Abstract-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 marrowderived 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, PϽ0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts, pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines.

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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.

Objective-To assess the importance of genetic background for collateral artery development. Methods and Results-C57BL/6, BALB/c and 129S2/Sv mice were studied after femoral artery ligation by laser Doppler imaging, visible light oximetry, time-of-flight-magnetic resonance imaging, and treadmill testing; C57BL/6 and BALB/c also underwent electron paramagnetic resonance (EPR) oximetry, x-ray angiography, and histology. C57BL/6 had the least initial distal ischemia and most complete recovery. BALB/c had the most severe initial ischemia and poorest recovery. BALB/c had some vasodilatory reserve in their ligated limbs not seen in the other strains at 3 weeks. By in vivo TOF-magnetic resonance angiography, C57BL/6 had larger preexistent and developed collaterals. By x-ray angiography, C57BL/6 had a higher collateral-dependent filling score and number of visible collaterals immediately after femoral ligation and a higher number of visible collaterals at 1 week but not at 4 weeks. EPR oximetry and histology revealed hypoxia and tissue damage in regions of collateral growth of BALB/c but not C57BL/6 mice. In C57BL/6 BrdUrd uptake in the thigh was limited to larger vessels and isolated perivascular cells. Proliferative activity in collateral arterioles was similar in both strains. Conclusions-Genetic differences in preexistent collateral vasculature can profoundly affect outcome and milieu for compensatory collateral artery growth after femoral artery occlusion. Key Words: angiogenesis Ⅲ collateral circulation Ⅲ hypoxia Ⅲ mouse strains Ⅲ vascular biology P re-existent interarterial anastomoses have been identified in the coronary, cerebral, and peripheral circulation of various species. 1 The number and size of these anastomoses varies between species and tissues, resulting in different degrees of protection after arterial occlusion. 1 Pre-existing collateral arterial connections have been found in human hearts without evidence for coronary artery disease. 1-3 Remodeling of preexistent arterioles into mature collateral arteries has been observed in dog hearts, 4 hindlimbs of rabbits, 5-7 rats, 8 and mice, 9 and this process has been suggested to be the dominant mechanism responsible for the restoration of blood flow after arterial occlusion. 10 -12 Previously, we identified differences in perfusion recovery by laser Doppler imaging (LDI) after femoral artery occlusion in inbred strains of mice and attributed these to marginal differences in growth rates of collateral arterioles. 9 However, this study was limited by the fact that LDI and visible light oxygen spectrometry were the only comparative methods of in vivo assessment and histology was confined to isolated gracilis muscle collaterals with minimal surrounding tissue. Diameters of preexistent collaterals on the gracilis muscle were not significantly different between strains.In this study we examined collateral artery development and related parameters after femoral artery ligation in C57BL/6, 129S2 and BALB/c mice using the most comprehensive approach ever attempt...

Abstract-Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. Induced macrophage migration in vivo is driven by the binding of monocyte chemoattractant protein-1 (MCP-1, or CCL2 in the new nomenclature) to the CCR2-chemokine receptor on macrophages. To determine whether the CCL2-CCR2 signaling pathway is involved in the accumulation of macrophages in growing collateral vessels, we used mice that are deficient in CCR2 in a model of experimental arterial occlusion and collateral vessel growth. In an in vitro CCL2-driven chemotaxis assay, mononuclear cells isolated from wild-type BALB/c mice exhibited CCL2 concentration-dependent migration, whereas this migration was abolished in cells from CCR2 Ϫ/Ϫ mice on a BALB/c genetic background. In vivo, blood flow recovery as measured by laser Doppler (LDI) and MRI (MRI) was impaired in CCR2 Ϫ/Ϫ mice on either the BALB/c or C57BL/6 genetic backgrounds. Three weeks after femoral artery ligation, LDI perfusion ratio of operated versus nonoperated distal hindlimb in BALB/c wild-type mice increased to 0.45Ϯ0.06 and in CCR2 Ϫ/Ϫ animals only to 0.21Ϯ0.03 (PϽ0.01). In C57BL/6 mice, ratio increased to 0.96Ϯ0.09 and 0.85Ϯ0.08 (PϽ0.05), respectively. MRI at 3 weeks (0.76Ϯ0.06 versus 0.62Ϯ0.01; PϽ0.05) and hemoglobin oxygen saturation measurements confirmed these findings. Active foot movement score significantly decreased and gastrocnemius muscle atrophy was significantly greater in CCR2 Ϫ/Ϫ mice. Morphometric analysis showed a lesser increase in collateral vessel diameters in CCR2 Ϫ/Ϫ mice. Importantly, the number of invaded monocytes/macrophages in the perivascular space of collateral arteries of CCR2 Ϫ/Ϫ animals was dramatically reduced in comparison to wild-type mice. In conclusion, our results demonstrate that the CCR2 signaling pathway is essential for efficient collateral artery growth.

In human hearts a continuous turnover of the extracellular matrix occurs during the progression from compensated hypertrophy to HF that is characterized by the upregulation of MMPs and inadequate inhibition by TIMPs. The altered balance between proteolysis/antiproteolysis with accompanying proliferation of fibroblasts results in fibrosis progression.

Abstract-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. (Circ Res. 2007;100:363-371.)

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