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Peripheral arterial disease (PAD) of the limbs can progress to critical limb ischemia (CLI), characterized by rest pain and tissue loss, including nonhealing ulceration and gangrene. , The body compensates via neovascularization, either the formation of collateral circulation to bypass the obstructed vessel (arteriogenesis) or increasing capillary density (angiogenesis) to deliver oxygen and nutrients to ischemic tissue. Despite these responses, disease progression can lead to amputation, decreased quality of life, comorbidity, and death and is an economic burden to the healthcare system.
Traditional treatment of PAD includes risk factor modification (tobacco abuse, diabetes, hypertension, and hyperlipidemia), exercise programs, and medical therapy (antiplatelet agents, anticoagulants, and phosphodiesterase inhibitors). As atherosclerosis progresses, more invasive intervention may be necessary, including endovascular and open surgical therapies. Patients with CLI may not be candidates for these interventions because of severe medical comorbidity or nonreconstructable vascular disease. This patient population may be candidates for biologic treatments, including gene-based, molecular, and cell-based therapies designed to promote healing and prevent amputation.
Advances in basic science research have developed these biologic therapies over the past two decades. Early clinical trials focusing on gene and molecular therapies, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), have demonstrated limited benefit. More promising results have been observed using cell-based therapies, including endothelial progenitor cells (EPCs) and bone marrow–derived mononuclear cells (BM-MNCs).
Herein, the basic science and processes involved in neovascularization (specifically arteriogenesis and angiogenesis), as well as recent human clinical trials designed to promote neovascularization for CLI, are discussed.
Neovascularization refers to the formation of new blood vessels and includes vasculogenesis, arteriogenesis, and angiogenesis.
Vasculogenesis is the de novo formation of embryonic blood vessels from vascular progenitor cells or hemangioblasts, which develop into hematopoietic precursors and endothelial cells. These cells induce differentiation of discrete vascular layers as cells transform into endothelial cells, smooth muscle cells (SMCs), and adventitial pericytes. Although vasculogenesis primarily occurs during the embryonic stages of development, postembryonic adaptive vasculogenesis results from either arteriogenesis or angiogenesis. ,
Arteriogenesis involves growth of collateral vessels and remodeling from preexisting arterial–arteriolar connections. , Arteriogenesis is induced by a change in hemodynamic forces (fluid shear stress [FSS]) resulting from pressure differences within an artery narrowed by atherosclerosis. FSS activates the endothelium, leading to increased transcription of the promoter regions of a variety of proteins contributing to vessel growth ( Table 7.1 ). With progressive arterial stenosis, blood follows the path of least resistance and is shunted into collateral vessels. Collateral vessel formation maintains a degree of flow beyond the obstruction.
Factor | Characteristics | Role in Arteriogenesis and Angiogenesis | Induced by |
---|---|---|---|
Transcription Factors | |||
HIF-1 | Helix:loop:helix structure Mainly involved in angiogenesis but possible role in VEGF stimulation in arteriogenesis |
Induces gene transcription to promote angiogenesis in ischemic environment Is involved in hypoxic vasodilation, cell growth, proliferation, migration, sprouting, recruitment of pericytes/SMCs, vascular remodeling, mobilization of angiogenic cells and EPCs Also induces MMPs for ECM digestion |
Hypoxia |
EGR-1 | Increased expression in endothelial cells, SMC, fibroblasts, leukocytes | Required for expression of cyclin D1, a regulator of the cell cycle in vascular cells | Shear stress, mechanical injury, hypoxia, PDGF, FGF-1, FGF-2 |
Post-Transcriptional Regulators | |||
MicroRNAs , , , , | ∼22 nucleotides in length, binds to 3′ UTR of target mRNA, single stranded RNA Post-transcriptional gene regulation |
Promote and/or inhibit angiogenesis and arteriogenesis through interactions with various transcription factors, cytokines, and cell adhesion molecules | Varies with each miRNA |
Growth Factors/Cytokines | |||
VEGF | Survival factor for endothelial cells Involved in angiogenesis and arteriogenesis |
Promotes proliferation, migration, lumen formation Induces monocyte chemotaxis via binding to VEGFR1 on monocytes |
Hypoxia and shear stress |
FGF , , | Perivascular macrophages are source of FGF-2 during collateral growth Role in arteriogenesis and angiogenesis |
Induces endothelial/SMC proliferation Stimulates endothelial cell migration/differentiation, and increases EGR-1 expression Potentiates VEGF and may be synergistic with VEGF-B |
Shear stress, endothelial activation |
TGF-β , , , | Expressed in areas of collateral formation | Expressed in developing collateral arteries Stimulates arteriogenesis by effects on endothelial cells, vascular SMCs, monocytes, macrophages |
Shear stress, endothelial activation |
TNFα | Proximal mediator of inflammation | Angiogenic effects from TNFR2R Enhances activation and adhesion of monocytes by upregulating cell adhesion molecules Upregulates GM-CSF Necessary for migration/adhesion of BM-hematopoietic cells to endothelium via NO synthase-dependent mechanisms |
Stimulated by endothelial activation by shear stress and LPS Lipopolysaccharide Ischemia stimulated TNFR2 expression |
GM-CSF , , , | Enhances arteriogenesis through effects on circulating cells | Enhances release, proliferation, and differentiation of hematopoietic stem cells, mobilization of endothelial progenitor cells Amplifies effects of MCP-1, 11 promotes survival of monocytes and macrophages |
Stimulated by endothelial activation by shear stress |
HGF | Augments arteriogenesis by enhancing endothelial cell function Role in angiogenesis and arteriogenesis |
Activates Dll4-Notch-Hey2 pathway for inducing proliferation and migration of endothelial cells | Hypoxia, shear stress |
ECM Proteins | |||
Del-1 , | Involved with angiogenesis | Ischemia | |
Chemokines and Chemokine Receptors | |||
MCP-1 (or CCR2) , | CCR2 axis: MCP-1 binds CCR2 receptor on monocytes Potent stimulator of arteriogenesis and angiogenesis |
Increases attraction/adherence of monocytes and tube formation. May attract endothelial progenitor cells to sites of vascular injury | Hypoxia and shear stress |
ELR-containing CXC , | Binds receptors on CXCR1/2/3 Potent stimulator of angiogenesis, some forms inhibit angiogenesis via inhibition of VEGF/FGF |
Binds chemokines MIG, IP-10, I-TAC, and PF-4 Decreases formation of collaterals and restoration of perfusion in knockout mice Perfusion improved with infusion of bone marrow mononuclear cells |
Hypoxia |
Cell Adhesion Molecules | |||
ICAM, VCAM-1, PECAM-1 | Enhance attraction and adhesion of monocytes | Support diapedesis of monocytes, while enhancing cell signaling and activation of mechanosensory complexes that activate intracellular changes in response to shear stress | Shear stress |
Proteases | |||
MMPs , , , | Macrophages and monocytes are source of MMPs in ischemic/nonischemic tissue Involved in angiogenesis and arteriogenesis |
Allow for ECM remodeling via proteolytic degradation of ECM/BM, enabling collateral vessel/capillary growth and endothelial cell migration Liberate growth factors and stimulate endothelial proliferation ECM/BM breakdown by MMPs promotes SMC proliferation and migration |
Shear stress and hypoxia; MCP-1 activates macrophages to secrete MMPs MMPs activate release of more MMPs from macrophages |
Immune Cells | |||
Monocytes/Macrophages , , , , , , , , | Induce vascular cell proliferation and wall remodeling via paracrine effects | Promote vascular growth and secrete growth factors (MMP, NO, VEGF, FGF-2, GM-CSF, TGF-β, TNF-α) that stimulate arteriogenesis Monocytes accumulate in wall of growing collateral and differentiate into macrophages, liberating MMPs to digest ECM; encourages migration and proliferation of endothelial cells and SMCs |
Shear stress-activated endothelium expresses MCP-1, leading to adhesion of monocytes |
T cells/NK cells , | Immune cells | CD4 and CD8 mononuclear cells migrate to collateral vessel, initiating arteriogenesis/angiogenesis by cytokine activation Athymic mice have higher rates of autoamputation than heterozygotes NK cell deficient mice are unable to form collaterals |
Shear stress |
Mast cells , , | Present in adventitia of collateral arteries | Release TGF-β, VEGF, FGF-2, MMPs, histamine, serotonin | |
Other Cells | |||
BM-EPCs , , | BM-derived cells | Attracted to sites of neovascularization, differentiate into endothelial cells | Shear stress, ischemia Release stimulated by VEGF, SDF-1 |
Pericytes | BM-derived cells | Release VEGF, FGF-2, MCP-1 and MMPs, promoting endothelial cell migration, proliferation, and survival | Shear stress |
Vascular SMCs , | Derived from endothelial cells, mesenchymal cells, and BM-derived cells | Collateral vessel stabilization via ECM production | SMCs attracted by PDGF/VEGF Proliferation stimulated by ECM breakdown |
Angiogenesis, which refers to the sprouting of new capillaries from preexisting ones, is stimulated by decreases in oxygen tension secondary to reduced tissue perfusion. Local ischemia stimulates an increase in hypoxia-inducible factor-1 (HIF-1), which results in increased production of VEGF, a potent angiogenic factor. VEGF induces endothelial cell proliferation and increases endothelial permeability. Matrix metalloproteinases (MMPs) locally degrade basement membrane and extracellular matrix (ECM), allowing vessel growth and increased tissue perfusion. Although this increased flow provides greater delivery of oxygen and nutrients to ischemic tissues, it is often not sufficient to overcome major arterial obstruction. , ,
Postembryonic arteriogenesis and angiogenesis occur over a continuum of vascular adaptations ( Fig. 7.1 ).
Chronic, progressive arterial stenosis leads to generation of a collateral network by remodeling preexisting arterial–arteriolar connections ( Fig. 7.2 ). The formation of this collateral circulation is primarily in response to an increase in FSS with a contribution from increased circumferential wall stress. As an example, femoral artery occlusion results in up to a 200-fold increase in shear stress in the arteriolar network.
FSS is proportional to flow velocity and inversely proportional to the radius cubed, whereby small changes in radius can normalize shear stress. Increased FSS activates the endothelium. , , Nitric oxide (NO) is liberated by endothelial cells (via endothelial NO synthase [eNOS]) as well as by macrophages and SMCs in the adventitia (via inducible NOS). NO induces SMC relaxation and vasodilatation beyond the arterial occlusion, thereby improving blood flow. , , NO also stimulates endothelial VEGF secretion, leading to the release of endothelial cell adhesion molecules (CAMs) and monocyte chemotactic protein-1 (MCP-1) by endothelial and SMCs. , , Both molecules mobilize to the cell surface, generating a “sticky” endothelium that enhances leukocyte attraction, adhesion, and invasion of arteriolar collaterals and periadventitia. ,
Monocytes either attach to CAMs to be incorporated into the lumen of the developing vessel or accumulate in the adventitia. Activated monocytes release tumor necrosis factor-α (TNF-α), further enhancing monocyte attraction. Platelet adherence and activation stimulates growth factor and interleukin-4 production, enhancing monocyte adhesion. Monocytes stimulate production of growth factors, chemokines, and cytokines, in addition to immune cells, leading to the proliferation of collateral arterioles ( Fig. 7.3 ). Macrophages contribute to remodeling of the collateral vessel by liberating proteases. , , Endothelial cell proliferation and endothelial permeability increase, while vascular SMCs proliferate and change from a contractile to a proliferative phenotype.
Circumferential wall stress also plays a role in inducing arteriogenesis. As part of a second phase of arteriogenesis, vascular SMC growth is induced by circumferential wall stress. Increased intravascular pressure leads to SMC proliferation and increased vessel thickness. Increased vessel thickness enables normalization of circumferential wall stress at low blood pressure, which can lead to cessation of collateral vessel growth prior to the complete resolution of ischemia.
The final phase of arteriogenesis involves “pruning” or regression of vessels. Poiseuille’s law states that flow is proportional to radius and predicts that greater flow is observed within fewer larger arterioles rather than many smaller ones. As such, the process of “pruning” leads to regression of smaller collaterals, with persistence of a few larger collateral vessels. Vessel regression is the result of proliferation of the intima that leads to occlusion of the collateral.
A variety of disease states perturb arteriogenesis, leading to impairment of endothelial or macrophage function. The recruitment of EPCs or growth factor production may also be impaired ( Fig. 7.4 ). Aging itself affects arteriogenesis secondary to an overall decrease in endothelial production of NO and by a higher rate of HIF degradation, along with decreased levels of VEGF, platelet-derived growth factor (PDGF), FGF-2, and chemokine signaling. Growth factor release is also impaired with aging. ,
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) and to impaired monocyte chemotaxis in response to VEGF. , , , This overall decrease in circulating EPCs leads to endothelial dysfunction and poor collateral artery formation. , In addition, eNOS is inhibited by an increase in free radicals related to diabetes.
Hypertension can have a variety of effects on arteriogenesis. Elevated blood pressure can cause an increase in FSS, which stimulates arteriogenesis. Hypertension is also associated with activation of the renin–angiotensin system and subsequent activation of arteriogenesis. Angiotensin’s role in regulating the inflammatory response can lead to initiation of arteriogenesis induced by inflammation. Angiotensin also stimulates increases in circulating VEGF, PDGF, 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 radicals uncouple eNOS, thereby reducing the availability of NO. , ,
Hyperlipidemia affects various steps in arteriogenesis and has direct toxic effect on both endothelial cells and vascular SMCs. Oxidized low-density lipoprotein (LDL) cholesterol interferes with VEGF function, leading to disordered endothelial cell migration via eNOS inhibition. , In addition, endothelial cell FGF and T-lymphocyte migration are reduced, as is endothelial cell replication. Expression of FGF receptors, HIF-1, and VCAM-1 is impaired, leading to impairment of monocyte chemotaxis.
Lastly, tobacco abuse impairs EPC number, function, migration, and adherence. Monocyte migration in response to VEGF is disordered with smoking. Decreased levels of VEGF and HIF-1 further impair EPC function. , ,
Angiogenesis involves new capillary formation induced by distal tissue ischemia via sprouting and nonsprouting (intussusceptive microvascular growth [IMG]) mechanisms).
Sprouting angiogenesis involves endothelial cell projections into surrounding connective tissue. Breakdown of the basement membrane occurs along with inter-endothelial junction formation, enabling endothelial cell projection. Endothelial cells migrate 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. 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; this is accomplished with minimal 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. , , 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. Initially, the process of sprouting requires minimal endothelial cell proliferation, although this demand increases with continued sprouting. , ,
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. ,
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. In addition, 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. , , 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. , , , 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. , , , These adjacent cells transform into a stalk cell phenotype and express Notch, which is induced by Dll4. , , 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. 7.5 ). , ,
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, three proposed mechanisms may 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 for lumen formation 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. , The negative charges of sialomucins lead to repulsion of adjacent surfaces of the endothelial cells, inducing lumen slit formation ( Fig. 7.6 ). , 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 non-muscle 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. , Beyond tubulogenesis, further increases in lumen diameter are primarily related to FSS. ,
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