Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.

We have previously shown that monocytes adhere to the vascular wall during collateral vessel growth (arteriogenesis) and capillary sprouting (angiogenesis). In this study we investigated the association of monocyte accumulation with both the production of the cytokines-basic fibroblast growth factor (bFGF) and TNF-alpha-and vessel proliferation in the rabbit after femoral artery occlusion. In particular, we studied the effects of an increase in monocyte recruitment by LPS on capillary density as well as collateral and peripheral conductance after 7 d of occlusion. Monocytes accumulated around day 3 in collateral arteries when maximal proliferation was observed, and stained strongly for bFGF and TNF-alpha. In the lower limb where angiogenesis was shown to be predominant, macrophage accumulation was also closely associated with maximal proliferation (around day 7). LPS treatment significantly increased capillary density (424+/-26.1 n/mm2 vs. 312+/-20.7 n/mm2; P < 0.05) and peripheral conductance (109+/-33.8 ml/min/100 mmHg vs. 45+/-6.8 ml/min/100 mmHg; P < 0.05) as compared with untreated animals after 7 d of occlusion. These results indicate that monocyte activation plays a major role in angiogenesis and collateral artery growth.

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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j Abstract Following an arterial occlusion outward remodeling of pre-existent inter-connecting arterioles occurs by proliferation of vascular smooth muscle and endothelial cells. This is initiated by deformation of the endothelial cells through increased pulsatile fluid shear stress (FSS) caused by the steep pressure gradient between the high pre-occlusive and the very low post-occlusive pressure regions that are interconnected by collateral vessels. Shear stress leads to the activation and expression of all NOS isoforms and NO production, followed by endothelial VEGF secretion, which induces MCP-1 synthesis in endothelium and in the smooth muscle of the media. This leads to attraction and activation of monocytes and T-cells into the adventitial space (peripheral collateral vessels) or attachment of these cells to the endothelium (coronary collaterals). Mononuclear cells produce proteases and growth factors to digest the extra-cellular scaffold and allow motility and provide space for the new cells. They also produce NO from iNOS, which is essential for arteriogenesis. The bulk of new tissue production is carried by the smooth muscles of the media, which transform their phenotype from a contractile into a synthetic and proliferative one. Important roles are played by actin binding proteins like ABRA, cofilin, and thymosin beta 4 which determine actin polymerization and maturation. Integrins and connexins are markedly up-regulated. A key role in this concerted action which leads to a 2-to-20 fold increase in vascular diameter, depending on species size (mouse versus human) are the transcription factors AP-1, egr-1, carp, ets, by the Rho pathway and by the Mitogen Activated Kinases ERK-1 and -2. In spite of the enormous increase in tissue mass (up to 50-fold) the degree of functional restoration of blood flow capacity is incomplete and ends at 30% of maximal conductance (coronary) and 40% in the vascular periphery. The process of arteriogenesis can be drastically stimulated by increases in FSS (arterio-venous fistulas) and can be completely blocked by inhibition of NO production, by pharmacological blockade of VEGF-A and by the inhibition of the Rho-pathway. Pharmacological stimulation of arteriogenesis, important for the treatment of arterial occlusive diseases, seems feasible with NO donors.j Key words arteriogenesis -growth factor -integrins -shear stress -MAP kinases INVITED EDITORIAL

Monocytes are activated during collateral artery growth in vivo, and monocyte chemotactic protein-1 (MCP-1) has been shown to be upregulated by shear stress in vitro. In order to investigate whether MCP-1 enhances collateral growth after femoral artery occlusion, 12 rabbits were randomly assigned to receive either MCP-1, PBS, or no local infusion via osmotic minipump. Seven days after occlusion, isolated hindlimbs were perfused with autologous blood at different pressures, measuring flows at maximal vasodilation via flow probe and radioactive microspheres, as well as peripheral pressures. This allowed the calculation of collateral (thigh) and peripheral (lower limb) conductances from pressure-flow tracings (slope of the curve). Collateral growth on postmortem angiograms was restricted to the thigh and was markedly enhanced with MCP-1 treatment. Both collateral and peripheral conductances were significantly elevated in animals with MCP-1 treatment compared with the control group, reaching values of nonoccluded hindlimbs after only 1 week of occlusion (collateral conductance, 70.6 +/- 19.23 versus 25.1 +/- 2.59 mL/min per 100 mm Hg; P < .01; peripheral conductance, 119.3 +/- 22.37 versus 45.4 +/- 6.80 mL/min per 100 mm Hg; P < .05). These results suggest that activation of monocytes plays an important role in collateral growth as well as in capillary sprouting.

Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 microm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation.