Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis

OBJECTIVE: Animal models of hindlimb ischemia are critical to our understanding of peripheral vascular disease and allow us to evaluate therapeutic strategies aimed to improve peripheral collateral circulation. To further elucidate the processes involved in revascularization following ischemia, we evaluated the temporal association between tissue ischemia, vascular endothelial cell growth factor (VEGF) release, angiogenesis (capillary sprouting), arteriogenesis (growth of the larger muscular arteries), and reserve blood flow (functional collateral flow). METHODS: New Zealand White rabbits (male 3-4 kg) were evaluated at specific days (0, 5, 10, 20 or 40) following femoral artery removal for measurement of hindlimb blood flow, skeletal muscle lactate production and VEGF content, capillary density (a marker of angiogenesis), and angiographic score (a marker of arteriogenesis). RESULTS: Maximal capillary sprouting occurred within 5 days of femoral artery removal and was temporally associated with reduced resting hindlimb blood flow, increased lactate release and detectable levels of skeletal muscle VEGF. The growth of larger angiographically visible collateral vessels occurred after 10 days and was not temporally associated with ischemia or skeletal muscle VEGF content, but did coincide with a large functional improvement in the reserve blood flow capacity of the limb. CONCLUSIONS: Following femoral artery removal in the rabbit, the time course of angiogenesis and arteriogenesis were clearly distinct. Tissue ischemia and/or VEGF may stimulate capillary sprouting, but this response does not translate to a significant improvement in collateral flow. The growth and development of the larger collateral vessels was correlated with a large functional improvement in collateral flow, and occurred at a time when VEGF levels were undetectable.     

Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice

The goal of this study was to examine the mechanisms of vascular growth that lead to the restoration of perfusion in a peripheral vascular disease model in mice. We monitored blood flow recovery and measured vascular growth in inbred strains of mice following femoral artery occlusion. Acute collateral blood flow to the hindlimb was lowest in Balb/C mice, causing intense ischemia, and showed a slower recovery (more than 21 days to 50% normal) than C57Bl/6 which had a 7-fold higher acute collateral flow and a fast recovery (3 days). Collateral vessels were enlarged by proliferation of ECs and SMCs. Capillary density increased in the lower limbs of Balb/Cs (1.7-fold) and of sv129s. Tissue oxygen saturation recovered faster than flow in all strains. Morphometry of mature collaterals showed a diameter increase of 2.1-2.4 fold. The increase in total vessel wall area exceeded that of the femoral artery by 1.4-fold and the common lumenal area by 1.6-fold. Infusion of the growth factor peptide FGF-2 by osmotic minipump accelerated arteriogenesis but inhibited the angiogenic response probably because it prevented ischemia. Conclusion: the speed of arteriogenesis is inversely related to the intensity of ischemia, and arteriogenesis is by far the most efficient mechanism to increase blood flow after femoral artery occlusion. De novo arteriogenesis was not observed.     

Pathophysiology of collateral development

The formation of collateral arteries in patients suffering from occlusive atherosclerotic vascular diseases has been frequently reported. The growth of these collateral arteries has been termed 'arteriogenesis'. Clinical observations and investigations using various animal models support the hypothesis that the mechanism of arteriogenesis is based on the remodelling of pre-existing collateral anastomoses. This process seems to be mainly triggered by fluid shear stress which is induced by the altered blood flow conditions after an arterial occlusion. Early arteriogenesis involves the activation of collateral endothelial cells, the attraction of leukocytes to the collateral vascular wall and subsequently their invasion into the perivascular space of the collateral vessel. In a second phase, proliferation of vascular cells is initiated by growth factors mainly released from accumulated leukocytes. Furthermore, tissue degradation and changes in the extracellular matrix are observed. Unravelling the mechanisms of arteriogenesis is crucial to the development of successful therapeutic approaches for the treatment of patients with ischemic vascular diseases.     

Arteriolar remodeling following ischemic injury extends from capillary to large arteriole in the microcirculation

Objective: Skeletal muscle vasculature undergoes arteriogenesis to restore tissue perfusion and function following loss of blood flow. This process has been shown to occur in large vessels following ischemia, although recent studies suggest this may occur in the microcirculation as well. We tested the hypothesis that ischemia induces microvascular remodeling in the skeletal muscle microcirculation on the scale of capillary to sub‐35 μm diameter arterioles. Methods: Ligations of a feeding arteriole to the caudal‐half of the spinotrapezius muscle were performed on C57BL/6 mice. At 5 days, microvascular remodeling responses were quantified using intravital and whole‐mount confocal microscopy. Immunohistochemistry was performed to visualize vessels, incorporated leukocytes, and regions of hypoxia. Results: Ischemic tissue underwent localized microvascular remodeling characteristic of arteriogenesis, including pronounced vessel tortuosity. In patent microvessels (diameters 15–35 μm), we observed increases in vascular density (38%), branching (90%) and collateral development (36.5%). The formation of new arterioles (diameters 6–35 μm) increased by 24.3%, while chronic hypoxia was absent from all tissues. Conclusions: Ischemic injury induces arteriogenesis in skeletal muscle microcirculation. Furthermore, this surgical model enables en face analysis of microcirculatory adaptations with single‐cell resolution and can provide investigators with morphometric data on a microscale that is difficult to achieve using other models.     

Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease

After birth two forms of vessel growth can be observed; angiogenesis and arteriogenesis. Angiogenesis refers to the formation of capillary networks. Arteriogenesis refers to the growth of preexistent collateral arterioles leading to formation of large conductance arteries that are well capable to compensate for the loss of function of occluded arteries. The process of arteriogenesis is initiated when shear stresses increase in the preexistent collateral pathways upon narrowing of a main artery. The increased shear stress leads to an upregulation of cell adhesion molecules for circulating monocytes, which accumulate subsequently around the proliferating arteries and provide the several required cytokines and growth factors. Several strategies are currently tested for their potential to stimulate the process of arteriogenesis. These strategies focus either at shear stress, at direct stimulation of endothelial and smooth muscle cell growth or at the monocytic pathway and promising results were obtained from experimental studies. However, some important questions remain to be answered before arteriogenesis can be brought from bench to bedside.     

Arteriogenesis: the development and growth of collateral arteries

In patients with atherosclerotic vascular diseases, collateral vessels bypassing major arterial obstructions have frequently been observed. This may explain why some patients remain without symptoms or signs of ischemia. The term "arteriogenesis" was introduced to differentiate the formation of collateral arteries from angiogenesis, which mainly occurs in the ischemic, collateral flow-dependent tissue. Many observations in various animal models and humans support that the remodeling of preexisting collateral vessels is the mechanism of collateral artery formation. This remodeling process seems to be mainly flow-mediated. It involves endothelial cell activation, basal membrane degradation, leukocyte invasion, proliferation of vascular cells, neointima formation (in most species studied), and changes of the extracellular matrix. The contribution of ischemia to arteriogenesis is still unclear, but arteriogenesis clearly can occur in the absence of any significant ischemia. It is questionable, whether collateral arteries also form de novo in ischemic vascular diseases. A better understanding of the mechanisms of arteriogenesis will be important for the design of more effective strategies for the treatment of patients with ischemic vascular diseases.     

Coronary flow velocity reserve measurement in three major coronary arteries using transthoracic Doppler echocardiography

BACKGROUND: Measurement of the coronary flow velocity reserve (CFVR) by transthoracic Doppler echocardiography (TTDE) has been reported to be useful for the noninvasive assessment of significant coronary artery stenosis or myocardial ischemia. The purpose of this study was to evaluate the value of this method in three major coronary arteries for detecting myocardial ischemia in the clinical setting. METHODS: We studied 89 consecutive patients who were referred to our outpatient clinic because of chest pain. We measured CFVR using TTDE in three major coronary arteries. We defined CFVR<2.0 in at least one vessel as being positive for myocardial ischemia. The accuracy of CFVR measurements for detecting myocardial ischemia was determined in comparison with exercise thallium-201 (Tl-201) single photon emission computed tomography (SPECT) as a reference standard. RESULTS: CFVR in at least one vessel was successfully measured in 87 of 89 patients (98%). The sensitivity and specificity of CFVR<2.0 in at least one coronary vessel, in any of the coronary territories, was 86% and 89%, respectively. In terms of assessing myocardial ischemia in each coronary artery territory, the agreement between CFVR<2.0 and Tl-201 SPECT for the left anterior descending coronary artery, the posterior descending coronary artery, and the left circumflex coronary artery territories was 95%, 81%, and 73%, respectively. CONCLUSION: Noninvasive CFVR measurement by TTDE may be useful for detecting myocardial ischemia, as well as for identifying ischemic territories in the clinical setting.     

Bone marrow-derived cells do not incorporate into the adult growing vasculature

Bone marrow-Derived cells have been proposed to form new vessels or at least incorporate into growing vessels in adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow-Derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) and tumor growth. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells from littermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation, the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelial growth factor or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Seven and 21 days after surgery, proximal hindlimb muscles with growing collateral arteries and ischemic gastrocnemius muscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFP signals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocal microscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells. Nevertheless, we observed accumulations of GFP-positive cells around growing collateral arteries (3-fold increase versus nonoccluded side, P<0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts, pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines. Our findings suggest that in the adult organism, bone marrow-Derived cells do not promote vascular growth by incorporating into vessel walls but may function as supporting cells.     

Influence of inflammatory cytokines on arteriogenesis

Blood vessel growth after birth is limited to two major processes. Angiogenesis is the growth of new capillaries by sprouting or intussusception. The major stimulus for angiogenesis is ischemia. In contrast, arteriogenesis describes the remodeling and growth of collateral arteries from a preexisting arteriolar network. Arteriogenesis is induced after the occlusion of a major artery which induces hemodynamic and mechanical effects on the collateral vessel wall which occur with increasing blood flow velocity due to the low pressure at the reentrant site of the collateral vessel. A variety of different cytokines that act by stimulating endothelial and smooth muscle cell proliferation and migration or recruitment and activation of monocytes have been identified to stimulate angiogenesis and/or arteriogenesis (i.e., MCP-1, FGF-2, TGF-beta, VEGF, and GM-CSF). Several clinical trials have been published in that field to suggest the feasibility and safety of treatment with such cytokines or their genes. However, the results indicate that further studies are needed before proangiogenic and proarteriogenic therapies are ready for clinical application.     

Generalized coronary arterio-systemic (left ventricular) fistula. Case report and review of literature

A coronary artery-to-left ventricular fistula is a rare finding; to the best of our knowledge, a total of only 35 cases have been reported. Only 5 cases of a generalized arterio-systemic fistula with three vessel involvement have been reported in the literature. We describe another case involving all major coronary arteries. A review of the literature is presented and the data of the reported cases are analyzed. A 55 year old woman was examined because of recurrent chest pain which had persisted for 2 years. On physical examination, the only abnormal finding was a fourth heart sound. Exertional chest pain, a positive exercise stress test, and the results of a lactate extraction study suggested severe myocardial ischemia. Thallium myocardial scintigraphy showed no evidence of a perfusion defect. Cardiac catheterization revealed an irregular left ventricular endocardial pattern (Thebesian veins). Selective coronary angiography showed communicating fistulae of all three major coronary arteries with the left ventricular cavity. We assume that this vascular anomaly causes a coronary steal phenomenon and subsequent myocardial ischemia.     

Coordinated activation of VEGFR-1 and VEGFR-2 is a potent arteriogenic stimulus leading to enhancement of regional perfusion

OBJECTIVE: The process of arteriogenesis is driven by various growth factors including vascular endothelial growth factor (VEGF)-A, which mediates its activity through VEGFR-2 (Flk-1/KDR) on endothelial cells and through VEGFR-1 (Flt-1) on endothelial cells and monocytes. The purpose of this study was to identify which of the VEGF receptors are involved in arteriogenesis in vivo. METHODS: Collateral vessel growth was induced by femoral artery ligation in a mouse model of hindlimb ischemia. Following ligation, Balb/c mice were treated with different growth factors (VEGF-A, VEGF-E, PlGF-2, VEGF-E plus PlGF-2 or VEGF-A plus PlGF-2, activating either VEGFR-1, VEGFR-2, or both). After 1 week of treatment, hindlimb perfusion was assessed by perfusion scintigraphy using Tc-99m-MIBI. RESULTS: The strongest improvement of regional perfusion was achieved by simultaneous activation of VEGFR-1 and VEGFR-2, using either VEGF-A or VEGF-A plus PlGF-2, with elevation of relative perfusion in the ischemic limbs from 0.61 to 0.83. The partial restoration in perfusion was associated with morphological changes typical for arteriogenesis. Moreover, specific inhibition of both VEGF-receptors using ZK 202650 resulted in a significant inhibition of arteriogenesis, indicating an active role of the VEGF system in compensatory arteriogenesis. CONCLUSION: The coordinated activation of both VEGFR-1 and VEGFR-2 represents a more potent arteriogenic stimulus compared to the isolated activation of either one of these two receptors. These data imply that the activation of both monocytes and endothelial cells is necessary to obtain a maximal VEGF-induced activation of arteriogenesis.     

Notch ligand Delta-like 1 is essential for postnatal arteriogenesis

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.     

Basic Concepts of (Myocardial) Angiogenesis: Role of Vascular Endothelial Growth Factor and Angiopoietin

Blood vessels are essential for the supply of oxygen and nutrients to the heart. An imbalance between oxygen demand and supply (ischemia), as occurs when coronary arteries become obstructed by atherosclerotic plaques, triggers a response to improve myocardial perfusion by the formation of new capillaries (angiogenesis) and by the enlargement of preexisting collateral vessels (arteriogenesis). Recently, novel insights have been obtained in the molecular mechanisms of angiogenesis and in its control by hypoxia. This has lead to the design of strategies to improve myocardial perfusion. However, rational design of therapeutic angiogenesis mandates a better understanding of the molecular basis of angiogenesis. This review discusses the role of two prime classes of angiogenic molecules, namely of vascular endothelial growth factor (VEGF) and angiopoietin (Ang), and addresses novel insights in the regulation of angiogenesis by hypoxia. In addition, a novel mouse model of ischemic cardiomyopathy with signs of hibernation is presented. Possible implications for therapeutic myocardial angiogenesis are discussed.     

Histopathological study of vascular changes after intra‐arterial and intravenous injection of N‐butyl‐2‐cyanoacrylate

OBJECTIVE: To assess the histopathological vascular changes after injection of N‐butyl‐2‐cyanoacrylate into the vessels of adult rabbits. METHODS: The animals used were 42 pure‐blood New Zealand white rabbits weighing 2–3 kg. 0.2 mL cyanoacrylate with lipiodol was injected into the external jugular vein and femoral artery of each rabbit. Tissue specimens were obtained for histopathological study at 3 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months and 3 months after injection. RESULTS: The vessels were obliterated immediately after the injection. The main manifestation of histopathology at 3 days to 2 weeks was an acute inflammatory reaction; this progressed to subacute vasculitis at 3 weeks and a chronic granulomatous foreign body reaction developed at 4 weeks. The glue mass essentially disappeared in 2–3 months, replaced by fibrotic tissue with partial vascular recanalization. At 3 weeks after injection, the elastic fibrils of the arterial wall proliferated distinctly, resulting in narrowing of the lumen with subsequent obliteration, whereas the venous wall still showed inflammation and necrosis without hyperplasia of elastic fibrils. Extrusion of glue was observed over 1–3 months in both arteries and veins and was obvious in the latter. CONCLUSIONS: The histopathological changes after injection of N‐butyl‐2‐cyanoacrylate were similar in the arteries and the veins with the exception of hyperplasia of elastic fibrils in the arterial wall and inflammation and necrosis in the venous wall at 2–3 weeks. Glue extrusion was seen in both arteries and veins.     

Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy.

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients (P<0.05), accompanied by a marked reduction in HGF mRNA. On the other hand, a novel therapeutic strategy for ischemic diseases that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently been proposed. Thus in view of the decreased endogenous vascular HGF, rhHGF (500 micrograms/animal) was intra-arterially administered through the internal iliac artery of rabbits in which the femoral artery was excised to induce unilateral hind limb ischemia, to evaluate the angiogenic activity of HGF, which could potentially have a beneficial effect in hypoxia. Administration of rhHGF twice on days 10 and 12 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemic model as assessed by angiography (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. In addition, we examined the feasibility of intravenous administration of rhHGF in a moderate ischemia model. Importantly, intravenous administration of rhHGF also resulted in a significant increase in angiographic score as compared with vehicle (P<0.01). Overall, a decrease in vascular HGF might be related to the pathogenesis of peripheral arterial disease. In the presence of decreased endogenous HGF, administration of rhHGF induced therapeutic angiogenesis in the rabbit ischemic hind limb model, as potential cytokine supplement therapy for peripheral arterial disease.     

Vascular endothelial growth factor-A specifies formation of native collaterals and regulates collateral growth in ischemia

The density of native (preexisting) collaterals and their capacity to enlarge into large conduit arteries in ischemia (arteriogenesis) are major determinants of the severity of tissue injury in occlusive disease. Mechanisms directing arteriogenesis remain unclear. Moreover, nothing is known about how native collaterals form in healthy tissue. Evidence suggests vascular endothelial growth factor (VEGF), which is important in embryonic vascular patterning and ischemic angiogenesis, may contribute to native collateral formation and arteriogenesis. Therefore, we examined mice heterozygous for VEGF receptor-1 (VEGFR-1(+/-)), VEGF receptor-2 (VEGFR-2(+/-)), and overexpressing (VEGF(hi/+)) and underexpressing VEGF-A (VEGF(lo/+)). Recovery from hindlimb ischemia was followed for 21 days after femoral artery ligation. All statements below are P<0.05. Compared to wild-type mice, VEGFR-2(+/-) showed similar: ischemic scores, recovery of hindlimb perfusion, pericollateral leukocytes, collateral enlargement, and angiogenesis. In contrast, VEGFR-1(+/-) showed impaired: perfusion recovery, pericollateral leukocytes, collateral enlargement, worse ischemic scores, and comparable angiogenesis. Compared to wild-type mice, VEGF(lo/+) had 2-fold lower perfusion immediately after ligation (suggesting fewer native collaterals which was confirmed by angiography) and blunted recovery of perfusion. VEGF(hi/+) mice had 3-fold greater perfusion immediately after ligation, more native collaterals, and improved recovery of perfusion. These differences were confirmed in the cerebral pial cortical circulation where, compared to VEGF(hi/+) mice, VEGF(lo/+) formed fewer collaterals during the perinatal period when adult density was established, and had 2-fold larger infarctions after middle cerebral artery ligation. Our findings indicate VEGF and VEGFR-1 are determinants of arteriogenesis. Moreover, we describe the first signaling molecule, VEGF-A, that specifies formation of native collaterals in healthy tissues.