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.

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.

The R-N and R-L BAVs are different etiological entities. The R-N BAVs are the product of a morphogenetic defect that happens before the OT septation and that probably relies on an exacerbated nitric oxide-dependent epithelial-to-mesenchymal transformation. The R-L BAVs result from the anomalous septation of the proximal portion of the OT, likely caused by a distorted behavior of neural crest cells. Care should be taken in further work on BAV genetics because R-N and R-L BAVs might rely on different genotypes. Detailed screening for R-N and R-L BAVs should be performed for a better understanding of the relationships between these BAV morphologic phenotypes and other heart disease.

Abstract-Vascular endothelial growth factor (VEGF) is known to play an important role in angiogenesis. Its place in collateral artery growth (arteriogenesis), however, is still debated. In the present study, we analyzed the expression of VEGF and its receptors (Flk-1 and Flt-1) in a rabbit model of collateral artery growth after femoral artery occlusion. Hypoxia presents the most important stimulus for VEGF expression. We therefore also investigated the expression level of distinct hypoxia-inducible genes (HIF-1␣, LDH A) and determined metabolic intermediates indicative for ischemia (ATP, creatine phosphate, and their catabolites). We found that arteriogenesis was not associated with an increased expression of VEGF or the mentioned hypoxia-inducible genes. Furthermore, the high-energy phosphates and their catabolites were entirely within normal limits. Despite the absence of an increased expression of VEGF and its receptors, collateral vessels increased their diameter by a factor of 10. The speed of collateral development could be increased by infusion of the chemoattractant monocyte chemotactic protein-1 but not by infusion of a 30 times higher concentration of VEGF. From these data, we conclude that under nonischemic conditions, arteriogenesis is neither associated with nor inducible by increased levels of VEGF and that VEGF is not a natural agent to induce arteriogenesis in vivo.

Abstract-Two signaling receptors for vascular

Abstract-Fibroblast growth factors (FGFs) have been applied in a variety of therapeutic and experimental studies to improve collateral blood flow. However, the pathophysiological role and the temporospatial expression of the FGFs and their receptors during arteriogenesis have never been elucidated in vivo. Here, we report that collateral artery growth in its early phase is associated with an increased expression of FGF receptor-1 (FGFR-1) and syndecan-4 on mRNA and protein levels as well as with an increased kinase activity of FGFR-1 in a rabbit model of arteriogenesis. However, the mRNA levels of FGF-1 and -2 remained constant. Our data suggest that these growth factors are supplied by endothelial attracted monocytes that, in turn, produce and deliver the FGFs to growing collateral arteries. Monocyte chemoattractant protein-1-stimulated arteriogenesis was strongly reduced in rabbits by application of the FGF inhibitor polyanetholesulfonic acid, indicating that the monocyte-related arteriogenesis (as well as the unstimulated adaptation proper) is promoted by FGFs. In summary, this study shows that arteriogenesis is associated with an increased expression of the FGFRs at the site of the vessel, whereas the growth-promoting ligands are supplied by monocytes in a paracrine way. Key Words: arteriogenesis Ⅲ fibroblast growth factors Ⅲ fibroblast growth factor receptor-1 Ⅲ syndecan-4 Ⅲ monocytes C ollateral artery growth (arteriogenesis) occurs spontaneously as an adaptive response to vascular occlusion. However, the speed of growth can be stimulated by factors such as monocyte chemoattractant protein-1 (MCP-1), 1 transforming growth factor-␤1, 2 or granulocyte-macrophage colony-stimulating factor, 3,4 which either attract or prolong the lifespan of monocytes. In a variety of studies, fibroblast growth factors (FGFs) have also been applied for their potency to induce vascular growth and to promote the recovery of blood flow after arterial occlusion.The FGFs are a family of Ͼ20 polypeptides that mediate a broad range of biological activities (hematopoiesis, development, and wound repair) in a variety of cell types of mesenchymal and neuroectodermal origin. These activities are initiated through 4 structurally related membraneassociated tyrosine kinase FGF receptors (FGFRs) that are derived from separate genes and exist in a multitude of isoforms. 5 Ligand-induced dimerization of these high-affinity receptors, which is mediated by low-affinity receptors, is a key event in transmembrane signaling. It leads to an increase in kinase activity, resulting in the autophosphorylation of the receptor and the induction of diverse biological responses. 6 -8 Special attention is focused on FGF-1 and FGF-2, potent mitogens playing an important role in cell proliferation, migration, and differentiation. 9 -11 These FGFs differ from most of the other FGFs because they lack hydrophobic signal peptides and therefore are concentrated within their cell of origin. 12 Exogenously administrated, these growth factors have been shown to incr...

Background: Bicuspid aortic valve is the most frequent congenital cardiac malformation in humans. However, the morphogenesis of the defect is still unknown. Previous work showed that, in the Syrian hamster, congenital bicuspid aortic valves with the aortic sinuses arranged in ventrodorsal orientation are expressions of a trait the variation of which takes the form of a continuous phenotypic spectrum, ranging from a tricuspid aortic valve with no fusion of the ventral commissure to a bicuspid aortic valve devoid of any raphe. The present study was designed to elucidate the mechanism involved in the formation of bicuspid aortic valves in Syrian hamsters as a possible starting point for further investigation of this process in humans.Methods: The sample examined consisted of 80 embryos, aged between 10 days, 16 hours and 13 days, 1 hour postcoitum. Most (n = 59) of the embryos belonged to a laboratory-inbred family of Syrian hamsters with a high incidence of bicuspid aortic valves. The study was carried out using scanning electron microscopy and histological techniques for light microscopy.Results: Twenty-three embryos showed a still undivided conotruncus. In all of these cases there were six mesenchymal semilunar valve primordia protruding into the lumen of the conotruncus. In a further 29 embryos, the conotruncus had just divided into the aortic and pulmonary channels; the embryos were at the beginning of the valvulogenesis. In 13 of these 29 embryos there were three well-defined aortic valve cushions, right, left, and dorsal, whereas in the other 16, the right and left valve cushions were more or less fused toward the lumen of the aorta; when they were completely fused, only two aortic valve cushions, a ventral and a dorsal, could be identified. In the remaining 28 embryos, the aortic valve cushions showed a marked degree of excavation. In 23 of these cases, the valve exhibited a basically tricuspid architecture, whereas it was unequivocally bicuspid in the other five.Conclusions: All variants of the aortic valve morphologic spectrum occurring in the Syrian hamster develop from three mesenchymal valve cushions, right, left, and dorsal, after normal septation of the conotruncus. The bicuspid condition of the aortic valve is not the consequence of improper development of the conotruncal ridges, conotruncal malseptation, valve cushion agenesis, or lesions acquired after a normal valvulogenesis. Fusion of the right and left valve cushions at the beginning of the valvulogenesis appears to be a key factor in the formation of bicuspid aortic valves. Each aortic valve acquires its specific morphology prior to the end of the valvulogenetic process.

This International Consensus Classification and Nomenclature for the congenital bicuspid aortic valve condition recognizes 3 types of bicuspid valves: 1. The fused type (right-left cusp fusion, right-non-coronary cusp fusion and left-non-coronary cusp fusion phenotypes); 2. The 2-sinus type (latero-lateral and antero-posterior phenotypes); and 3. The partial-fusion (forme fruste) type. The presence of raphe and the symmetry of the fused type phenotypes are critical aspects to describe. The International Consensus also recognizes 3 types of bicuspid valve-associated aortopathy: 1. The ascending phenotype; 2. The root phenotype; and 3. Extended phenotypes.