Nitric Oxide Synthase Expression and Functional Response to Nitric Oxide Are Both Important Modulators of Circulating Angiogenic Cell Response to Angiogenic Stimuli

Abstract

Objective-Circulating angiogenic cells (CACs), also termed endothelial progenitor cells, play an integral role in vascular repair and are functionally impaired in coronary artery disease (CAD). The role of nitric oxide (NO) in CAC function is poorly understood. We hypothesized that CAC migration toward angiogenic signals is modulated by both NO synthase (NOS) expression and functional response to NO. Methods and Results-Similar to endothelial cells, CAC chemotaxis to vascular endothelial growth factor (VEGF) was blocked by inhibition of NOS, phosphatidylinositol 3-kinase, or guanylyl cyclase or by treatment with an NO scavenger. Addition of an NO donor (S-nitroso-N-acetylpenicillamine) and the NOS substrate L-arginine increased random cell migration (chemokinesis) and enhanced VEGF-dependent chemotaxis. Healthy CACs expressed endothelial NOS, but endothelial NOS was not detected in CAD patient CACs. Both chemokinesis and chemotaxis to VEGF of patient CACs were decreased compared with healthy CACs but were restored to healthy values by S-nitroso-N-acetylpenicillamine. In parallel, CAD patients exhibited lower flow-mediated vasodilation and plasma NO source nitrite than young, healthy subjects, indicating endothelial dysfunction with reduced NO bioavailability. Conclusion-NOS activity is required for CAC chemotaxis. In CAD patients, impairment of NOS expression and NO bioavailability, rather than response to NO, may contribute to dysfunction of CACs and limit their regenerative capacity. Physiologically, many integral functions of the vascular endothelium are modulated by endothelial nitric oxide synthase (eNOS)-derived NO, including the inhibition of platelet and leukocyte adhesion, smooth muscle relaxation, and proliferation. Newer literature shows that NO not only acts in paracrine manner but may also exert systemic effects via reversible formation of more stable storage forms, including nitrite and nitoso-adducts. The disruption of this pathway in endothelial cells is associated with chronic vascular disease. 2 Risk factors appear to selectively damage the vascular endothelium, leading to a dysfunctional, maladaptive endothelial phenotype. 3,4 Studies suggest that eNOS activity and expression as well as circulating NO storage forms in blood are progressively decreased with cardiovascular risk factors including aging, hypertension, hypercholesterolemia, diabetes, and smoking and cigarette smoke exposure. 2,[5][6][7] Over time, chronic endothelial dysfunction leads to intimal hyperplasia and enhanced plaque formation in predisposed areas of the vascular tree. Notably, the functional capacity of the vascular endothelium not only depends on the degree of damage but also on the presence and status of repair systems, including circulating angiogenic cells (CACs). 8 Vascular repair involves not only local migration and proliferation of mature endothelial cells but also angiogenic cells that circulate in blood and the recruitment of the latter cells to sites of injury. Literature from the last 10 years suggests tha...

Central Role of eNOS in the Maintenance of Endothelial Homeostasis

“…Interestingly, eNOS appears to be an important modulator of CAC function similar to ECs (40) and mobilization from the bone marrow (2). We have shown that eNOS activity is required for CAC chemotaxis (40).…”

“…We have shown that eNOS activity is required for CAC chemotaxis (40). Furthermore, in CAD patients, impairment of NOS expression and NO bioavailability, rather than response to NO, may contribute to dysfunction of CACs and limit their regenerative capacity.…”

“…It is expressed mainly in the endothelium of large arteries with the expression decreasing in smaller arteries and not expressed in capillary ECs. As depicted in Figure 2, eNOS is also expressed in essentially all blood cells, including erythrocytes, leucocytes, platelets, and CAC (previously termed endothelial progenitor cells [EPCs]) (21,40,42). We have recently shown the expression of a functional eNOS enzyme in circulating microparticles (MP) (47).…”

“…(32, 36, 38-40, 46, 52, 63, 65, 74, 75, 82, 88) (green symbols) studied in our institution using the identical standardized methodology. Decreased values in smokers (34,37) and non-smokers exposed to second-hand smoke (32) and patients with coronary artery disease (CAD) (40,47,75), with diabetes mellitus (10, 50) and on optimal medical therapy (OMT) (35,40). (b) Relative cardiovascular risk (RR) associated with continuous (e.g., 1% absolute difference) and categorical differences (''high vs. low'' populations) in FMD values according to a recent meta-analysis (73).…”

The genetics of exceptional longevity identifies new druggable targets for vascular protection and repair

“…In addition to endothelial cells, eNOS is present on circulating blood cells, including circulating angiogenic MNCs, and on circulating microparticles, therefore its regulation impacts on the overall homeostasis of the vascular tree controlling blood flow restoration via angiogenesis, vasculogenesis, and arteriogenesis (23)(24)(25)(26). Together with neuronal NOS (nNOS), eNOS is constitutively expressed.…”