Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [L-N 6 -(1-iminoethyl) lysine, L-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) > WT with L-NIL or iNOS ؊/؊ > eNOS ؊/؊ > eNOS ؊/؊ with L-NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS-and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS ؊/؊ mice but not in eNOS ؊/؊ mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.

Endothelial progenitor cells (EPCs) are essential in vasculogenesis and wound healing, but their circulating and wound level numbers are decreased in diabetes. This study aimed to determine mechanisms responsible for the diabetic defect in circulating and wound EPCs. Since mobilization of BM EPCs occurs via eNOS activation, we hypothesized that eNOS activation is impaired in diabetes, which results in reduced EPC mobilization. Since hyperoxia activates NOS in other tissues, we investigated whether hyperoxia restores EPC mobilization in diabetic mice through BM NOS activation. Additionally, we studied the hypothesis that impaired EPC homing in diabetes is due to decreased wound level stromal cell-derived factor-1α (SDF-1α), a chemokine that mediates EPC recruitment in ischemia. Diabetic mice showed impaired phosphorylation of BM eNOS, decreased circulating EPCs, and diminished SDF-1α expression in cutaneous wounds. Hyperoxia increased BM NO and circulating EPCs, effects inhibited by the NOS inhibitor N-nitro-l-arginine-methyl ester. Administration of SDF-1α into wounds reversed the EPC homing impairment and, with hyperoxia, synergistically enhanced EPC mobilization, homing, and wound healing. Thus, hyperoxia reversed the diabetic defect in EPC mobilization, and SDF-1α reversed the diabetic defect in EPC homing. The targets identified, which we believe to be novel, can significantly advance the field of diabetic wound healing.

One of the most important functions of the blood circulation is O 2 delivery to the tissue. This process occurs primarily in microvessels that also regulate blood f low and are the site of many metabolic processes that require O 2 . We measured the intraluminal and perivascular pO 2 in rat mesenteric arterioles in vivo by using noninvasive phosphorescence quenching microscopy. From these measurements, we calculated the rate at which O 2 diffuses out of microvessels from the blood. The rate of O 2 eff lux and the O 2 gradients found in the immediate vicinity of arterioles indicate the presence of a large O 2 sink at the interface between blood and tissue, a region that includes smooth muscle and endothelium. Mass balance analyses show that the loss of O 2 from the arterioles in this vascular bed primarily is caused by O 2 consumption in the microvascular wall. The high metabolic rate of the vessel wall relative to parenchymal tissue in the rat mesentery suggests that in addition to serving as a conduit for the delivery of O 2 the microvasculature has other functions that require a significant amount of O 2 .

We hypothesized that exposure to hyperbaric oxygen (HBO(2)) would mobilize stem/progenitor cells from the bone marrow by a nitric oxide (*NO) -dependent mechanism. The population of CD34(+) cells in the peripheral circulation of humans doubled in response to a single exposure to 2.0 atmospheres absolute (ATA) O(2) for 2 h. Over a course of 20 treatments, circulating CD34(+) cells increased eightfold, although the overall circulating white cell count was not significantly increased. The number of colony-forming cells (CFCs) increased from 16 +/- 2 to 26 +/- 3 CFCs/100,000 monocytes plated. Elevations in CFCs were entirely due to the CD34(+) subpopulation, but increased cell growth only occurred in samples obtained immediately posttreatment. A high proportion of progeny cells express receptors for vascular endothelial growth factor-2 and for stromal-derived growth factor. In mice, HBO(2) increased circulating stem cell factor by 50%, increased the number of circulating cells expressing stem cell antigen-1 and CD34 by 3.4-fold, and doubled the number of CFCs. Bone marrow *NO concentration increased by 1,008 +/- 255 nM in association with HBO(2). Stem cell mobilization did not occur in knockout mice lacking genes for endothelial *NO synthase. Moreover, pretreatment of wild-type mice with a *NO synthase inhibitor prevented the HBO(2)-induced elevation in stem cell factor and circulating stem cells. We conclude that HBO(2) mobilizes stem/progenitor cells by stimulating *NO synthesis.

The objective of this study was to investigate the mechanism of S-nitrosothiol formation under physiological conditions. A mechanism is proposed by which nitric oxide ( S-Nitrosothiols are important physiological regulators capable of producing vasodilation and inhibition of platelet aggregation (1-4). An increasing number of proteins such as albumin, glyceraldehyde-3-phosphate dehydrogenase, hemoglobin, and p21 ras have been found to be S-nitrosylated in vivo (5-9). With the discovery of new S-nitrosylated proteins, it becomes evident that the formation of S-nitrosothiols may be important in such diverse processes as signal transduction, DNA repair, and blood-pressure regulation. However, at present the mechanism of the biosynthetic pathway for the formation of Snitrosothiols is unclear (10).It has been shown previously that the reaction of ⅐ NO 1 with sulfhydryl groups under anaerobic conditions at neutral pH does not produce S-nitrosothiol (11-13). This has led to the conclusion that S-nitrosothiols are formed by the autoxidation of ⅐ NO, a second order reaction with respect to ⅐ NO, to higher oxides of nitrogen (NO x ), by metal catalysis (12,14,15), or by the action of dinitrosyl-iron complexes (16). However, the reaction of ⅐ NO and oxygen is slow, approximately 3 to 300 pmol/s, at physiological concentrations of ⅐ NO (0.1-1.0 M), and the availability of redox metal is unclear (17, 18). Although dinitrosyl-iron complexes represent one possible mechanism for the formation of S-nitrosothiols, the reaction mechanism under physiological conditions also remains unclear (10).Here we propose a novel mechanism for S-nitrosothiol formation that would operate at physiological concentrations of ⅐ NO. In this mechanism ⅐ NO reacts directly with a reduced thiol to produce a radical intermediate, R-S-N ⅐ -O-H (Equation 1).In the presence of an electron acceptor, such as oxygen, this intermediate can be converted to S-nitrosothiol by the reduction of the acceptor.Therefore, the reaction of ⅐ NO and cysteine in buffer under aerobic conditions will form superoxide via the reduction of O 2 by R-S-N ⅐ -O-H (Equation 2). Superoxide reacts at a nearly diffusion-limited rate with ⅐ NO to form peroxynitrite (19) (Equation 3). The overall reaction mechanism is shown below (Equation 4).From this reaction mechanism the following testable predictions can be made: first, that free thiol will accelerate the decomposition of ⅐ NO and will result in the formation of a ⅐ NO donor; second, that thiol will accelerate the consumption of O 2 by ⅐ NO and generate H 2 O 2 in the presence of Cu,Zn-superoxide dismutase; and third, that the reaction will proceed under anaerobic conditions in the presence of an electron acceptor. Here experimental evidence is provided for each of the above predictions in support of the proposed mechanism. EXPERIMENTAL PROCEDURESMaterials-Cu,Zn-superoxide dismutase was obtained from Fluka (Switzerland), and DEANO was obtained from Cayman Chemical (Ann Arbor, MI). All other chemicals were obtained from Sigma. Spec...

We tested the hypothesis that high-viscosity (HV) plasma in extreme hemodilution causes wall shear stress to be greater than low-viscosity (LV) plasma, leading to enhanced production of nitric oxide (NO). The perivascular concentration of NO was measured in arterioles and venules and the tissue of the hamster chamber window model, subjected to acute extreme hemodilution, with a hematocrit (Hct) of 11% using Dextran 500 (n = 6) or Dextran 70 (n = 5) with final plasma viscosities of 1.99 +/- 0.11 and 1.33 +/- 0.04 cp, respectively. HV plasma significantly increased the periarteriolar, perivenular, and tissue NO concentration by 2.0, 1.9, and 1.4 times the control (n = 7). The NO concentration with LV plasma was not statistically different from control. Arteriolar shear stress was significantly increased in HV plasma relative to LV plasma in arterioles but not in venules. Aortic endothelial NO synthase (eNOS) protein expression was increased with HV plasma but not with LV plasma. There was a weak correlation between perivascular NO concentration and the locally calculated shear stress induced by the procedures, when blood viscosity was corrected according to Hct values previously determined in studies of microvascular Hct distribution. The finding that the periarteriolar and venular NO concentration in HV plasma was the same although arteriolar shear stress was significantly greater than venular shear stress maybe be due to differences in vessel wall metabolism between arterioles and venules and the presence of NO transport through the blood stream in the microcirculation. Results support the concept that in extreme hemodilution HV plasma maintains functional capillary density through a NO-mediated vasodilatation.

Vascular immunotargeting may facilitate the rapid and specific delivery of therapeutic agents to endothelial cells. We investigated whether targeting of an antioxidant enzyme, catalase, to the pulmonary endothelium alleviates oxidative stress in an in vivo model of lung transplantation. Intravenously injected enzymes, conjugated with an antibody to platelet-endothelial cell adhesion molecule-1, accumulate in the pulmonary vasculature and retain their activity during prolonged cold storage and transplantation. Immunotargeting of catalase to donor rats augments the antioxidant capacity of the pulmonary endothelium, reduces oxidative stress, ameliorates ischemia-reperfusion injury, prolongs the acceptable cold ischemia period of lung grafts, and improves the function of transplanted lung grafts. These findings validate the therapeutic potential of vascular immunotargeting as a drug delivery strategy to reduce endothelial injury. Potential applications of this strategy include improving the outcome of clinical lung transplantation and treating a wide variety of endothelial disorders.

NO has been shown to mediate angiogenesis; however, its role in vessel morphogenesis and maturation is not known. Using intravital microscopy, histological analysis, α-smooth muscle actin and chondroitin sulfate proteoglycan 4 staining, microsensor NO measurements, and an NO synthase (NOS) inhibitor, we found that NO mediates mural cell coverage as well as vessel branching and longitudinal extension but not the circumferential growth of blood vessels in B16 murine melanomas. NO-sensitive fluorescent probe 4,5-diaminofluorescein imaging, NOS immunostaining, and the use of NOS-deficient mice revealed that eNOS in vascular endothelial cells is the predominant source of NO and induces these effects. To further dissect the role of NO in mural cell recruitment and vascular morphogenesis, we performed a series of independent analyses. Transwell and under-agarose migration assays demonstrated that endothelial cell-derived NO induces directional migration of mural cell precursors toward endothelial cells. An in vivo tissue-engineered blood vessel model revealed that NO mediates endothelial-mural cell interaction prior to vessel perfusion and also induces recruitment of mural cells to angiogenic vessels, vessel branching, and longitudinal extension and subsequent stabilization of the vessels. These data indicate that endothelial cell-derived NO induces mural cell recruitment as well as subsequent morphogenesis and stabilization of angiogenic vessels.