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.angiogenesis ͉ arteriogenesis ͉ genetics

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-Plaque vascularity has been implicated in its growth and stability. However, there is a paucity of information regarding the origin of plaque vasculature and the role of vasa vasorum in plaque growth. To inhibit growth of vasa vasorum in atherogenic mice and assess its effect on plaque growth, we used a truncated plasminogen activator inhibitor (PAI)-1 protein, rPAI-1 23 , that has significant antiangiogenic activity. Female LDLR Ϫ/Ϫ ApoB-48 -deficient mice fed Paigen's diet without cholate for 20 weeks received rPAI-1 23 treatment (nϭ21) for the last 6 weeks. Plaque size and vasa vasorum density were compared to 2 controls: mice fed Paigen's diet and treated with saline for the last 6 weeks (nϭ16) and mice fed Paigen's diet until the onset of treatment (nϭ14). The rPAI-1 23 treatment significantly reduced plaque area and plaque cholesterol in the descending aorta and plaque area in the innominate artery. Measurements of reconstructed confocal microscopy images of vasa vasorum demonstrate that rPAI-1 23 treatment decreased vasa vasorum area and length, which was supported by microCT images. Confocal images provide evidence for vascularized plaque in the saline-treated group but not in rPAI-1 23 -treated mice. The increased vessel density in saline-treated mice is attributable, in part, to upregulated fibroblast growth factor-2 expression, which is inhibited by rPAI-1 23 . In conclusion, rPAI-1 23 inhibits growth of vasa vasorum, as well as vessels within the adjacent plaque and vessel wall, through inhibition of fibroblast growth factor-2, leading to reduced plaque growth in atherogenic female LDLR Ϫ/Ϫ ApoB-48 -deficient mice.

Background and Aims Cholangiocytes are the target of a heterogeneous group of liver diseases, known as the cholangiopathies. An evolving understanding of the mechanisms driving biliary development provides the theoretical underpinnings for rational development of induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs). Therefore, the aims of this study were to develop an approach to generate iDCs and to fully characterize the cells in vitro and in vivo. Methods Human iPSC lines were generated by forced expression of the Yamanaka pluripotency factors. We then pursued a step-wise differentiation strategy toward iDCs using precise temporal exposure to key biliary morphogens and we characterized the cells using a variety of morphologic, molecular, cell biologic, functional, and in vivo approaches. Results Morphology shows a stepwise phenotypic change toward an epithelial monolayer. Molecular analysis during differentiation shows appropriate enrichment in markers of iPSC, definitive endoderm, hepatic specification, hepatic progenitors, and ultimately cholangiocytes. Immunostaining, Western blotting, and flow cytometry demonstrate enrichment of multiple functionally relevant biliary proteins. RNA sequencing reveals that the transcriptome moves progressively toward that of human cholangiocytes. iDCs generate intracellular calcium signaling in response to ATP, form intact primary cilia, and self-assemble into duct-like structures in 3-dimensional culture. In vivo, the cells engraft within mouse liver following retrograde intra-biliary infusion. Conclusions In summary, we have developed a novel approach to generate mature cholangiocytes from iPSCs. In addition to providing a model of biliary differentiation, iDCs represent a platform for in vitro disease modelling, pharmacologic testing, and individualized, cell-based, regenerative therapies for the cholangiopathies.

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Objective. Systemic sclerosis (SSc; scleroderma) is a systemic connective tissue disease with an extensive vascular component that includes aberrant microvasculature and impaired wound healing. The aim of this study was to investigate the presence of antiangiogenic factors in patients with SSc.Methods. Plasma samples were obtained from 30 patients with SSc and from 10 control patients without SSc. The samples were analyzed for the ability of plasma to affect endothelial cell migration and vascular structure formation and for the presence of antiangiogenic activity.Results. Exposure of normal human microvascular dermal endothelial cells to plasma from patients with SSc resulted in decreased cell migration (mean ؎ SEM 52 ؎ 5%) and tube formation (34 ؎ 6%) compared with that in plasma from control patients (P < 0.001 for both). SSc plasma contained 2.9-fold more plasminogen kringle 1-3 fragments (angiostatin) than that in control plasma. The addition of angiostatin to control plasma resulted in inhibition of endothelial cell migration and proliferation similar to that observed in SSc plasma. In vitro studies demonstrated that granzyme B and other proteases contained in T cell granule content cleave plasminogen and plasmin into angiostatin fragments.Conclusion. Plasminogen conformation in patients with SSc enables granzyme B and granule content protease to limit the proangiogenic effects of plasmin and increase the levels of antiangiogenic angiostatin. This increase in angiostatin production may account for some of the vascular defects observed in patients with SSc.

Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrixbound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-1 23 , that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-1 23 effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. The vascular endothelium is normally maintained in a differentiated, quiescent state. Pro-angiogenic factors destabilize the quiescent endothelium into migratory, proliferative endothelial cells that are attenuated by anti-angiogenic factors. The pro-and anti-angiogenic molecules have cell survival and death functions that are tightly controlled to maintain a balance (1-4).Many negative regulators of angiogenesis are cleavage products of an existing cellular protein that is not inhibitory in its normal intact conformation (5-7). Induction of endothelial cell apoptosis is one characteristic common to these inhibitors (8). Endothelial cells are more susceptible to apoptosis when they are activated (angiogenic) as compared with quiescent (9, 10). Survival of the latter is dependent upon angiogenic growth factors in the local environment, which can be blocked by antiangiogenic factors. If growth factor functions are blocked, endothelial cells are removed by apoptosis (4, 9, 11).Apoptosis can be induced by diverse stimuli that initiate specific signal transduction pathways. The apoptosis pathways are regulated by pro-and anti-apoptotic molecules that are controlled in part by kinases (12) and proteasomal degradation (13). The c-Jun NH 2 -terminal kinase (JNK) 2 signaling pathway is activated by cellular stress, and its role in apoptosis versus survival remains unclear (14). The role of JNK in cellular proliferation, apoptosis, differentiation, and motility is dependent upon the activity and stability of JNK isoforms and their associated substrates (15-18). The cellular function associated with JNK-substrate complexes is tightly regulated by proteasomal activity (19,20).Plasminogen activator inhibitor-1 (PAI-1) has been shown to have both pro-and anti-angiogenic activity (21-24). It has been suggested that PAI-1 pro-angiogenic versus anti-angiogenic activity is based on the relative amounts of the inhibitor that are in active versus inactive conformations (22,25). Proteolytic molecules plasmin (26) and matrix metalloproteinase 3 (27) cleave and inactivate PAI-1. Potential functions of cleaved PAI-1 have not been studied. We made truncated PAI-1 cDNAs and produced truncated PAI-1 proteins (rPAI-1) to investigate potential PAI-1 functions in the absen...

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