Cardiovascular Risk Factors Associated with Impaired Arteriogenesis

A variety of disease states perturb arteriogenesis, leading to impairment of endothelial or macrophage function or to recruitment of endothelial progenitor cells or of growth factor production (Fig. 8-4). Aging itself affects arteriogenesis secondary to an overall decreased endothelial production of NO¹⁶ and migration by a higher rate of HIF degradation and decreased levels of VEFG, platelet-derived growth factor (PDGF), FGF-2, and chemokine signaling. Growth factor release is also impaired with aging.^16,17

Diabetes mellitus retards vessel formation in relation to an attenuated response to the mobilization of mononuclear cells induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)¹⁸ and to impaired monocyte chemotaxis in response to VEGF.^16,17,19,20 This overall decrease in circulating EPCs leads to endothelial dysfunction and poor collateral artery formation.^16,20 Additionally, eNOS is inhibited by an increase in free radicals related to diabetes.¹⁷

Hypertension can have a variety of effects on arteriogenesis. Elevated blood pressure can cause an increase in fluid shear stress, which stimulates arteriogenesis. Hypertension is also associated with activation of the renin-angiotensin system and subsequent activation of arteriogenesis. Angiotensin’s role in regulating inflammatory response can lead to initiation of arteriogenesis induced by inflammation. Angiotensin also stimulates increases in circulating VEGF, platelet-derived growth factor, and FGF, all of which stimulate arteriogenesis. Conversely, activation of angiotensin by hypertension is associated with endothelial cell dysfunction as a result of increased oxidative stress due to activation of NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase activity. The increase in oxidative stress increases reactive oxygen and superoxide levels. These oxygen radicles uncouple eNOS, thereby reducing the availability of NO.^16,21,22

Hyperlipidemia affects various steps in arteriogenesis and has direct toxic effect on both endothelial cells and vascular smooth muscle cells.¹⁷ Oxidized low-density lipoprotein cholesterol (LDL) interferes with VEGF function, leading to disordered endothelial cell migration via eNOS inhibition.^17,22 Additionally, endothelial cell FGF and T-lymphocyte migration is reduced, as is endothelial cell replication. Expression of FGF receptors, HIF-1, and VCAM-1 is impaired, leading to impairment of monocyte chemotaxis.²³

Lastly, tobacco abuse impairs endothelial progenitor cell number, function, migration, and adherence. Monocyte migration in response to VEGF is disordered with smoking. Decreased levels of VEFG and HIF-1 further impair EPC function.^16,17,22

Sprouting Angiogenesis

Sprouting angiogenesis involves endothelial cell projections into surrounding connective tissue. Breakdown of the basement membrane occurs, along with interendothelial junction formation, enabling endothelial cell projection.^26–28 Endothelial migration occurs along the projection’s front with further sprouting, ultimately developing a complex capillary meshwork.

Sprouting angiogenesis requires specialization of cells along the migrating projection into “tip,” “stalk,” and “phalanx” cell phenotypes on the basis of the interaction of factors promoting or inhibiting angiogenesis.^29–34 Tip cells are polarized migratory cells that are at the forefront of the endothelial sprout. These cells branch at the tip of the stalk as they extend filopodia toward the stimulus, yet accomplish this with hardly any proliferation. Stalk cells conversely exhibit a proliferative phenotype responsible for the lengthening of the endothelial sprout. These cells are also responsible for secretion of basement membrane along the stalk and formation of vascular lumina from the initial luminal slit.^32,34,35 Additional stability to the proliferating stalk is provided by pericytes, which surround the basement membrane and provide further vessel coverage and decrease leakage from the vessel.^35–37 Initially, the process of sprouting requires only minimal endothelial cell proliferation, although this demand increases with continued sprouting.^29,30,35

A third cell type, the “phalanx” cells, are endothelial cells that become quiescent after completion of the vascular branch. These cells deposit basement membrane and form tight cellular junctions via increased expression of vascular endothelial cadherin (VE-cadherin). These cells are ultimately responsible for delivery of oxygen and nutrients to surrounding tissues.^35,38

Regulation of Angiogenesis

Angiogenesis is initiated by ischemia, leading to increased VEGF expression. VEGF is a potent angiogenic factor, serving to encourage endothelial cell binding to VEGF receptor 2 (VEGFR-2), which promotes endothelial chemotaxis. VEGF expression induces extension of tip cells and proliferation of stalk cells with concomitant synthesis of basement membrane components.^39–41 Additionally, pericytes are attracted and contribute to capillary network formation. Whereas VEGF induces sprouting, Notch signaling pathways function to limit tip migration. Notch signaling occurs by increasing expression of VEGFR-1, competitively binding VEGF and thereby limiting its availability.^{25,31–33,42} The balance of VEGF and Notch signaling therefore regulates sprouting-related vessel development.

Transformation to a tip cell phenotype is induced by exposure of endothelial cells to VEGF. Delta-like ligand 4 (Dll4), a Notch binding ligand, is highly expressed by tip cells, increasing sensitivity to VEGF and binding to VEGFR-2.^{31,32,39,43–46} Increased VEGF–VEGFR-2 binding upregulates Dll4, leading to downregulation of VEGFR-2 on adjacent endothelial cells. This process allows the tip cell to competitively maintain its position.^29,33,41,47 These adjacent cells transform into a stalk cell phenotype and express Notch, which is induced by Dll-4.^31,46,48 Whereas tip cells have low Notch signaling, stalk cells express higher Notch signaling and higher expression of the jagged protein-1 (JAG-1), counteracting Notch-Dll4 activity and limiting tip cell migration (Fig. 8-5).^{31,39,47–50}

Tubulogenesis, or lumen formation, is responsible for transforming endothelial stalks into vessels capable of carrying blood and nutrients to surrounding tissue. This process initially involves establishing endothelial cell apical-basal polarity, mediated by VE-cadherin. Apical borders face apposing cells, whereas the basal border faces the periphery. Beyond this first step, there are three proposed mechanisms to explain lumen development. The first process involves development of intracellular pinocytotic vesicles and vacuoles, which progressively fuse within the endothelial cell and then with adjacent cells, leading to lumen formation along the length of the stalk. The second mechanism is similar but involves exocytosis of these vacuoles between endothelial cells along the length of the growing stalk. These vacuoles then coalesce and form a lumen.

The third mechanism by which lumen formation may occur is by reorganization of intracellular junctions, mediated by VE-cadherin. Endothelial cells adhere to each other and become polar cells as VE-cadherin localizes CD34-sialomucins to the apical cell surface.^32,51–55 The negative charges of sialomucins leads to repulsion of adjacent surfaces of the endothelial cells, inducing lumen slit formation (Fig. 8-6).^32,51 As the lumen develops, the CD34-sialomucins are rearranged to the lateral surfaces of the cells and F-actin is attracted to the exposed lumen. VEGF attracts nonmuscle myosin II to the cell surface, with formation of an actinomyosin complex along the apical endothelial cell surface. This cytoskeletal interaction encourages cellular morphologic shape changes and further luminal expansion.^32,51–53 Beyond tubulogenesis, further increases in lumen diameter are primarily related to fluid shear stress.^29,51

Intussusceptive angiogenesis involves the formation of transcapillary tissue pillars that fuse to form new vessels. This leads to a rapid increase in the complexity of the capillary plexus and increased surface area for gas and nutrient exchange.^56–59 Initially, cell contact is created between opposing capillary cells, followed by formation of a bilayer with reorganization of endothelial cell junctions. A pillar core then develops as the result of this activity. Pericytes, myofibrils, and interstitial cells are responsible for perforating the endothelial bilayer to define discrete vessels,^24,59,60 while collagen fibrils are deposited as a foundation for the developing meshwork.⁶⁰ After initial capillary formation by either vasculogenesis or sprouting angiogenesis, intussusception is initiated to allow for rapid capillary expansion and remodeling by three basic mechanisms: intussusceptive microvascular growth, arborization, and intussusceptive branching remodeling.^24,59–61

Intussusceptive microvascular growth initially involves formation of tissue pillars, which provides an increase in the surface area available for exchange of oxygen, carbon dioxide, and nutrients.^24,58–60

The next phase, intussusceptive arborization, involves the development of “vertical pillars,” which transform well-perfused capillary segments into arterioles and venules. This process decreases the distance between arteries and veins as the capillary plexus expands. Pillars form from endothelial cell reorganization and merging of developing tissue septa (Fig. 8-7). Horizontal folds develop and lead to pillar detachment from any remaining connections, ultimately separating the feeding vessel from the capillary plexus. Hemodynamic forces therefore have a role in further development of the arterial tree.^59,60,62