The art of arteriogenesis

The identification of collateral artery growth (arteriogenesis) as the only mechanism to compensate for the loss of an occluded artery forced us to define the mechanisms responsible for this type of vessel growth. To achieve this, a variety of coronary as well as peripheral models of arteriogenesis have been developed. Based on these studies it is obvious that arteriogenesis obeys different mechanisms than angiogenesis, the sprouting of capillaries. Upon occlusion of an artery, the blood flow is redirected into preexisting arteriolar anastomoses that experience increased mechanical forces such as shear stress and circum ferential wall stress. The endothelium of the arteriolar connections is then activated, resulting in an increased release of monocyte-attracting proteins as well as an upregulation of adhesion molecules. Upon adherence and extravasation, monocytes promote arteriogenesis by supplying growth factors and cytokines that bind to receptors that are expressed on vascular cells within a limited time frame. Animal studies evidenced that factors, such as monocyte chemoattractant protein-1, granulocyte-monocyte colony-stimulating factor, or transforming growth factor-β₁, that either attract or prolong the lifetime of monocytes efficiently enhance collateral artery growth, an effect that was seen only to a minor degree after application of a single growth factor. Bone marrow-derived stems cells and endothelial progenitor cells do not incorporate in growing arteries but, rather, function as supporting cells. Complete elucidation of the mechanisms of arteriogenesis may lead to efficacious therapies counteracting the devastating consequences of vascular occlusive diseases.     

Experimental models of arteriogenesis: differences and implications

Cardiovascular and cerebrovascular disease represent the two most common causes of mortality and morbidity in western countries, and the treatment for these is generally by the mechanical restoration of blood flow in the affected tissues. Stimulation of collateral artery growth (arteriogenesis) provides a potential alternative option for the treatment of patients suffering from occlusive artery disease. Therefore, researchers have established several angiogenesis and arteriogenesis animal models to investigate basic mechanisms and pharmacological modulation of collateral artery growth. The authors highlight the most important aspects of vascular growth, discuss different methods and techniques for examining the process, and review the advantages and disadvantages associated with the animal models available for studying this phenomenon.     

Anti-tumor necrosis factor-{alpha} therapies attenuate adaptive arteriogenesis in the rabbit

The specific antagonists of tumor necrosis factor-alpha (TNF-alpha), infliximab and etanercept, are established therapeutic agents for inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Although the importance of TNF-alpha in chronic inflammatory diseases is well established, little is known about its implications in the cardiovascular system. Because proliferation of arteriolar connections toward functional collateral arteries (arteriogenesis) is an inflammatory-like process, we tested in vivo the hypothesis that infliximab and etanercept have antiarteriogenic actions. Sixty-three New Zealand White rabbits underwent femoral artery occlusion and received infliximab, etanercept, or vehicle according to clinical dosage regimes. After 1 wk, collateral conductance, assessed with fluorescent microspheres, revealed significant inhibition of arteriogenesis (collateral conductance): 52.4 (SD 8.1), 35.2 (SD 7.7), and 33.3 (SD 10.1) ml x min(-1) x 100 mmHg(-1) with PBS, infliximab, and etanercept, respectively (P < 0.001). High-resolution angiography showed no significant differences in number of collateral arteries, but immunohistochemical analysis demonstrated a decrease in mean collateral diameter, proliferation of vascular smooth muscle cells, and reduction of leukocyte accumulation around collateral arteries in treated groups. Infliximab and etanercept bound to infiltrating leukocytes, which are important mediators of arteriogenesis. Infliximab induced monocyte apoptosis, and neither substance affected monocyte expression of the adhesion molecule Mac-1. We demonstrated that TNF-alpha serves as a pivotal modulator of arteriogenesis, which is attenuated by treatment with TNF-alpha inhibitors. Reduction of collateral conductance is most likely due to inhibition of perivascular leukocyte infiltration and subsequent lower vascular smooth muscle cell proliferation. This is the first report showing a negative influence of TNF-alpha inhibitors on collateral artery growth.     

Enhanced arteriogenesis in mice overexpressing erythropoietin

After permanent occlusion of the femoral artery, the survival of ischemic limb tissue depends on collateral artery growth (arteriogenesis). In previous work, we have shown that shear stress triggers arteriogenesis. To test whether increased shear stress results in enhanced arteriogenesis, we compared arteriogenesis in transgenic mice overexpressing erythropoietin (EPO), which possessed increased blood viscosity through the higher hematocrit (thereby providing increased shear stress), with wild-type mice. The right femoral artery was occluded proximal to the origin of the arteria poplitea. Distal blood flow was assessed by laser Doppler imaging, and the growth and remodeling of collateral arteries was examined by light and electron microscopy and morphometry. After occlusion of the femoral artery, EPO mice demonstrated enhanced arteriogenesis: their collateral arteries developed a 1.7-fold diameter and a 2-fold wall thickness compared with wild-type. However, the blood flow recovery in EPO mice was markedly retarded. Structural remodeling and growth of collateral arteries was markedly enhanced in EPO mice, presumably as a result of increased blood viscosity and shear stress.     

Collateral arteries grow from preexisting anastomoses in the rat hindlimb

Previous findings have suggested that collateral arteries grow from preexisting arteriolar anastomoses ("arteriogenesis"). To investigate whether collateral growth occurs without preceding angiogenesis, we obtained vascular casts and postmortem angiographies 3, 7, and 21 days after unilateral femoral artery occlusion in the rat. Proliferation kinetics were determined after 5'-bromo-2'-desoxyuridin infusion. A preexisting anastomosis was identified. Proliferation of this vessel began 24 h after femoral artery occlusion, increased maximally during the first 3 days, and reached 60% at day 7. Cell division was restricted to preexisting anastomoses and occurred neither in directly neighboring arterial vessels nor in capillaries. Collateral vessels doubled their diameter within 7 days and assumed a typical corkscrew appearance (increase of length: 21%). After 7 days of occlusion, we measured a further increase of length (14%) but no proliferation or increase of diameter. We conclude that arteriogenesis is a biphasic process involving rapid proliferation of preexisting arteriolar shunts followed by pronounced remodeling processes. Arteriogenesis occurs independently of angiogenesis and denotes a separate entity of vascular proliferation.     

VEGF receptor antagonism blocks arteriogenesis, but only partially inhibits angiogenesis, in skeletal muscle of exercise-trained rats

Both collateral vessel enlargement (arteriogenesis) and capillary growth (angiogenesis) in skeletal muscle occur in response to exercise training. Vascular endothelial growth factor (VEGF) is implicated in both processes. Thus we examined the effect of a VEGF receptor (VEGF-R) inhibitor (ZD4190, AstraZeneca) on collateral-dependent blood flow in vivo and collateral artery size ex vivo (indicators of arteriogenesis) and capillary contacts per fiber (CCF; an index of angiogenesis) in skeletal muscle of both sedentary and exercise-trained rats 14 days after bilateral occlusion of the femoral arteries. Total daily treadmill run time increased appreciably from approximately 70 to approximately 100 min (at 15-20 m/min, twice per day) and produced a large (approximately 75%, P < 0.01) increase in calf muscle blood flow and a greater size of the collateral artery (wall cross-sectional area). ZD4190, which previously has been shown to inhibit the activity of VEGF-R2 and -R1 tyrosine kinase in vitro (IC50 = 30 and 700 nM, respectively), completely blocked the increase in collateral-dependent blood flow and inhibited collateral vessel enlargement. Thus exercise-stimulated collateral arteriogenesis appears to be completely dependent on VEGF-R signaling. Interestingly, enhanced mRNA expression of the VEGF family ligand placental growth factor (2- to 3.5-fold), VEGF-R1 (approximately 2-fold), and endothelial nitric oxide synthase (2- to 3.5-fold) in an isolated collateral artery implicates these factors as important in arteriogenesis. Training of ischemic muscle also induced angiogenesis, as shown by an increase (approximately 25%, P < 0.01) in CCF in white gastrocnemius muscle. VEGF-R inhibition only partially blocked (P < 0.01) but did not eliminate the increase (P < 0.01) in capillarity. Our findings indicate that VEGF-R tyrosine kinase activity is essential for collateral arteriogenesis and important for the angiogenesis induced in ischemic muscle by exercise training; however, other angiogenic stimuli are also important for angiogenesis in flow-limited active muscle.     

Nitrite anion stimulates ischemic arteriogenesis involving NO metabolism

Nitric oxide (NO) is a potential regulator of ischemic vascular remodeling, and as such therapies augmenting its bioavailability may be useful for the treatment of ischemic tissue diseases. Here we examine the effect of administering the NO prodrug sodium nitrite on arteriogenesis activity during established tissue ischemia. Chronic hindlimb ischemia was induced by permanent unilateral femoral artery and vein ligation. Five days postligation; animals were randomized to control PBS or sodium nitrite (165 μg/kg) therapy twice daily. In situ vascular remodeling was measured longitudinally using SPY angiography and Microfil vascular casting. Delayed sodium nitrite therapy rapidly increased ischemic limb arterial vessel diameter and branching in a NO-dependent manner. SPY imaging angiography over time showed that nitrite therapy enhanced ischemic gracillis collateral vessel formation from the profunda femoris to the saphenous artery. Immunofluorescent staining of smooth muscle cell actin also confirmed that sodium nitrite therapy increased arteriogenesis in a NO-dependent manner. The NO prodrug sodium nitrite significantly increases arteriogenesis and reperfusion of established severe chronic tissue ischemia.     

Regulation of collateral blood vessel development by the innate and adaptive immune system

The development of collateral circulation is an inherent compensatory mechanism to restore impaired blood perfusion following artery stenosis and/or occlusion. This process, termed arteriogenesis, is driven by inflammation and involves a complex remodeling of pre-existing conduit vessels running in parallel to the occluded artery. Recent studies have unveiled roles for different immune cell subsets as regulators of arteriogenesis, including natural killer (NK) cells, T helper 17 (Th17) cells, regulatory T lymphocytes (Tregs), and functional subsets of macrophages (e.g., M2 macrophages). This review summarizes recent findings and discusses future research needed to better define the time during which each cellular subset is active and reveal further critical regulatory switches.     

Blood monocyte concentration is critical for enhancement of collateral artery growth

Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. We tested the hypothesis that arteriogenesis is functionally linked to the concentration of circulating blood monocytes. Monocyte concentrations in peripheral blood were manipulated by single injections of the antimetabolite 5-fluorouracil (5-FU), resulting in a marked rebound effect in New Zealand White rabbits. Collateral artery growth was assessed by the use of a model of acute femoral artery ligation. Seven days after ligation, collateral conductance and the number of visible collateral arteries were increased in the rebound group. This increase was accompanied by an increased monocyte accumulation as demonstrated by immunohistology in the thigh 3 days after surgery. In a second animal model (129S2/SvHsd mice), 5-FU treatment caused a remarkable decrease in blood monocyte numbers at day 4, followed by a rebound effect at day 12. Foot blood flow, assessed by laser-Doppler imaging before and at various time points after surgery, increased from day 7 through day 21 in mice from the rebound group. In contrast, ligation during the phase of monocyte depletion resulted in a reduction of blood flow reconstitution. This inhibition could be reversed by an injection of isolated monocytes. In conclusion, we have demonstrated a functional link between the monocyte concentration in the peripheral blood and the enhancement of arteriogenesis.     

Placenta growth factor expression is regulated by hydrogen peroxide in vascular smooth muscle cells

When supply arteries become occluded, blood is diverted through preexisting collateral vessels. Shear stress arising from this increase in blood flow provides the initial physiological stimulus for expansion of the collateral circulation, a process termed arteriogenesis. Endothelial cells (EC) respond to increased shear stress by releasing a variety of mediators that can act on underlying smooth muscle cells (SMC). Placenta growth factor (PLGF) is known to mediate certain aspects of arteriogenesis, such as recruitment of monocytes to the vessel wall. Therefore, we tested whether SMC PLGF expression is influenced by mediators released by EC. We used A10 SMC cultured with medium that had been conditioned by EOMA EC for 4 days as a model. We found that EC-conditioned medium is able to upregulate PLGF gene expression in A10 SMC. Further experiments identified hydrogen peroxide (H(2)O(2)) as a key mediator of this response. We confirmed the physiological relevance of this mechanism in primary human coronary artery SMCs by demonstrating that exogenous H(2)O(2) specifically upregulates PLGF gene and protein expression. We also demonstrated that the physiological stimulus of shear stress raises endogenous H(2)O(2) levels in media into the range found to increase PLGF expression. In this study, we demonstrate that EC-released H(2)O(2) acts as a positive regulator of PLGF gene and protein expression in vascular SMC. To our knowledge, this is the first study to describe H(2)O(2) as a regulator of PLGF expression and therefore an upstream mediator of PLGF-driven arteriogenesis.     

Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury

Transgenic mouse models are increasingly being used to investigate the functions of specific growth factors or matrix proteins to design therapeutic strategies for controlling blood vessel growth. However, the available methodologies for evaluating angiogenesis and arteriogenesis in these models are limited by animal size, user subjectivity, the power to visualize the three-dimensional vessel networks, or the capability to employ a vigorous quantitative analysis. In this study, we employed contrast-enhanced microcomputed tomography imaging to assess collateral development after induction of hindlimb ischemia in the mouse. The morphological parameters vessel volume, connectivity, number, thickness, thickness distribution, separation, and degree of anisotropy were evaluated in control and surgery limbs 0, 3, and 14 days postsurgery. Results indicate that the vascular volume of the surgically manipulated limb was reconstituted as early as 3 days after femoral artery excision through development of a series of highly connected, small caliber, closely spaced, and isotropically oriented collateral vessels. Parametric analyses were completed to assess the sensitivity of the calculated morphological parameters to variations in image binarization threshold and voxel size. Images taken at the 36-microm voxel size were found to be optimal for evaluating collateral vessel formation, whereas 8- to 16-microm voxel sizes were needed to resolve smaller vascular structures. This study demonstrates the utility of microcomputed tomography as a robust method for quantitative, three-dimensional analysis of blood vessel networks. Whereas these initial efforts focused on the mouse hindlimb ischemia model, the developed techniques may be applied to a variety of model systems to investigate mechanisms of angiogenesis and arteriogenesis.     

Vascular endothelial growth factor (VEGF) stimulates monocyte migration through endothelial monolayers via increased integrin expression

Monocytes play an important role in collateral vessel formation (arteriogenesis) by attaching to activated endothelium and by invading the walls of innate collateral vessels where they produce growth factors. Previous studies have demonstrated that this process can be promoted by several chemokines and growth factors. In this study we examined the interaction between monocytes and endothelium under stimulation of the angiogenic agent vascular endothelial growth factor (VEGF). We report here the novel finding that VEGF stimulates the expression of the alphaL-, alphaM- and beta2-integrin monomers. In functional assays and by using neutralizing antibodies it was shown that VEGF stimulates adhesion of monocytes to human umbilical vein endothelial cells (HUVEC), and increased transmigration through endothelial monolayers is dependent on interaction of monocyte beta2-integrins with its endothelial counter ligand ICAM-1. Based on these in vitro data we hypothesize that the positive effect of VEGF on arteriogenesis may involve monocyte activation.     

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis.     

MAP-Kinase Activated Protein Kinase 2 Links Endothelial Activation and Monocyte/macrophage Recruitment in Arteriogenesis

Arteriogenesis, the growth of natural bypass arteries, is triggered by hemodynamic forces within vessels and requires a balanced inflammatory response, involving induction of the chemokine MCP-1 and recruitment of leukocytes. However, little is known how these processes are coordinated. The MAP-kinase-activated-proteinkinase-2 (MK2) is a critical regulator of inflammatory processes and might represent an important link between cytokine production and cell recruitment during postnatal arteriogenesis. Therefore, the present study investigated the functional role of MK2 during postnatal arteriogenesis. In a mouse model of hindlimb ischemia (HLI) MK2-deficiency (MK2KO) significantly impaired ischemic blood flow recovery and growth of collateral arteries as well as perivascular recruitment of mononuclear cells and macrophages. This was accompanied by induction of endothelial MCP-1 expression in wildtype (WT) but not in MK2KO collateral arteries. Following HLI, MK2 activation rapidly occured in the endothelium of growing WT arteries in vivo. In vitro, inflammatory cytokines and cyclic stretch activated MK2 in endothelial cells, which was required for stretch- and cytokine-induced release of MCP-1. In addition, a monocyte cell autonomous function of MK2 was uncovered potentially regulating MCP-1-dependent monocyte recruitment to vessels: MCP-1 stimulation of WT monocytes induced MK2 activation and monocyte migration in vitro. The latter was reduced in MK2KO monocytes, while in vivo MK2 was activated in monocytes recruited to collateral arteries. In conclusion, MK2 regulates postnatal arteriogenesis by controlling vascular recruitment of monocytes/macrophages in a dual manner: regulation of endothelial MCP-1 expression in response to hemodynamic and inflammatory forces as well as MCP-1 dependent monocyte migration.     

Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback

In developing limb skin, peripheral nerves are required for arterial differentiation, and guide the pattern of arterial branching. In vitro experiments suggest that nerve-derived VEGF may be important for arteriogenesis, but its role in vivo remains unclear. Using a series of nerve-specific Cre lines, we show that VEGF derived from sensory neurons, motoneurons and/or Schwann cells is required for arteriogenesis in vivo. Arteriogenesis also requires endothelial expression of NRP1, an artery-specific coreceptor for VEGF(164) that is itself induced by VEGF. Our results provide the first evidence that VEGF is necessary for arteriogenesis from a primitive capillary plexus in vivo, and show that in limb skin the nerve is indeed the principal source of this signal. They also suggest a model in which a 'winner-takes-all' competition for VEGF may control arterial differentiation, with the outcome biased by a VEGF(164)-NRP1 positive-feedback loop. Our results also demonstrate that nerve-vessel alignment is a necessary, but not sufficient, condition for nerve-induced arteriogenesis. Different mechanisms therefore probably underlie these endothelial patterning and differentiation processes.     

bFGF enhances the development of the collateral circulation after acute arterial occlusion.

An adequate collateral circulation is crucial to tissue survival subsequent to proximal major arterial occlusion. The precise mechanism of collateral blood vessel development and the biochemical mediators involved in this process are unknown. To evaluate the influence of a number of agents on the development of the collateral circulation, we developed a rat model of severe hind limb ischaemia. The recovery of blood flow after acute arterial occlusion was increased by exogenous basic fibroblast growth factor and heparin, and decreased by protamine. Erucamide (cis-13-docosenamide), an angiogenic lipid, had no effect on collateral blood flow. These results indicate that basic fibroblast growth factor and heparin are potential therapeutic agents in the treatment of peripheral vascular disease.     

Expression of endothelial nitric oxide synthase in the vascular wall during arteriogenesis

Nitric oxide (NO) has been demonstrated to play an important role in angiogenesis, and also to be involved in collateral vessel growth. The expression of endothelial NO synthase (eNOS) is moderated partly by blood flow-induced mechanical factors, i.e., shear stress. The purpose of this study was to evaluate how the expression of eNOS correlates with the development of collateral vessels in dog heart, induced by chronic occlusion of the left circumflex artery. Immunoconfocal microscopy using an antibody against eNOS was used to detect expression of eNOS in different stages of arteriogenesis. Collateral vessels were classified into normal, growing and mature vessels by using the cytoskeleton marker desmin. Expression of the growth factors bFGF and metallproteinase-2 (MMP-2) was also examined. The data show that in normal arteriolar vessels, expression of eNOS is very low, but in growing collateral vessel there is a 6.2-fold increase, which, however, returned to normal levels in mature collateral vessels. The expression of eNOS was localized only in endothelium, either in normal or growing vessels. bFGF was very weakly stained in normal vessels, but highly expressed in growing collateral vessels. MMP-2 was strongly stained in neointima, but very weak in endothelium. In addition, we also examined expression of iNOS because iNOS may be induced in vessel injury or in disease states, but it was not detected in either normal or growing collateral vessels. Our findings indicate that the expression pattern of eNOS is closely associated with the development of collateral vessels, suggesting that eNOS plays an important role in arteriogenesis. (Mol Cell Biochem 264: 193–200, 2004)     

Angiogenic synergism,vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2

The establishment of functional and stable vascular networks is essential for angiogenic therapy. Here we report that a combination of two angiogenic factors, platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2, synergistically induces vascular networks, which remain stable for more than a year even after depletion of angiogenic factors. In both rat and rabbit ischemic hind limb models, PDGF-BB and FGF-2 together markedly stimulated collateral arteriogenesis after ligation of the femoral artery, with a significant increase in vascularization and improvement in paw blood flow. A possible mechanism of angiogenic synergism between PDGF-BB and FGF-2 involves upregulation of the expression of PDGF receptor (PDGFR)-alpha and PDGFR-beta by FGF-2 in newly formed blood vessels. Our data show that a specific combination of angiogenic factors establishes functional and stable vascular networks, and provides guidance for the ongoing clinical trials of angiogenic factors for the treatment of ischemic diseases.     

Collateral vessel growth induced by femoral artery ligature is impaired by denervation

Innervation plays an important role in development and remodeling of blood vessels. However, very little is known whether innervation is involved in arteriogenesis. In the present study, we tested the hypothesis that innervation may contribute to the process of arteriogenesis induced by ligature of femoral artery in rat/rabbit hind limb with or without denervation. We found that: (1) angiography showed more collateral vessels in the ligature side than that in ligature plus denervation side; (2) collateral vessels in denervation side was characterized by an inward remodeling; (3) in both collateral vessels (CVs) from only femoral ligature side as well as the ligature plus denervation side, ICAM-1 and VCAM-1 expression was up-regulated but increased VCAM-1 was more evident in the adventitia of collateral vessels of only femoral ligature side; (4) 7 days after surgery, in CVs from the femoral ligature side only, numerous macrophages (RAM11 positive cells) and high cell proliferation ratio (ki67 positive cells) were detected, but they were less in the denervation side. In conclusion, our data demonstrate for the first time that neural regulation is one of the factors that contributes to collateral vessel growth in rat/rabbit hind limb ischemic model by showing collateral vessel growth induced by femoral artery ligature is impaired by denervation.     

Combined administration of naked DNA vectors encoding VEGF and bFGF enhances tissue perfusion and arteriogenesis in ischemic hindlimb

Few studies have examined in detail the combined effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene delivery on collateral development. Here, we evaluated the potential synergism of naked DNA vectors encoding VEGF and bFGF using a skeletal-muscle based ex vivo angiogenesis assay and compared tissue perfusion and limb loss in a murine model of hindlimb ischemia. In the ex vivo angiogenesis assay, the VEGF+bFGF combination group had a larger capillary sprouting area than those of the LacZ, VEGF, and bFGF groups. Consistent with these results, regional blood flow recovery on day 14 was also highest in the VEGF+bFGF combination group, followed by the bFGF, VEGF, and LacZ groups. The limb loss frequency was 0% in the combination group, whereas the limb loss frequencies of the other groups were 7-29%. The ischemic muscles of the combination group revealed evidence of increased angiogenesis and arteriogenesis and the upregulated expression of genes that may be associated with arteriogenesis, such as those for cardiac ankyrin repeat protein, early growth response factor-1, and transforming growth factor-beta1. Our study has implications for the development of a combined gene therapy for the vascular occlusive diseases.