**4. Extracellular vesicles as biological vectors modulating diabetic cardiovascular diseases**

#### **4.1 Role of extracellular vesicles in coronary artery disease**

Individuals with type 2 diabetes mellitus develop cardiovascular disorders, including coronary artery disease, more frequently than healthy controls, mainly through the chronic, damaging exposure of the vascular system to hyperglycemia. Therefore, it is important to understand the exact mechanisms through which diabetes contributes to the development and severity of these complications.

EVs generated in patients with diabetes mellitus promote inflammation and contribute to the development of atherosclerotic lesions, stimulating monocyte adhesion and their infiltration in the subendothelial layer, promoting the migration and proliferation of vascular smooth muscle cells (VSMCs) and also the calcification of the atherosclerotic plaque.

#### *4.1.1 Extracellular vesicles and the coronary atherosclerotic plaque*

Recent studies have shown that atherosclerotic lesions of all stages contain MVs. Higher levels of circulating MVs have been discovered in individuals with cardiovascular risk factors, such as smoking [38], dyslipidemia [39], diabetes mellitus [40], and arterial hypertension [41], probably through activation or from apoptosis of different cells being exposed to a damaging stimulus. Data extracted from in vitro studies suggest that MVs can have both pro-inflammatory and antiinflammatory effects, depending on the stimulus that induces their formation [42]. MVs increase the release of proinflammatory cytokines, mainly interleukin-6 and -8 (IL-6 and IL-8), from ECs and leukocytes, promoting the adhesion of monocytes to the endothelium and their migration to the atherosclerotic plaque [42, 43]. Also, endothelial MVs can activate monocytes by transferring miR-10a and thus interfering with the nuclear factor-κB inflammatory pathway. Another effect of MVs is the decrease of the nitric oxide (NO) production by ECs, consequently impairing endothelial vasodilating properties [44]. Endothelial-derived MVs and platelet-derived MVs increase endothelial permeability by delivering two enzymes (caspase 3 and Rho-kinase) to target cells and inducing apoptosis [45]. MVs promote adhesion of monocytes to the endothelium by increasing endothelial expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), or adhesion molecule receptors, such as CD11a, on monocytes [46]. ICAM1 expression can also be regulated by miR-222 in MVs [42, 47].

Various MVs contribute to foam cell formation in the atherosclerotic plaque by stimulating lipid and cholesterol formation in macrophages. Macrophages and foam cells undergo afterward apoptosis, forming a core of extracellular lipids. Increased monocyte and macrophage apoptosis contributes to augmented MV release in the plaque. MVs of monocyte and macrophage origin are the largest population of MVs in human atherosclerotic lesions [48].

Infiltration of LDL particles in the vascular wall during the atherosclerotic process can induce the formation and release of tissue factor-enriched MVs from the VSMCs, which in turn influence VSMC proliferation and migration [49].

EVs of different origins, with different miRNA content, contribute to VSMC proliferation and migration; for example, MVs with miR-223 induce a decrease in atherosclerotic plaque size by inhibiting VSMS proliferation and migration, while endothelial vesicles with miR-143 and miR-145 prevent VSMC dedifferentiation [50].

#### **4.2 Role of extracellular vesicles in peripheral arterial disease**

#### *4.2.1 Atherosclerosis and vascular calcification in diabetes mellitus*

Adipose tissue-derived EVs were shown to have immunomodulatory effects on macrophages which in turn, after contacting said EVs, secreted factors that interfered with insulin signaling in human adipocytes [51]. Moreover, EVs released from adipocytes from obese individuals act in a paracrine manner on neighboring adipocytes and impair insulin-mediated glucose transport [52]. In turn, the exosomes derived from insulin resistant adipocytes carry sonic hedgehog (SHH) protein that increases the expression levels of TNF-α, IL-1β, IL-6, VEGF-A, ICAM-1, MMP2, and MMP9 in the atheroma plaque and promotes vasa vasorum angiogenesis, leading to greater plaque burden and vulnerability [53]. Thus, EVs provide a link between obesity, low-grade inflammation, insulin resistance, and atherosclerosis progression.

EVs also play a key role in the cross talk between ECs and macrophages that can either act in the direction of vascular homeostasis or promote atherosclerosis, depending on their composition. It was shown that EVs secreted by Kruppel-like factor 2-treated ECs show anti-inflammatory actions, while oxidized-LDLtreated ECs produce EVs with high levels of miR-155, directing macrophage differentiation toward pro-inflammatory M1 macrophages [54]. In M1, but not in M2 macrophages, the inflammasome is known to be activated [55] and the inflammasome signaling leads to the secretion of pro-inflammatory exosomes [56], further favoring atherosclerosis progression. Furthermore, atherosclerotic patients have high numbers of monocyte/macrophage-derived miR-150-rich EVs that enhance EC migration, a major component of atherosclerosis [57]. Thus, circulating endothelial microparticles (EMPs) were shown to be an independent risk factor for endothelial dysfunction which occurs early in atherosclerosis, and the fact that in type 2 diabetes mellitus their number is increased [58] and their miRNA composition is altered containing miRNAs mainly involved in angiogenesis [59] proves the involvement of EVs in cardiovascular complications of diabetes mellitus.

However, exosomes from other sources can alleviate diabetes mellitus as shown at rats treated with exosomes from human umbilical cord derived multipotent mesenchymal stromal cells (MSCs) that have the ability to reverse peripheral insulin resistance and relieve β-cell destruction [60].

Atherosclerosis, old age, diabetes, and hyperphosphatemia induce VSMC transdifferentiation to osteoblasts [61] characterized as loss of SM22a and SMA markers and gain of Runx2, SP7, osteopontin, osteocalcin, alkaline phosphatase (ALP), transcription factor Sox9, collagen II, and collagen X [62]. These trans-differentiated cells secrete 50–150 nm calcium-phosphorus-rich exosomes that serve as calcification nuclei, in the same manner that osteoblast-secreted matrix vesicles lead to bone mineralization [63]. However, extracellular calcium leads to Fetuin-A uptake in VSMCs mediated by annexins, and high Fetuin-A levels in secreted exosomes prevent further calcification [64]. This control mechanism is affected when Fetuin-A

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**Figure 2.**

*Part One: Extracellular Vesicles as Valuable Players in Diabetic Cardiovascular Diseases*

levels are low due to chronic dialysis [65] and higher than normal plasma Fetuin-A levels can lead to insulin resistance and diabetes through the direct inhibition of the insulin receptor [66], thus only worsening the cardiovascular diseases (CVDs). EVs found in intima and media of calcified vascular wall (**Figure 2A–D**) [14] seem to be different of matrix vesicle with role in physiological and pathological calcification [19]. Vascular cell-derived EVs may serve as a continuous source of damaging microcalcifications in atherosclerotic plaques [20]. These vesicles have been described as exosomes from endosomal compartment, plasma membranederived ectosomes, and vesicles shielded by a plasma membrane (multivesicular cargos) that are released into extracellular space as a cargo [14]. As the first two types are intense investigated and described, the last type derived from multive-

The prevalence of type 2 diabetes is rapidly increasing worldwide, in parallel with the current obesity epidemic, posing a major healthcare expenditure burden. Obesity increases the risk for development of diabetes, cancer, and CVDs. In the course of events leading from obesity to type 2 diabetes, several mechanisms are currently outlined. Among them, low-grade systemic inflammation in adipose tissue of obese persons has been proposed as a possible link between insulin resistance and altered functions of vascular cells by increased cytokines production. Furthermore, it has been shown that the molecules that are released by adipose

*Transmission electron microscopy of the diabetic arteries in diabetic kidney. (A) Large spaces with vesicular content [square marked area in (A) is magnified in (B)] increase the vascular wall thickness. (B) Numerous extracellular vesicles accumulated between vascular smooth muscles cells (VSMCs) and small calcifications (c) may be seen. (C) Concentric amorphous deposits (d) are located between endothelium (E) and vascular smooth muscle cells (VSMCs). (D) Higher magnification of square marked area in (C) shows also numerous* 

*vesicles in the extracellular space (#) between vascular smooth muscle cells (VSMCs).*

*DOI: http://dx.doi.org/10.5772/intechopen.85225*

sicular cargos is less investigated.

**4.3 Role of extracellular vesicles in insulin resistance**

### *Part One: Extracellular Vesicles as Valuable Players in Diabetic Cardiovascular Diseases DOI: http://dx.doi.org/10.5772/intechopen.85225*

levels are low due to chronic dialysis [65] and higher than normal plasma Fetuin-A levels can lead to insulin resistance and diabetes through the direct inhibition of the insulin receptor [66], thus only worsening the cardiovascular diseases (CVDs).

EVs found in intima and media of calcified vascular wall (**Figure 2A–D**) [14] seem to be different of matrix vesicle with role in physiological and pathological calcification [19]. Vascular cell-derived EVs may serve as a continuous source of damaging microcalcifications in atherosclerotic plaques [20]. These vesicles have been described as exosomes from endosomal compartment, plasma membranederived ectosomes, and vesicles shielded by a plasma membrane (multivesicular cargos) that are released into extracellular space as a cargo [14]. As the first two types are intense investigated and described, the last type derived from multivesicular cargos is less investigated.
