**3. Mechanical factors involved in coronary collateral growth**

The precise stimulation of arteriogenesis is yet to be found; however, both mechanical and chemical influences are required to induce the formation of collaterals in the heart. Mechanical shear stress occurs due to increased pressure gradients that form when an occlusion is present [32]. A stenotic artery will increase the pressure prior to the occlusion while decreasing the pressure distal to the occlusion. The increased pressure above the occlusion will cause an increase in blood flow into capillary beds prior to the occlusion increasing the shear stress [32]. The increased movement of blood into pre-existing collaterals and the resultant increased shear stress leads to several changes in the capillary endothelium. The first of which includes an increase in MCP-1 that serves to attract more monocytes to the proliferative site in order to transform them into the subsequent macrophages. The macrophages play a vital role in releasing cytokines and growth factors required for arteriogenesis. TNF-α, released by macrophages, helps form the inflammatory environment required for the growth of collaterals [33]. Another major factor includes basic fibroblast growth factor (bFGF), which helps with the actual development of collaterals [32]. A more in depth analysis on chemical inducers will be discussed later on in this chapter.

Although mechanical shear stress is thought to be a major contributor to arteriogenesis, it cannot be the sole solution due to the inability of fluid shear stress to completely replace the conducting artery. Fluid shear stress (FSS) has been found to only reach 35–40% of the maximal conductance possessed by the original stenotic artery [34]. An explanation for this phenomenon can be found in the relationships: FSS and blood flow velocity and FSS and cube of the vessel radius. FSS and blood flow velocity have a proportional relationship, while FSS is inversely related to the cube of the vessel radius [34]. The increase in blood flow velocity in the pre-existing collaterals leads to an increase in FSS. Since the shear stress causes growth in the collaterals (meaning an increase in the vessel radius), the FSS begins to decline preventing full recovery of the stenotic artery [34]. This indicates the need for both mechanical and chemical effectors for the production of proper coronary collaterals.

## **4. Chemical factors involved in coronary collateral growth**

In addition to mechanical mechanisms of arteriogenesis, there are several chemical mediators involved in regulation of the process. Many of these chemical factors modulate the functions of the various cell types involved in arteriogenesis, including induction of cell proliferation, chemotaxis, and cellular remodeling. In this section, we will outline the various chemical mediators that are currently known to play a role in arteriogenesis.

#### **4.1. Vascular endothelial growth factor (VEGF)**

Vascular endothelial growth factor (VEGF) is known to play a major role in development of new vasculature. Under hypoxic conditions, VEGF production and release stimulates new capillary formation (angiogenesis) via endothelial cell sprouting, proliferation, and migration [35]. Alternatively, under different conditions, it can instead stimulate growth of new arteries, formation of collateral vessels, and modulation of lumen expansion—these actions are collectively referred to as arteriogenesis [35].

In VEGF signaling, there are three primary cell-surface receptors to which it binds: two tyrosine kinase receptors VEGFR-1 and VEGFR-2 and a nonkinase receptor neuropilin-1 (NRP-1) [35–37]. There are multiple isoforms of VEGF, with VEGF-A playing the major role in endothelial cell function via binding to VEGFR-2 [35]. VEGFR-2 is also involved in signaling pathways that lead to arteriogenesis via stimulation of proliferation, migration, survival, and lumenization of endothelial cells [35].

The first of these signaling cascades is activation of phosphatidylinositol 3-kinase (PI3K)/Akt that inhibits apoptosis in endothelial cells thus promoting cell survival [35]. The cascade is initiated by binding of VEGF-A to VEGFR-2 (Flk-1), which initiates receptor internalization via clathrin-coated pits followed by receptor autophosphorylation [35]. Active VEGFR-2 then phosphorylates PI3K, which goes on to phosphorylate the serine/threonine kinase Akt, which will go on to phosphorylate targets to inhibit apoptosis [38].

The second signal cascade is phosphorylation of profilin-1, indirectly via Src/FAK as well as directly via VEGFR-2, which stimulates migration of endothelial cells [35, 37]. Like the previous mechanism, this cascade is initiated by binding of VEGF-A to VEGFR-2. Activated VEGFR-2 then goes on to phosphorylate profilin-1 as well as Src kinase, which also phosphorylates profilin-1 [37]. Phosphorylated profilin-1 then goes on to catalyze the exchange of ADP for ATP on G-actin that stimulates polymerization of actin and resultant remodeling of the endothelial cell cytoskeleton [37]. This remodeling results in formation of actin-rich filopodia extending in the direction of the concentration gradient of VEGF, thus stimulating endothelial cell migration [37].

The third signal cascade is activation of the Raf-MEK-ERK signal cascade, which stimulates proliferation of endothelial cells, network formation, and increase in lumen size via phosphorylation of ERK1/2 [35]. While the exact mechanism of ERK1/2 action on cell proliferation and motility is not yet well understood, it has been suggested that a major component of this signaling cascade is downregulation of Rho-Kinase activity [39].

Finally, it is important to note that VEGF has been shown to be a critical factor in the process of coronary collateral growth. In a study of collateral growth following myocardial infarction in rats, it was observed that when the endogenous functions of VEGF were blocked by anti-VEGF neutralizing antibody, the result was a complete lack of collateral growth and subsequently no increase in coronary flow in the anti-VEGF group [40]. Additionally, upon treatment with dipyridamole (a potent vasodilator), it was observed that the increased coronary flow seen in the control group was in fact due to collateral growth, as there was no observed increase in coronary flow in the anti-VEGF group after the dipyridamole [40]. This study solidifies the importance of VEGF in the process of angiogenesis, particularly as it relates to coronary collateral growth.

## **4.2. bFGF and PDGF**

shear stress leads to several changes in the capillary endothelium. The first of which includes an increase in MCP-1 that serves to attract more monocytes to the proliferative site in order to transform them into the subsequent macrophages. The macrophages play a vital role in releasing cytokines and growth factors required for arteriogenesis. TNF-α, released by macrophages, helps form the inflammatory environment required for the growth of collaterals [33]. Another major factor includes basic fibroblast growth factor (bFGF), which helps with the actual development of collaterals [32]. A more in depth analysis on chemical inducers will be

Although mechanical shear stress is thought to be a major contributor to arteriogenesis, it cannot be the sole solution due to the inability of fluid shear stress to completely replace the conducting artery. Fluid shear stress (FSS) has been found to only reach 35–40% of the maximal conductance possessed by the original stenotic artery [34]. An explanation for this phenomenon can be found in the relationships: FSS and blood flow velocity and FSS and cube of the vessel radius. FSS and blood flow velocity have a proportional relationship, while FSS is inversely related to the cube of the vessel radius [34]. The increase in blood flow velocity in the pre-existing collaterals leads to an increase in FSS. Since the shear stress causes growth in the collaterals (meaning an increase in the vessel radius), the FSS begins to decline preventing full recovery of the stenotic artery [34]. This indicates the need for both mechanical and chemical

In addition to mechanical mechanisms of arteriogenesis, there are several chemical mediators involved in regulation of the process. Many of these chemical factors modulate the functions of the various cell types involved in arteriogenesis, including induction of cell proliferation, chemotaxis, and cellular remodeling. In this section, we will outline the various chemical

Vascular endothelial growth factor (VEGF) is known to play a major role in development of new vasculature. Under hypoxic conditions, VEGF production and release stimulates new capillary formation (angiogenesis) via endothelial cell sprouting, proliferation, and migration [35]. Alternatively, under different conditions, it can instead stimulate growth of new arteries, formation of collateral vessels, and modulation of lumen expansion—these actions are

In VEGF signaling, there are three primary cell-surface receptors to which it binds: two tyrosine kinase receptors VEGFR-1 and VEGFR-2 and a nonkinase receptor neuropilin-1 (NRP-1) [35–37]. There are multiple isoforms of VEGF, with VEGF-A playing the major role in endothelial cell function via binding to VEGFR-2 [35]. VEGFR-2 is also involved in signaling pathways that lead to arteriogenesis via stimulation of proliferation, migration, survival, and

discussed later on in this chapter.

effectors for the production of proper coronary collaterals.

138 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**4. Chemical factors involved in coronary collateral growth**

mediators that are currently known to play a role in arteriogenesis.

**4.1. Vascular endothelial growth factor (VEGF)**

collectively referred to as arteriogenesis [35].

lumenization of endothelial cells [35].

In addition to VEGF, other growth factors are known to play a role in arteriogenesis—notably basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) [41]. Basic FGF and PDGF are known to induce mitosis in both endothelial and smooth muscle cells and also exert other mitogenic effects such as promoting cell migration and differentiation [41, 42]. Basic FGF stimulates these mitogenic effects via binding to FGF receptors (FGFRs) expressed on cell surfaces [43]. These FGFRs are a part of the tyrosine kinase receptor family, and following binding of bFGF dimerize are autophosphorylated to become activated [43]. Activated FGFRs, notably FGFR-2 (FGFR-1 is thought to be a regulator of bFGF concentration available to bind FGFR-2), then continue the signal cascade by activation of cytoplasmic mitogen-activated protein kinase (MAPK), which then is translocated to the nucleus to initiate transcription promoting the aforementioned mitogenic effects [43]. PDGF, on the other hand, is known to activate multiple other downstream targets including PI3K, phospholipase C (PLC), as well as MAPK to mediate its mitogenic effects [44].

#### **4.3. MCP-1 and macrophages**

In addition to endothelial cells, macrophages are also heavily involved in arteriogenesis, but in order to do so must be directed to the correct location [36]. The primary molecule that has been studied as part of this mechanism is monocyte chemotactic protein 1 (MCP-1) [36]. Secretion of MCP-1 is initiated by activation of endothelial cell MAP-kinase-protein-kinase-2 (MK2) by elevation of fluid shear stress [45]. Released MCP-1 subsequently activates monocyte MK2, initiating migration to the correct location [45]. In the last step of the cycle, release of inflammatory cytokines by recruited monocytes cause increased secretion of MCP-1 from the endothelium, resulting in further monocyte recruitment [45]. Of similarly significant importance to MCP-1 are two adhesive molecules, intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), which serve to bind the surface of migrating monocytes allowing them to roll along the luminal surface of the vasculature [36].

Once the macrophages have reached their destination, the correct phenotype must be expressed to stimulate new vessel growth [36]. There are two primary phenotypes of monocyte macrophages: M1 macrophages that secrete inflammatory molecules and help fight pathogens and M2 macrophages that play a role in vascular growth and wound healing [36]. These two phenotypes are induced by different cytokines, with interferon-γ causing a shift toward the M1 phenotype, while IL-4, IL-13, and several other factors such as IL-10 and IL-33 causing M2 differentiation. In histological analysis of hypoxia-induced arteriogenesis, the number of M2 macrophages was shown to increase indicating their essential role in development of new vasculature [36].

## **4.4. NO and eNOS**

Nitric oxide (NO) is a potent vasodilator that is produced via the activity of endothelial nitric oxide synthase (eNOS). eNOS has been shown to function in stimulating production of new vasculature, and its expression is also known to be upregulated in response to elevated fluid shear stresses—a principal mechanical stimulus of arteriogenesis [46]. While the exact effects of NO and eNOS on arteriogenesis are still controversial, it has been shown that one contribution of elevated NO due to increased expression of eNOS is a reduction of vascular endothelial cadherin (VE-cadherin), resulting in increased vascular permeability and indirect promotion of macrophage invasion [46]. Additionally, eNOS activity and pharmacological inhibition of eNOS were shown to play a role in mediating vascular remodeling during collateral growth [47, 48].

## **4.5. Catestatin**

Catestatin is a neuroendocrine peptide derived from a specific cleavage of the larger protein human chromogranin A (CgA) [49]. It functions in many different processes within the body including secretion of histamine from mast cells, defense against microbes, vasodilation, and attraction of monocytes. It has also been observed that catestatin acts in a proangiogenic capacity, involved in inducing proliferation and migration of endothelial cells as well as formation of capillary tubes [49, 50]. This is accomplished through stimulating release of bFGF, which in turn will activate MAPK via binding to FGFR-1 as previously discussed [49, 50]. It has also been shown that catestatin activates other signal cascades such as PI3K/Akt, serving an anti-apoptotic role to promote cell survival [50]. Finally, catestatin influences effects in both endothelial progenitor cells (EPCs) and vascular smooth muscle cells (VSMCs) in addition to its direct effects on endothelial cells, inducing chemotaxis to incorporate these cell types into formation of new vasculature [50].

## **4.6. Neuregulins**

transcription promoting the aforementioned mitogenic effects [43]. PDGF, on the other hand, is known to activate multiple other downstream targets including PI3K, phospholipase C

In addition to endothelial cells, macrophages are also heavily involved in arteriogenesis, but in order to do so must be directed to the correct location [36]. The primary molecule that has been studied as part of this mechanism is monocyte chemotactic protein 1 (MCP-1) [36]. Secretion of MCP-1 is initiated by activation of endothelial cell MAP-kinase-protein-kinase-2 (MK2) by elevation of fluid shear stress [45]. Released MCP-1 subsequently activates monocyte MK2, initiating migration to the correct location [45]. In the last step of the cycle, release of inflammatory cytokines by recruited monocytes cause increased secretion of MCP-1 from the endothelium, resulting in further monocyte recruitment [45]. Of similarly significant importance to MCP-1 are two adhesive molecules, intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), which serve to bind the surface of migrating

monocytes allowing them to roll along the luminal surface of the vasculature [36].

Once the macrophages have reached their destination, the correct phenotype must be expressed to stimulate new vessel growth [36]. There are two primary phenotypes of monocyte macrophages: M1 macrophages that secrete inflammatory molecules and help fight pathogens and M2 macrophages that play a role in vascular growth and wound healing [36]. These two phenotypes are induced by different cytokines, with interferon-γ causing a shift toward the M1 phenotype, while IL-4, IL-13, and several other factors such as IL-10 and IL-33 causing M2 differentiation. In histological analysis of hypoxia-induced arteriogenesis, the number of M2 macrophages was shown to increase indicating their essential role in development of new vasculature [36].

Nitric oxide (NO) is a potent vasodilator that is produced via the activity of endothelial nitric oxide synthase (eNOS). eNOS has been shown to function in stimulating production of new vasculature, and its expression is also known to be upregulated in response to elevated fluid shear stresses—a principal mechanical stimulus of arteriogenesis [46]. While the exact effects of NO and eNOS on arteriogenesis are still controversial, it has been shown that one contribution of elevated NO due to increased expression of eNOS is a reduction of vascular endothelial cadherin (VE-cadherin), resulting in increased vascular permeability and indirect promotion of macrophage invasion [46]. Additionally, eNOS activity and pharmacological inhibition of eNOS were shown to play a role in mediating vascular remodeling during collateral growth [47, 48].

Catestatin is a neuroendocrine peptide derived from a specific cleavage of the larger protein human chromogranin A (CgA) [49]. It functions in many different processes within the body including secretion of histamine from mast cells, defense against microbes, vasodilation, and attraction of monocytes. It has also been observed that catestatin acts in a proangiogenic capacity, involved in inducing proliferation and migration of endothelial cells

(PLC), as well as MAPK to mediate its mitogenic effects [44].

140 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**4.3. MCP-1 and macrophages**

**4.4. NO and eNOS**

**4.5. Catestatin**

Neuregulins (NRG) are another class of molecules produced by endothelial cells. These growth factor ligands bind to erbB receptors expressed on the surface of endothelial cells this action has been shown to induce angiogenesis [51]. NRG involvement in angiogenesis and arteriogenesis is tied to regulation of α<sup>v</sup> β3 integrin, thus playing a role in cell migration, proliferation, and differentiation mechanisms as shown in a NRG-erbB knockout mouse model. The mechanism by which this occurs involves another proangiogenic protein, Cyr61, the expression of which is upregulated by NRG-erbB signaling, in addition to mediation via induction of VEGF release and subsequent activation of the ERK signaling cascade [51]. ErbB receptors have also been found to be expressed on EPCs, playing a role in increasing cell survival, and on certain types of VSMCs, though the role NRG-erbB signaling plays here is not well known [51].

## **4.7. Early growth response 1 (Egr-1)**

Early growth response 1 (Egr-1) is a transcription factor of the zinc-finger family that has been shown to be upregulated during arteriogenesis [52]. It plays a major role in modulating the levels of other growth factors that are involved in the process of collateral growth, including playing a role in the recruitment and proliferation of leukocytes [52, 53]. Specific genes that are upregulated by Egr-1 include PDGF and transforming growth factor β (TGF-β), which then indirectly upregulates other factors involved in collateral growth such as VEGF and metalloproteinases [53]. Interestingly, despite the fact that most of these factors have been primarily shown to affect angiogenesis, it has been observed that Egr-1 primarily affects the growth of arterioles rather than capillaries, indicating its primary role in regulation of arteriogenesis [53].

Much like the other factors mentioned here, Egr-1 production is stimulated primarily by elevations in fluid shear stress, in this case by activating the Egr-1 gene promoter [54]. It has been suggested that this is mediated by the Ras-MEK-ERK1/2 signal cascade in which shear stress leads to activation of MEK1, which proceeds to activate ERK1/2 of the MAPK family, and finally ERK1/2 activate the protein Elk-1 that induces transcription of Egr-1 [54]. Interestingly, this pathway can be activated by very low levels of shear stress due to the sensitivity of ERK1/2 [54].

There are many varying chemical mediators of arteriogenesis, many of which share similar signaling pathways leading to their involvement. While some of these mediators have been studied extensively and are relatively well understood, there are others whose mechanisms have not yet been elucidated and require more investigation. There are likely even more chemical mediators involved that have not yet been studied. Going forward, more research will be extremely valuable in understanding the overall chemical mechanism behind collateral vessel growth and how to apply this knowledge to a clinical setting.
