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Stefania Lucia Magda1,2, Elisa Serban2

1 „Carol Davila”University of Medicine and Pharmacy, Bucharest, Romania
2 Department of Cardiology, Emergency University Hospital, Bucharest,

Abstract: Individuals with type 2 diabetes mellitus develop more frequently than healthy controls cardio- and cerebro-vascular disorders. It is important to understand the mechanisms through which diabetes contributes to the development and severity of these complications. Extracellular vesicles (EV) are important mediators of cell to cell communication in several diseases, including diabetes mellitus and vascular disease. Three populations of EVs are described presently: exo-somes, microvesicles and apopototic bodies. Recent studies have shown that atherosclerotic lesions of all stages contain microvesicles. Higher levels of circulating EVs have been discovered in individuals with cardiovascular risk factors, with both pro-atherogenic and anti-atherogenic effects. Regarding cerebrovascular disease, studies have shown that exosomes, espe-cially those derived from stem cells, play an important role, preventing post-ischemic suppression. Different cell types in the heart contribute to the pathogenesis of diabetic cardiomyopathy and EV seem to be essential in the intercellular crosstalk between heart cells. According to recent research EVs could play an important role in different cardiac and cerebral rege-nerative therapies and could also be used as therapeutic vectors in cardiovascular medicine. Further large animal and human studies are necessary to validate EVs as diagnostic and therapeutic tools.
Keywords: Diabetes, cardiovascular disease, cerebrovascular disease, cardiomyopathy.

INTRODUCTION
Diabetes mellitus is a common chronic disease all over the world, with increasing incidence and prevalence, due to contemporary lifestyle with reduced physical activity, processed food and increased obesity. A me-ta-analysis published in 2009, including studies from 91 countries, reported an estimate for 2010 of 285 million people with diabetes worldwide, with significant differences between populations and regions, and a predicted increase from 2010 to 2030 of 54%, cor-responding to an annual growth of 2.2%, nearly twice as high as the annual growth of the total world adult population1.
Individuals with type 2 diabetes mellitus develop more frequently than healthy controls cardiovascular disorders, including coronary heart disease, stroke, peripheral arterial disease, and diabetic cardiomyo-pathy, mainly through the chronic, damaging exposure of the vascular system to hyperglycemia2. Therefore, it is important to understand the exact mechanisms through which diabetes contributes to the develop-ment and severity of these complications.
Extracellular vesicles (EV) represent small cell-se-creted structures naturally released into the extra-cellular space by all eukaryotes and many prokaryo-tes, containing proteins, lipids, nuclear material and noncoding RNAs2. They were first described in the late 1960s and are important mediators of cell to cell communication in several diseases, including diabetes mellitus and cardiovascular disease. Most cell types can release vesicles into the interstitial space. These vesicles can be found in body fluids, both human and animal, such as blood, urine, tears and saliva, as well as in cell cultures3.
Three populations of extracellular vesicles are de-scribed presently: 1. Exosomes are the smallest, with a diameter around 30-150 nm in diameter. They are released through exocytosis after fusion of multivesi-cular bodies with the plasma membrane; 2. Microve-sicles (microparticles/ectosomes) are larger vesicles, with a diameter of 100-1000 nm, which are formed by the outward budging and scission of the extracellular membrane; 3. Apoptotic bodies are the largest subtype of microvesicles with a diameter of 1-5 μm, generated by the plasma membrane of apoptotic cells4.
Several strategies are currently available for the quantification of extracellular vesicles, the most po-pular being ultracentrifugation, among others, such as: density gradient, precipitation, field flow, chromato-graphy and affinity based capture and microfluidic te-chniques4. All available isolation methods are time in-tensive, require expensive equipment, and are limited by the fact that they do not purify specific populations of vesicles, probably due to lack of standardization of the techniques and methods4. In the last 8 years, the International Society of Extracellular Vesicles has constantly tried to update the topics of nomenclature, separation, characterization and functional analysis of EV5.

EV AND CORONARY ARTERY DISEASE
Recent studies have shown that atherosclerotic lesi-ons of all stages contain microvesicles6,7. Higher levels of circulating microvesicles have been discovered in individuals with cardiovascular risk factors, such as smoking8, dyslipidemia9, diabetes mellitus10 and arterial hypertension11, probably through activation or from apoptosis of different cells being exposed to a damaging stimulus.
Data extracted from in vitro studies suggest that microvesicles can have both pro-inflammatory and anti-inflammatory effects, depending on different si-tuations3. Microvesicles increase the release of pro-inflammatory cytokines (mainly interleukin 6 and 8) from endothelial cells and leukocytes, promoting the adhesion of monocytes to the endothelium and their migration to the atherosclerotic plaque3,12. Also, endothelial microvesicles can activate monocytes. Another effect of microvesicles is their interaction with the vascular endothelium and decreasing the NO production by endothelial cells-consequently impai-ring endothelial properties13. Endothelial microvesicles and platelet derived microvesicles increase endotheli-al permeability14. Microvesicles promote adhesion of monocytes to the endothelium by increasing endothe-lial expression of adhesion molecules15.
Various microvesicles contribute to foam cell for-mation in the atherosclerotic plaque by stimulating lipid and cholesterol formation in macrophages. Macro-phages and foam cell undergo afterwards apoptosis, forming a core of extracellular lipids. Increased mono-cytes and macrophage apoptosis contributes to incre-ased microvesicle release in the plaque. Microvesicles of monocyte and macrophage origin are the largest population of microvesicles in human atherosclerotic lesions16.
Infiltration of LDL particles in the vascular wall during the atherosclerotic process can induce the formation and release of tissue factor enriched micro-vesicles from the smooth muscle cell, microvesicles which in their turn influence smooth muscle cell pro-liferation and migration17.
Extracellular vesicles of different origins, with diffe-rent microRNA content, contribute to smooth muscle cell proliferation19.
Several studies of patients with stable coronary artery disease have reported increased levels of cir-culating microvesicles. Specifi c microvesicle subpo-pulations, especially those of endothelial origin, cha-racterized by CD 144+, CD 131+/annexin A5+, or microvesicles containing miR-199a and miR-126, are currently researched as interesting biomarkers for cardiovascular risk and mortality in these patients20-22.
Calcifications present in the atherosclerotic plaque have destabilizing effect in early lesions, favoring rup-ture, but gain a potential protective effect in advanced
lesions with heavy calcium deposits23. The calcification process is based on three mechanisms: 1. Cell apopto-sis, which releases microvesicles and necrotic debris, leading to nucleate apatite; 2. Deficiency of minerali-zation inhibitors (both tissue derived and circulating), leading to random apatite deposits; 3. An altered di-fferentiation of vascular smooth muscle cells and stem cells, leading to bone formation24. As several studies have shown, atherosclerotic plaque calcifications are associated with extracellular vesicles of endothelial, smooth muscle cell and macrophage origin.
Exposure of vascular smooth muscle cells to pro-infl ammatory cytokines can stimulate the release of exosomes which can mineralize when inhibitors of calcification are missing or not functioning25. Also, en-dothelial cells exposed to proinfl ammatory stimuli can release microvesicles rich in bone morphogenetic pro-tein 2, promoting calcifi cation in vascular smooth mus-cle cells26. Alterations in local homeostasis of calcium and phosphate lead to the formation of macrophage derived exosomes which stimulate mineralization27.
Recent studies have also noted that in humans, advanced atherosclerotic plaques have a high content of procoagulant microvesicles, originating form leuko-cytes, erythrocytes and smooth muscle cells. These microvesicles can actively initiate the coagulation cas-cade, either through the presence of tissue factor on their surface, or by exposing phosphatidylserine, whi-ch concentrates factors VII and VIIa on their outer membrane16,28.
Circulating leukocyte and platelet derived microve-sicles can affect the clotting29, but the actual magnitude of the prothrombotic effects of microvesicles in acute coronary syndromes is still under evaluation3,30.
In opposition to microvesicles, exosomes have shown antithrombotic effects. In animal studies plate-led-derived exosomes suppressed platelet aggregation and occlusive thrombosis31.
Microvesicles influence different mechanisms that lead to plaque destabilization and rupture. Intraplaque hemorrhages are produced by neovascularization ori-ginating from adventitial tissue, stimulated by plaque microvesicles, such as CD40+ vesicles of macrophage origin. Hemorrhages are also favored by leukocyte and endothelial microvesicles with fibrinolytic activity32,33.
Microvesicles can rise endothelial permeability 34 and also modulate inflammation in the plaque35, pro-moting fi brous cap rupture. Fibrous cap weakening is associated with smooth muscle cell apoptosis, indu-ced by the presence of microvesicles and exosomes, released in some pathological conditions36. Moreo-ver microvesicles can influence breakdown of matrix structural proteins through metalloproteinase (MMP) interactions3.
The final evolution of the atherosclerotic plaque is represented by plaque erosion or rupture with in situ thrombosis, clinically expressed as acute coronary syndrome3.
Circulating levels of procoagulant microvesicles are higher in patients with acute coronary syndromes compared to healthy controls or patients with stable coronary artery disease37, the origin of those microve-sicles being mostly endothelial cells, leukocytes, eryth-rocytes and platelets22,37. Circulating microvesicles al-terate endothelium dependent NO mediated vasodila-tion and endothelial microvesicles increase endothelial thrombogenicity14,38.
Circulating microvesicles have been also investiga-ted as prognostic markers in secondary prevention, in order to identify patients at high cardiovascular risk21,22. Increased levels of CD11b+/CD66+ leukocy-te derived microvesicles could be useful in identifying asymptomatic patients at high risk for plaque ruptu-re39, while CD3+/CD45+ microvesicles could identify individuals who will develop a major cardiovascular event40.
In patients with acute ST elevation myocardial in-farction, circulating microvesicles from the coronary arteries contain higher levels of oxidation specifi c epi-topes, linked to inflammatory responses involved in atherosclerosis, than microvesicles from the periphe-ral circulation41.
Circulating exosomes and microvesicles with speci-fic cardiac microRNAs, increase after coronary artery by-pass. Expression of miR-208a in circulating exoso-mes increases in patients with acute coronary syndro-mes42 and specific p-53 responsive microRNAs from plasma exosomes are predictive indicators for heart failure after myocardial infarction43.

EV AND CEREBROVASCULAR DISEASE
Regarding cerebrovascular disease, especially stroke, studies have shown that exosomes, especially those derived from stem cells, play an important role in ne-urological disease, preventing post-ischemic suppres-sion. Also, exosomes might be an interesting thera-peutic resource in the field of regenerative medicine after stroke44.
After stroke, exosomes are released from brain cells and can be detected in the peripheral blood or the cerebro-spinal fluid45,46. As a response to stroke, exosomes are released also from blood cells and endothelial cells47. Circulating exosomes could therefore be useful biomarkers for stroke progression and re-covery44.
Exosome levels of cystatin C and CD 14 have been good predictors in studies of vascular risk in patients with coronary artery disease and also they have been associated to the progression of cerebral atrophy in patients with vascular disease48.
Circulating exosomes can express different mi-croRNAs in various types of cerebrovascular disea-se. Serum exosomal miR-9 and miR-124 levels are higher in patients with stroke compared to controls49. Another study has reported higher levels of miR-223 in acute ischemic stroke, correlated to stroke severity and short term outcomes50. Finally miR-199b-3p, miR 27b-3p, miR-130a-3p, mi-R 221-3- and miR-24-3p are more expressed in patients with asymptomatic carotid artery stenosis progression51.
Exosomes derived from mesenchymal stem cells have enhanced in animal studies the restorative effects in the brain after stroke, reducing neurological impair-ment, promoting grey matter repair and white matter repair, as well as neurogenesis and reversing stroke-induced peripheral immunosuppression52-56.
Cardiovascular dysfunction has been proposed as one of the main causes of cognitive impairment in the elderly, this association being stronger in patients with diabetes mellitus57,58. In vitro studies have shown that extracellular vesicles in diabetic microvascular disease may increase the haemato-encephalic barrier perme-ability59,60.

EV AND DIABETIC CARDIOMYOPATHY (DCM)
Diabetic cardiomyopathy can be clinically defined by the presence of abnormal myocardial performance or structure in the absence of epicardial coronary artery disease, hypertension, and signifi cant valvular disease. Hyperglycemia is the cornerstone of the pathogenesis, inducing stimuli that result in myocardial fibrosis and collagen deposition. These processes are generating altered myocardial relaxation and determine diastolic dysfunction on ultrasound imaging61. Over time, the progression of diabetic cardiomyopathy can lead to clinically manifest heart failure. Different cell types in the heart (such as cardiomyocytes, endothelial cells, smooth muscle cells, hematopoietic derived cells and fibroblast cells) contribute to the pathogenesis of DCM and several studies have shown that EV are essential in the intercellular crosstalk between heart cells2,62.
Cardiomyocyte’s derived EV are implicated in dia-betic cardiomyocyte steatosis. Higher levels of miR-1 and miR-133a were noted in EV derived from lipid loa-ded cardiomyocytes, in the serum of mice fed with a high fat diet and in the circulation of diabetic patients63.
An animal study performed in 2014 has shown that the communication between cardiomyocyte derived EV and endothelium is altered in diabetes, inducing an altered angiogenesis64.
Endothelial cell death and dysfunctional angiogene-sis are frequent in diabetes mellitus. Several microR-NA based mechanisms have been studied in order to explain vascular dysfunction in diabetes2. Hyperglyce-mia increases levels of miR-503 in the endothelium, le-ading to low endothelial cell proliferation and angioge-nesis65. Also, hyperglycemia reduces miR-126 expres-sion in extracellular vesicles derived from endothelial cell, thus impairing endothelial cell repair66. Reduced miR-126 expression in circulating EV and endothelial progenitor cells derived EV in patients with uncontrolled diabetes altered endothelial repair, increased apo-ptosis and the production of reactive oxygen species67.
Cardiac fibroblasts are important components of the fibrotic response in diabetic cardiomyopathy. A potential mediator of the pro-fibrotic action induced by hyperglycemia in the cardiac fibroblasts is miR-21*. Inhibition of miR-21* in mice with cardiac hypertrophy suppressed the myocardial thickening68. Also, a model including in vitro cellular stretch and in vivo pressure overload has induced the release of extracellular ve-sicles form cardiomyocytes enriched with angiotensin type I receptor69.

THERAPEUTIC POTENTIAL OF EV
According to results from studies from the last 5 to 10 years, extracellular vesicles could play an important role in different cardiac regenerative therapies and co-uld also be used as therapeutic vectors in cardiovas-cular medicine.
Platelet derived vesicles induce vascular endotheli-al growth factor (VEGF) dependent angiogenesis and stimulate post-ischemic revascularization after chro-nic ischemia70. Also, plasma derived exosomes activate Toll like receptor 4 on cardiomyocytes and thus protect the myocardium from ischemia-reperfusion injury71.
Mesenchymal stem cell derived extracellular vesicles could be an alternative to stem cell transplantation after myocardial ischemia, by transfer of specific microRNAs through embryonic stem cell extracellular vesicles72.
The use of extracellular vesicles as therapeutic vectors could be done through bioengineering, either by modifying the cytosolic content of the vesicles which could be transferred to the target cell in order to in-fluence cell metabolism; or by loading of extracellular vesicles with molecules to be delivered to target cells. Studies regarding the use of extracellular vesicles as therapeutic vectors in cardiovascular disease are few and are only on animal models. For example, adminis-tration of apoptotic bodies containing miR-126 decre-ased atherosclerotic plaque formation in mice73 and stimulate vascular endothelial cell repair after vascular injury66.
Different cardiovascular medications can influen-ce the level of circulating microvesicles. Antiplatelet agents (ticlopidine, abiciximab) inhibit platelet activati-on and also the release of platelet-derived microvesi-cles74-76. Antihypertensive agents (such as angiotensin receptor inhibitors, beta blockers and calcium chan-nel blockers) lower the circulating levels of platelet and monocyte derived microvesicles77. The effects of statin treatment on circulating microvesicles of pla-telet and endothelial origin are still under debate31,78. Statins and antihypertensive medication are able to modify the properties of in vivo generated endothelial microvesicles and their effect on the expression of en-dothelial adhesion molecules, inhibiting the adhesion of monocytes to endothelial cells and improving endothelial function79. Exosomes from various cell types (such as embryo-nic stem cells, neural stem cells and mononuclear stem cells), have been tested as treatment for stroke in addition to mesenchymal stem cell derived exosomes and showed good results in animal models of stroke, with improvement in neurological scores and reduction in lesion volume and tissue loss44, showing a promising clinical applicability regarding neurological restorative effects and meeting also important safety considerations.

CONCLUSIONS
Extracellular vesicles are vectors of biological informa-tion that could influence cardio- and cerebro-vascular disease in diabetic patients, by transferring benefi cent or negative mediators/stimuli. Also, they have strong therapeutic potential, especially regarding regenerati-ve medicine. The issue of correct isolation of extracellular vesicles from circulation, liquid biopsies and different tissue still limit current knowledge on this subject. Also, there is still little information about in vivo dynamics of extracellular vesicles. Further large cohort animal and human studies are necessary to va-lidate extracellular vesicles as diagnostic and therape-utic tools.

Acknowledgements: Grant 83/PCCDI 2018

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