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.     

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.     

Bone marrow transplantation abolishes inhibition of arteriogenesis in placenta growth factor (PlGF) -/- mice

We studied the influence of placenta growth factor (PlGF) on arteriogenesis and tested the role of bone marrow (BM)-derived cells on PlGF-depleted vascular growth. METHODS: Right femoral artery was occluded in wild type (+/+), PlGF k.o. (-/-) and (-/-) mice receiving BM from (+/+) mice (-/- BM). Blood supply in paws was assessed by laser-Doppler imaging (LDI) measurements before, immediately after, as well as 3, 7, 14, 21 and 28 days after surgery. Seven and 28 days after occlusion, hindlimbs were perfusion fixed and filled with contrast medium. Angiograms were evaluated, collateral arteries were analysed histologically and morphometrically. RESULTS: Seven days after occlusion the blood flow and collateral growth in (-/-) were delayed in comparison to the (+/+) group. BM transplantation from +/+ donors prevented this delay. After 3 weeks the (-/-) mice reached values similar to the +/+ control. CONCLUSION: PlGF is an important promoter of arteriogenesis. BM transplantation abolishes inhibition of arteriogenesis in PlGF -/- mice.     

Arteriogenesis requires toll-like receptor 2 and 4 expression in bone-marrow derived cells

Adaptive collateral growth (arteriogenesis) is an important protective mechanism against ischemic injury in patients with cardiovascular disease. Arteriogenesis involves enlargement of pre-existent arterial anastomoses and shares many mechanistic similarities with inflammatory processes. Although infusion of the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) has shown to result in a significant stimulation of arteriogenesis and both Toll-like receptor 2 and 4 are involved in structural arterial adaptations, the requirement for TLRs in arteriogenesis has not yet been established. We therefore subjected TLR 2 null and TLR 4 defective mice to unilateral femoral artery occlusion. At 7 days, both TLR 2 null and TLR 4 defective mice showed a significant reduction (~ 35%) of collateral perfusion. Histological staining showed that TLR 2 and TLR 4 expression during arteriogenesis is mostly restricted to infiltrating leukocytes. To distinguish between the functional importance of vascular and leukocytic TLRs in arteriogenesis, cross-over bone marrow transplantation was performed 6 weeks before femoral artery occlusion. Perfusion measurements showed that transplantation of wild-type bone marrow into TLR 2 null and TLR 4 defective mice rescued the impaired arteriogenesis, while injection of TLR 2 null and TLR 4 defective bone marrow into wild-type mice significantly reduced collateral vessel growth to levels of TLR null/defective mice. RT-PCR analysis demonstrated a significant upregulation of two endogenous TLR ligands EDA and Hsp60 (91.7 fold and 1.9 fold respectively) in regions of collateral vessel formation. This study illustrates the involvement of TLR 2 and TLR 4 in adaptive collateral artery growth and shows the importance of TLR 2 and 4 expression by bone-marrow derived cells for this process.     

Tissue resident cells play a dominant role in arteriogenesis and concomitant macrophage accumulation

Collateral growth is characterized by macrophage accumulation, suggesting an important role of circulating cells. To study origin and function of macrophages during arteriogenesis, we related the extent of macrophage accumulation to vascular proliferation and investigated the fate of fluorescently (CMFDA) labeled blood cells that were injected at the time of femoral artery occlusion. The effect of bone marrow depletion via cyclophosphamide before femoral artery occlusion on collateral proliferation and macrophage accumulation was studied, and we looked for the presence of bone marrow-derived stem cells in the vicinity of growing collateral vessels. Finally, we investigated the arteriogenic effect of macrophage activation via MCP-1 in bone marrow-depleted animals. Maximal macrophage accumulation occurred during the first 3 days after femoral artery occlusion and paralleled the extent of vascular proliferation. Fluorescently labeled leukocytes homed to spleen and wound but they were absent in proliferating collateral arteries during maximal macrophage accumulation. Depletion of circulating cells did neither affect macrophage accumulation nor collateral growth. Staining of monocyte-depleted animals for BrdUrd and ED2, alphaSMA, or VE-Cadherin demonstrated local proliferation of macrophages and vascular cells, whereas C-Kit, SSEA1, or Thy1-positive bone marrow-derived stem cells were not detectable. Enhancement of macrophage accumulation via MCP-1 was independent of circulating monocytes and promoted arteriogenesis in the absence of direct effects on vascular cells. We propose that the initial phase of vascular growth is characterized by local proliferation of tissue resident precursors rather than by migration of blood born cells. The full text of this article is available online at http://circres.ahajournals.org.     

Preconditioning of arteriogenesis

OBJECTIVE: Last decade, the shear stress caused by increased blood flow in collateral circulation after occlusion of main artery was recognized as a trigger of vascular remodeling and collateral growth. The goal of this study was to differentiate whether the on-going increased blood flow is necessary for the vascular remodeling or the remodeling, once in progress, develops independently of flow. METHODS: Femoral artery occlusion was performed in C57B1/6 mice. After 1-3 days, the ligature was removed and normal limb perfusion was re-established, monitored by laser Doppler Imaging (LDI). Two weeks after the first occlusion, both femoral arteries were re-occluded to compare collateral growth on the "naive" and "preconditioned" sides. After perfusion fixation, ultrastructural studies and morphometry of the collateral vessels were performed. RESULTS: Blood flow fell after occlusion to about 15% of control levels and recovered to about 40% by day 3. The reperfusion normalized sustainable blood flow. After the second occlusion, blood flow on both sides fell again to about 15% but recovered to 70% in the "preconditioned" compared to 40% in the "naive" side during the following 3 days. 5-Bromo-2'-desoxy-uridine (BrdU) administered during reperfusion was detected mainly in the neointima that, in many cases, had markedly narrowed the lumen. Two to three days after re-occlusion, a statistically significant lumen enlargement on the "preconditioned" side was observed, while neointima disappeared. CONCLUSION: Cellular proliferation and remodeling of collateral arteries were induced by short period of increased blood flow (occlusion of the femoral artery) but realized mostly during the low blood flow (reperfusion of the femoral artery). The neointima developing as a result of this remodeling can be recruited as a functional part of the arterial wall if the collateral perfusion increases as a result of repetitive occlusion of the femoral artery. The "medialization" of the neointima might cause the observed quicker gain of collateral lumen diameter and conductance, saving distal muscle tissue from the ischemia.     

The range of adaptation by collateral vessels after femoral artery occlusion

Natural adaptation to femoral artery occlusion in animals by collateral artery growth restores only approximately 35% of adenosine-recruitable maximal conductance (C(max)) probably because initially elevated fluid shear stress (FSS) quickly normalizes. We tested the hypothesis whether this deficit can be mended by artificially increasing FSS or whether anatomical restraints prevent complete restitution. We chronically increased FSS by draining the collateral flow directly into the venous system by a side-to-side anastomosis between the distal stump of the occluded femoral artery and the accompanying vein. After reclosure of the shunt collateral flow was measured at maximal vasodilatation. C(max) reached 100% already at day 7 and had, after 4 weeks, surpassed (2-fold) the C(max) of the normal vasculature before occlusion. Expression profiling showed upregulation of members of the Rho-pathway (RhoA, cofilin, focal adhesion kinase, vimentin) and the Rho-antagonist Fasudil markedly inhibited arteriogenesis. The activities of Ras and ERK-1,-2 were markedly increased in collateral vessels of the shunt experiment, and infusions of L-NAME and L-NNA strongly inhibited MAPK activity as well as shunt-induced arteriogenesis. Infusions of the peroxinitrite donor Sin-1 inhibited arteriogenesis. The radical scavengers urate, ebselen, SOD, and catalase had no effect. We conclude that increased FSS can overcome the anatomical restrictions of collateral arteries and is potentially able to completely restore maximal collateral conductance. Increased FSS activates the Ras-ERK-, the Rho-, and the NO- (but not the Akt-) pathway enabling collateral artery growth.     

Arteriogenesis - is this terminology necessary?

The role of the collateral circulation has been controversially discussed over many decades. With the availability of purified growth factors such as the fibroblast growth factors several studies provided data, that the growth of collateral arteries, termed arteriogenesis, is not limited to its natural timecourse. When applied in experimental models FGF in particular led to a significant increase in collateral conductance upon arterial occlusion. Thus the proliferation of preexisting bypassing arterioles could be enhanced therapeutically. The purpose of this review is to discuss the physiological importance of different kinds of vascular growth (e.g. vasculogenesis, angiogenesis, arteriogenesis) to enhance blood flow to ischemic limbs or the heart. It is outlined that large conductance arteries rather than capillary networks are needed to compensate for perfusion deficits due to atherosclerotic stenosis or occlusion.     

Enhanced hindlimb collateralization induced by acidic fibroblast growth factor is dependent upon femoral artery extraction

Recent investigations have established the feasibility of using exogenously delivered angiogenic growth factors to increase collateral artery development in animal models of myocardial and hindlimb ischemia. OBJECTIVE: Our aim was to evaluate the ability of a stabilized form of acidic fibroblast growth factor (aFGF-S117) to stimulate collateralization and arteriogenesis in the rabbit hindlimb following the surgical induction of ischemia by femoral artery extraction. A secondary objective was to examine angiogenic and arteriogenic effects of aFGF-S117 in the absence of a peripheral blood flow deficit. METHODS AND RESULTS: Five days after femoral artery removal, aFGF-S117 (1, 3, or 30 microg/kg) was intramuscularly delivered into the hindlimb, three times per week for 2 consecutive weeks. End-point measurements performed on day 20 found that hindlimb reserve blood flow was significantly improved in rabbits that received 3 or 30 microg/kg of aFGF-S117, with no difference in efficacy between these two doses. These hemodynamic results were supported by angiographic evidence showing enhanced density of collateral vessels in the medial thigh region and histological findings of increased capillary density within the gastrocnemius muscle from rabbits treated with aFGF-S117. When an efficacious dose of 3 microg/kg of aFGF-S117 was administered to sham-operated rabbits with intact femoral arteries, there was no change in any of the blood flow, angiographic or histological parameters measured. CONCLUSIONS: These findings demonstrate that a stabilized form of aFGF stimulated the development of functional collateral arteries in the rabbit hindlimb, an effect which was dependent upon removal of the femoral artery. These results suggest that aFGF-S117 may have therapeutic potential for the treatment of arterial occlusive disorders.     

Combined treatment of sustained-release basic fibroblast growth factor and sarpogrelate enhances collateral blood flow effectively in rabbit hindlimb ischemia.

BACKGROUND: The effectiveness of sustained-release basic fibroblast growth factor (bFGF) in potentiating arteriogenesis and angiogenesis was evaluated, as well as determining whether chronic oral administration of sarpogrelate, a serotonin blocker, would further increase collateral blood flow in the rabbit hindlimb following surgical induction of ischemia by femoral artery extraction. METHODS AND RESULTS: Two weeks after femoral artery removal, the rabbits were assigned to 1 of 4 experimental groups and treated for 4 weeks: group A, no treatment; group B, supplemented with diet containing sarpogrelate; group C, single intramuscular injection of sustained-release form of bFGF microspheres; group D: combined treatment with sustained-release bFGF and sarpogrelate. Endpoint measurements performed at 6 weeks found that the ischemic hindlimb blood flow was significantly improved in the rabbits that received sustained-release bFGF, with a further significant improvement in those with the additional administration of sarpogrelate. Angiographic assessment revealed augmented density of collateral vessels in the medial thigh region in the rabbits given the combined treatment. CONCLUSIONS: The findings demonstrate that sustained-release bFGF stimulated the development of collateral vessels, and additional administration of sarpogrelate produced a further improvement in hindlimb blood flow in the rabbit hindlimb ischemia model.     

Factors regulating arteriogenesis

Growth of collateral vessels is potentially able to preserve structure and a variable degree of function in subtended tissues in the presence of arterial occlusions. The process of transformation of a small arteriole into much larger conductance artery is called arteriogenesis. Small arterioles that interconnect side branches proximal from the arterial occlusion with distal ones experience increased fluid shear stress because of the increased blood flow velocity attributable to the pressure gradient along the bridging collaterals. This activates the endothelium and leads to monocyte adhesion and infiltration with the subsequent production of growth factors and proteases. Preexistent arterioles are essential. Their presence is genetically determined. Arteriogenesis is not organ- or species-specific; coronary or peripheral collateral vessels develop following the same design principles in mice, rats, rabbits, or dogs. In contrast to angiogenesis, arteriogenesis is not dependent on the presence of hypoxia/ischemia.     

Time course of arteriogenesis following femoral artery occlusion in the rabbit

OBJECTIVE: We examined the time course of arteriogenesis (collateral artery growth) after femoral artery ligation and the effect of monocyte chemoattractant protein-1 (MCP-1). METHODS: New Zealand White rabbits received MCP-1 or phosphate buffered saline (PBS) for a 1-week period, either directly or 3 weeks after femoral artery ligation (non-ischemic model). A control group was studied with intact femoral arteries and another 1 min after acute femoral artery ligation. RESULTS: Collateral conductance index significantly increased when MCP-1 treatment started directly after femoral artery ligation (acute occlusion: 0.94+/-0.19; without occlusion: 168.56+/-15.99; PBS: 4.10+/-0.48; MCP-1: 33.96+/-1.76 ml/min/100 mmHg). However, delayed onset of treatment 3 weeks after ligation and final study of conductance at 4 weeks showed no significant difference against a 4-week control (PBS: 79.08+/-7.24; MCP-1: 90.03+/-8.73 ml/min/100 mmHg). In these groups increased conductance indices were accompanied by a decrease in the number of visible collateral vessels (from 18 to 36 identifiable vessels at day 7 to about four at 21 days). CONCLUSION: We conclude that the chemokine MCP-1 markedly accelerated collateral artery growth but did not alter its final extent above that reached spontaneously as a function of time. We show thus for the first time that a narrow time window exists for the responsiveness to the arteriogenic actions of MCP-1, a feature that MCP-1 may share with other growth factors. We show furthermore that the spontaneous adaptation by arteriogenesis stops when only about 50% of the vasodilatory reserve of the arterial bed before occlusion are reached. The superiority of few large arterial collaterals in their ability to conduct large amounts of blood flow per unit of pressure as compared to the angiogenic response where large numbers of small vessels are produced with minimal ability to allow mass transport of bulk flow is stressed.     

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.     

Arteriogenesis is associated with an induction of the cardiac ankyrin repeat protein (carp)

OBJECTIVE: Collateral artery growth (arteriogenesis) can be induced in rabbit and mice by occlusion of the femoral artery. We aimed to identify genes that are differentially expressed during arteriogenesis. METHODS: 24 h after femoral ligation or sham operation collateral arteries were isolated from New Zealand white rabbits, mRNAs were extracted and amplified using the SMART technique. cDNAs were subjected to suppression subtractive hybridization. The differential expression was confirmed by Northern blot, Real time PCR and Western blot. Additionally, the gene expression was modulated in vivo by application of cytokines via osmotic minipumps. RESULTS: We found the cardiac ankyrin repeat protein (carp) mRNA to be upregulated at 24 h and already at 6 h and 12 h after surgery as shown by Northern blot hybridization and real time PCR. The carp mRNA was also increased in our mouse model of arteriogenesis. Western blot results on nuclear extracts of rabbit collaterals 24 h after surgery indicated that carp, which we showed to be expressed in endothelial cells and smooth muscle cells of collateral arteries by immunohistochemistry, was also upregulated on the protein level. We infused MCP-1, TGF-beta1 or doxorubicin for 24 h in rabbits and found that only TGF-beta1 led to an additional increase of carp mRNA. Overexpression of carp in cos-1 cells resulted in a 3.7-fold increase of the immediate early gene egr-1. CONCLUSIONS: Our results implicate that carp is associated with the initiation and regulation of arteriogenesis.     

Endothelial nitric oxide synthase is critical for ischemic remodeling, mural cell recruitment, and blood flow reserve

The genetic loss of endothelial-derived nitric oxide synthase (eNOS) in mice impairs vascular endothelial growth factor (VEGF) and ischemia-initiated blood flow recovery resulting in critical limb ischemia. This result may occur through impaired arteriogenesis, angiogenesis, or mobilization of stem and progenitor cells. Here, we show that after ischemic challenge, eNOS knockout mice [eNOS (-/-)] have defects in arteriogenesis and functional blood flow reserve after muscle stimulation and pericyte recruitment, but no impairment in endothelial progenitor cell recruitment. More importantly, the defects in blood flow recovery, clinical manifestations of ischemia, ischemic reserve capacity, and pericyte recruitment into the growing neovasculature can be rescued by local intramuscular delivery of an adenovirus encoding a constitutively active allele of eNOS, eNOS S1179D, but not a control virus. Collectively, our data suggest that endogenous eNOS-derived NO exerts direct effects in preserving blood flow, thereby promoting arteriogenesis, angiogenesis, and mural cell recruitment to immature angiogenic sprouts.     

Differential effects of MCP-1 and leptin on collateral flow and arteriogenesis

OBJECTIVE: Strategies to therapeutically stimulate collateral artery growth in experimental models have been studied intensively in the last decades. However, the experimental methods to detect collateral artery growth are discussed controversially and vary significantly. We compared different methods in a model of arteriogenesis in the rabbit hind limb and determined the effects on collateral flow of a known pro-arteriogenic factor, monocyte chemoattractant protein-1 (MCP-1), and a cytokine not previously evaluated for its arteriogenic efficacy, the adipocytokine leptin. METHODS AND RESULTS: Forty-two New Zealand White rabbits received either MCP-1, leptin or PBS after ligation of the right femoral artery. The pro-arteriogenic effect of MCP-1 was confirmed by flow measurements during reactive hyperemia, as demonstrated by increased flow ratio (PBS 0.56+/-0.07 vs. MCP-1 0.77+/-0.06, no unit, p<0.0001), ankle-brachial index and microsphere-based conductance measurements (PBS 50.8+/-2.1 vs. MCP-1 225.8+/-8.8 ml/min/100 mm Hg, p<0.001). Biological activity of leptin on rabbit monocytes was shown by a dose dependent increase in Mac-1 expression. In-vivo administration of leptin also led to an increase in hyperemic flow and flow ratio (leptin 0.69+/-0.03, p<0.05 vs. PBS), but not to an increase in collateral conductance (leptin 54.7+/-4.1 ml/min/100 mm Hg, p=ns vs. PBS) or proliferation of vascular smooth muscle cells (Ki-67 staining: PBS 24.7+/-3.9%, leptin 22.7%+/-0.8% (p=ns), MCP-1 32.0+/-1.9% (p<0.01)). Ki-67 mRNA measured by real-time polymerase chain reaction increased (8.8+/-3.1-fold, p<0.01) during natural arteriogenesis, and was further enhanced (25.5+/-8.1-fold, p<0.005) after stimulation with MCP-1. CONCLUSION: MCP-1 and leptin increase collateral flow in the rabbit hind limb model. In contrast to MCP-1, leptin does not enhance direct markers of vascular proliferation such as collateral conductance under maximal vasodilation and proliferation indices. The observed increase in hyperemic collateral flow thus most probably can be attributed to the well-documented vasodilatory effects of leptin. These data stress the necessity of the use of proliferation markers and microsphere-based conductance measurements under maximal vasodilation in order to separate effects of substances on vascular proliferation from effects on vasodilation.     

GM-CSF: a strong arteriogenic factor acting by amplification of monocyte function.

We investigated the role of the colony stimulating factor for monocytes (GM-CSF) to test the hypothesis whether prolongation of the monocyte's life cycle will support arteriogenesis (rapid growth of preexisting collateral arteries). This appeared logical in view of our discovery that circulating monocytes play an important part in the positive remodeling of small preexisting arterioles into arteries to compensate for arterial occlusions (arteriogenesis) and especially following our findings that MCP-1 markedly increases the speed of arteriogenesis. The continuous infusion of GM-CSF for 7 days into the proximal stump of the acutely occluded femoral artery of rabbits by osmotic minipump produced indeed a marked arteriogenic response as demonstrated by an increase (2-fold) in number and size of collateral arteries on postmortem angiograms and by the increase of maximal blood flow during vasodilation measured in vivo by blood pump perfusion of the hindquarter (5-fold). When GM-CSF and MCP-1 were simultaneously infused the effects on arteriogenesis were additive on angiograms as well as on conductance. GM-CSF was also able to widen the time window of MCP-1 activity: MCP-1 treatment alone was ineffective when given after the third week following occlusion. When administered together with GM-CSF about 80% of normal maximal conductance of the artery that was replaced by collaterals were achieved, a result that was not reached before by any other experimental treatment. Experiments with cells isolated from treated animals showed that monocyte apoptosis was markedly reduced. In addition we hypothesize that GM-CSF may aid in releasing pluripotent monocyte (stem-) cells from the bone marrow into the circulation. In contrast to MCP-1, GM-CSF showed no activity on monocyte transmigration through- and also no influence on monocyte adhesion to cultured endothelial cells. In conclusion we have discovered a new function of the hemopoietic stem cell factor GM-CSF, which is also a powerful arteriogenic peptide that acts via prolongation of the life cycle of monocytes/macrophages.     

Statins augment collateral growth in response to ischemia but they do not promote cancer and atherosclerosis

3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, are widely prescribed to lower cholesterol. Recent reports suggest that statins may promote angiogenesis in ischemic tissues. It remains to be elucidated whether statins potentially enhance unfavorable angiogenesis associated with tumor and atherosclerosis. Here, we induced hind limb ischemia in wild-type mice by resecting the right femoral artery and subsequently inoculated cancer cells in the same animal. Cerivastatin enhanced blood flow recovery in the ischemic hind limb as determined by laser Doppler imaging, whereas tumor growth was significantly retarded. Cerivastatin did not affect capillary density in tumors. Cerivastatin, pitavastatin, and fluvastatin inhibited atherosclerotic lesion progression in apolipoprotein E-deficient mice, whereas they augmented blood flow recovery and capillary formation in ischemic hind limb. Low-dose statins were more effective than high-dose statins in both augmentation of collateral flow recovery and inhibition of atherosclerosis. These results suggest that statins may not promote the development of cancer and atherosclerosis at the doses that augment collateral flow growth in ischemic tissues.     

Biological bypass in cardiovascular surgery

Protein and gene therapy offer a tremendous opportunity to improve the care of critically ill patients with ischemic heart and peripheral artery occlusion disease. With the availability of purified growth factors such as vascular endothelial and fibroblast growth factors (FGF), several experimental and clinical studies provided data, that the growth of capillaries (angiogenesis) and of collateral arteries (arteriogenesis) is not limited to its natural time course. When applied in experimental models and in conjunction with coronary artery bypass operations, FGF in particular, led to a significant increase in endogenous rerouting of blood flow by collateral vessels inside the tissue itself. Thus, the proliferation of preexisting bypassing arterioles could be enhanced therapeutically (biological bypass). The purpose of this review is to discuss the physiological importance of different kinds of cytokines which are able to induce angio- and arteriogenesis in ischemic limbs or the heart. It is outlined that a combination of a sufficient amount of large arterioles and a capillary network are needed to compensate perfusion deficits. Each patient, who has an ischemic area and cannot be conventionally revascularized, is a potential candidate for the biological bypass.