**5. Endothelium and endothelial progenitor cells**

Endothelial cells constitute the innermost layer of blood vessel and promote vascular homeostasis and angiogenesis. Endothelial cells can secrete several mediators that can alternatively mediate vasoconstrictors, such as endothelin-1 and thromboxane A2, or vasodilators, such as NO, prostacyclin, and endotheliumderived hyperpolarizing factor. Since hyperglycemia and IR can negatively impact NO secretion from the endothelium, with vasoconstriction, vessel wall stiffness, platelet aggregation and diminished angiogenesis, there is a counter-regulatory reparatory cellular response by circulating endothelial progenitor cells (EPCs). These are immature bone marrow derived cells that can differentiate into mature endothelial cells. These cells home in on areas that experience vascular injury or ischemia by way of circulating growth factors and cytokines to initiate repair of the endothelial surface and stimulate neovascularization and angiogenesis. In conditions such as diabetes with vascular damage, the presence of diminished circulating EPCs constitute cellular biomarkers of compromised cardiovascular health [19]. In subjects with IR such as metabolic syndrome, decreased EPC number and impaired functionality prognosticates increased cardiovascular risk [20]. Interestingly, exercise promotes the production and numbers of EPCs [19, 21] putatively related to the anti-apoptotic effect of NO [22]. EPCs are also stimulated by exercise in aging studies. Moreover, the migratory function of EPCs is improved by exercise in subjects with IR [19, 23]. The degree of exercise dose appears to influence the overall EPC response [23]. In presence of insulin resistance but not overt diabetes, CPAP therapy improves endothelial health and EPC parameters [24].

Exercise, endothelium and fat derived mesenchymal stromal cells (MSCs): clinical trials are necessary to investigate the possible cellular and molecular pathways that may impact endothelium and fat metabolism. Identification of the pathways that influence crosstalk between endothelium and fat, and thereby improve cardiometabolic health in the elderly and young subjects is important to identify. The process will help to identify genes and cell differentiation pathways that may change fat derived stem cell differentiation, following exercise training in the elderly and the young subject cohorts, which will subsequently influence the mesenchymal

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

dense LDL), and hypertension termed the "insulin resistance syndrome". As discussed, during physiological conditions, insulin binding to endothelial receptors leads to phosphorylation of downstream substrates including activation of the IRS-1, PI3K pathway and subsequent recruitment of GLUT4 to mediate glucose transport into muscle and other tissues [7]. However, In the IR state, the IRS-1- PI3K-Akt-NO pathway is muted while the MAPK pathway remains intact [8]. The unopposed action of ET-1 leads to a shift towards vasoconstriction, increased arterial stiffness, hypertension, and tissue hypoxia. In addition to this decrease in NO bioavailability, increases in oxidative stress, inflammatory markers, and pro-thrombotic mediators (i.e., increased plasma von Willebrand factor, decreased lipoprotein lipase activity) are seen. More direct evidence for endothelial IR was shown in freshly isolated arterial endothelial cells where the insulin-induced eNOSphosphorylation was negatively associated with oxidative stress markers [9]. During states of IR each vascular site becomes affected and contributes to an increase in atherosclerosis. At the level of the conduit arteries, IR causes decreased compliance with a concomitant increase in vessel stiffness, which is a predictor of coronary artery disease and stroke [10]. The impaired vasodilatory action of insulin on the resistance arterioles leads to decrease in blood flow to the tissues it supplies. For example, Baron et al. demonstrated that the inhibition of NO production (similar to states of IR) causes a decrease in blood flow and glucose uptake in the leg. The terminal arterioles in patients with IR showed a blunted response to mixed meal in brachial blood flow and forearm microvascular recruitment compared to lean subjects [3]. *Oxidative stress:* hyperglycemia due to IR also induces generation of reactive oxygen species (ROS) by activation of the NADPH oxidase system. ROS activates multiple pathways linked with cell growth, proliferation and modifies NO bioavailability. One such pathway includes the renin-angiotensin system which is inappropriately activated in settings of IR. Interestingly, during continuous insulin infusion, Angiotensin 2 receptor antagonism resulted in whole-body insulin resistance and attenuation of microvasculature recruitment [5]. The mechanism may involve increased binding to Angiotensin 1 receptors, which have been shown to increases oxidative stress and cause vasoconstriction through decreased bioavailability of *e*NOS and increased ROS. Chai et al. has shown that when AT2R is blocked, there is decreased microvascular blood flow by 80% along with reduced

**4. The effect of exercise on insulin resistance and vasculature**

It is well established that exercise augments insulin signaling independent of PI3K, while the combination of skeletal muscle contraction and insulin additively enhances glucose transport via GLUT4 translocation. A plethora of studies have reported that regular physical activity is effective in patients with IR, such as type 2 diabetes, prediabetes and metabolic syndrome, in improving glucose tolerance, insulin sensitivity, glycosylated hemoglobin levels (HbA1c) and morbidity and mortality [12]. For instance, adults with IR were found to have improvements in hepatic and peripheral insulin sensitivity after 12 weeks of aerobic exercise. Shorter term studies (i.e., 7 days) have also demonstrated similar improvements in insulin sensitivity in obese patients [13]. Lifestyle interventions such as diet modifications added to 12 weeks exercise training showed further enhancements in addition to insulin sensitivity including fatty acid oxidation, post-prandial hyperinsulinemia

*Exercise and the endothelium*: exercise causes several adaptations to IR in the vasculature in both the skeletal muscle and endothelium. Vessel wall shear stress

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glucose extraction [11].

and systolic resting blood pressure [14].

structures of our body. Our study [25] appears to indicate that exercise promotes osteogenic differentiation but not myogenic differentiation in the middle-aged veteran population with mean age of 51 years. However, whether osteogenic differentiation of adipose tissue derived mesenchymal stromal cells (MSCs) also occurs in young and the elderly is unknown. Myogenic differentiation in response to exercise is well documented [26], and different types of exercise appear to influence the differentiation depending on plasma-based differentiation factors [26]. However, the exact mechanism of how exercise modifies mesenchymal stromal cell (MSC) differentiation in the body needs further investigation. Prior to our recent studies, we would have hypothesized that exercise will promote myogenic differentiation in all age groups, however the differentiation of stem cells may be dependent upon the need of the body to regenerate a particular tissue lineage at a particular age. For example, exercise promotes myogenic differentiation in the young [26] whereas endothelial function improvement and bone regeneration may be more important in the elderly [27, 28].
