the samples without collagen. Further, collagen degradation promotes _mierocakification. Localized collagen decay within the plaque al- lows extracellular vesicles to accumulate and initiate the caleifca- tdon process. Then, microcalcfications serve as bullding blocks for larger calcifications when normal collagen is deficient [20] 5, Summary Insulin resistance is associated with systemic impairment of collagen homeostasis that affects the extracellular matrix of human tissues including blood vessels of any size. Excessive deposition of abnormal collagen on the interstitial space contributes to cause ‘kin abnormalities, bone fractures, tendon disorders, lung ‘dysfunction, diastolic dysfunction, and liver disease. Accumulation of defective collagen on the capillary wall causes microvascular disease including retinopathy, peripheral neuropathy, and ne- phropathy. Collagen accumulation on the medial layer of large ar- {eties produces intima-media thickening, Fibrous plaques develop in regions of intima-media thickening with further collagen deposition. Both lesions are associated with calcification of the arterial wall,2 ene Ate-dany ea Babes Mtoe Syarome: Cn Reser & Redes 15 (2019) 1877-186 Funding ‘There was no financial support for this work Conflicts of interest ‘The authors declare that they have no conflict of interest. Acknowledgement We are pleased to acknowledge the continuous support from Ms. Gema Souto-Adeva, References (1 ng Fae Selective wn esse a te deepen Diaberes 2016 jum 65,6:1400- 71, bed PMID: 27222300, Pubmed Central Picib:Pucas73431.EpuD 2016105/26.ene [2] Wiliams AS Kang L Wassemnan DH The extaclblar mass and inslin feskance. Tents Endocrine Netabol TEM 2015 267) 137-ah. Pub Pb: 25059707. Pabed Cental PRICID: PMCHASOD3B. Eps 2015001. [a] Sor, vid Men Shaul¥, Rede Caf} Diab bilons A report of fives cases Am J Med 1970 jula{tio4—@. MbMied PMID 5431478 Epub is7oo7f0. eng. yb Hed. 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