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

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

#### **Figure 2.**

*Extracellular Vesicles and Their Importance in Human Health*

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

*4.2.1 Atherosclerosis and vascular calcification in diabetes mellitus*

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].

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

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

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

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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progression.

diabetes mellitus.

resistance and relieve β-cell destruction [60].

*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).*

tissue cells into circulation are enclosed in vesicles. EVs derived from adipose tissue may play a role in the paracrine cross talk between adipocytes and macrophages in adipose tissue in obesity [51], and in endocrine manner for transmission of signals to other cells from cardiovascular system [67]. There are the studies that support the idea that EVs are important mediators for metabolic organ cross talk. Thus, it was hypothesized that insulin-secreting beta (β) cells and insulin-sensitive tissues release exosomes that can be transferred to other metabolic organs, or to immune or endothelial cells. In this way, in an autocrine or paracrine manner, exosomes influence glucose homeostasis and insulin resistance [68].

When circulating miRNA profile of lean and obese individuals was compared, those miRNAs differentially expressed were predicted to regulate inflammatory and fibrotic signaling pathways [69]. Moreover, in obesity, exosomes from adipose tissue-derived MSCs have reduced pro-angiogenic properties due to decreased content in miR-126, VEGF, and MMP-2. A differential EV proteomic profile has also been observed between obese diabetic and obese nondiabetic rats [70]. In a recent study, the lean mice treated with exosomes from obese mice developed glucose intolerance and insulin resistance. In addition, using exosomes transfected with a specific siRNA targeting PPARα, the phenotype induced by obesity-associated miR-NAs was recapitulated. Importantly, it was demonstrated that obesity-associated exosomal miRNAs modulate glucose and lipid metabolism in mice [71].

In type 1 diabetes, the imbalances between effector T cells and regulatory T cells, as well as dendritic cell presentation of islet auto-antigens, play an important role in the destruction of islet β cells. It has been shown that MVs derived from endothelial progenitor cells (EPCs) combined with islets can activate angiogenesis, decrease leucocyte-endothelial interaction, and improve pancreatic β cell function [72]. Another study revealed that insulinoma-released exosomes or MPs are immunostimulatory and can activate autoreactive T cells spontaneously developed in nonobese diabetic mice [73]. Exosomes could also serve as triggering factors for specific autoimmunity events leading to diabetes, as shown in another study where in NOD mice exosomes released by islet-derived MSCs trigger autoimmune responses [74]. Thus, specific biological roles of EVs are dependent on functional state and the type of cells from which the EVs are released.
