Cells with endothelial phenotype generated from adult peripheral blood have emerging diagnostic and therapeutic potential. This study examined the lineage relationship between, and angiogenic function of, early endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) in culture. Culture conditions were established to support the generation of both EPCs and OECs from the same starting population of peripheral blood mononuclear cells (PBMCs). Utilizing differences in expression of the surface endotoxin receptor CD14, it was determined that the vast majority of EPCs arose from a CD14 ؉ subpopulation of PBMCs but OECs developed exclusively from the CD14 ؊ fraction. Human OECs, but not EPCs, expressed key regulatory proteins endothelial nitric oxide synthase (eNOS) and caveolin-1. Moreover, OECs exhibited a markedly greater capacity for capillary morphogenesis in in vitro and in vivo matrigel models, tube formation by OECs being in part dependent on eNOS function. Collectively, these data indicate lineage and functional heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype and provide rationale for the investigation of new celltherapeutic approaches to ischemic cardiovascular disease.

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In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.

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Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein Snitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimidesensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes.endothelial nitric oxide synthase ͉ Golgi ͉ targeting N itric oxide (NO), produced by the nitric oxide synthase (NOS) family of proteins, regulates a variety of important physiological responses, including vasodilation, respiration, cell migration, and apoptosis (1-5). NO has been considered to mediate these responses by activating the primary NO effector soluble guanylyl cyclase to produce cGMP (6) or by NO-based chemical modifications of proteins or perhaps lipids. One clear example of cGMPindependent actions of NO is protein thiol group modification by NO known as S-nitrosylation (7). This posttranslational control mechanism regulates important physiological activities of proteins in response to endogenously or exogenously generated NO (8). Thus, S-nitrosylation of proteins is an emerging area of investigation for NO-mediated physiological responses (8).NO is a lipophillic, highly diffusible, and short-lived physiological messenger (9). On the one hand, NO is thought to diffuse over a wide area (100 m), moving freely through membranes of neighboring cells (9). On the other hand, given the apparent promiscuity of NO, the question arises as to how S-nitrosylation might occur in a precisely regulated manner, i.e., protein S-nitrosylation occurs on specific thiol residues in proteins that are targeted to specific organelles in cells (10), and low concentrations of NO activate the ryanodine receptor via thiol modification, whereas higher concentrations inhibit the receptor (11). There are several compelling arguments in favor of the concept that all sources of NO are not bioequivalent. (i) In cardiac myocytes, which express two forms of NOS, gene deletion of either neuronal NOS or endothelial NOS (eNOS) exerts isoform-specific phenotypes, arguing that the source of NO favors local control of different cellular functions (12). Moreover, in many circumstances, the actions of endogenously gener...

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Angiogenesis, a process resulting in the formation of new capillaries from the pre-existing vasculature plays vital role for the development of therapeutic approaches for cancer, atherosclerosis, wound healing, and cardiovascular diseases. In this report, the synthesis, characterization, and angiogenic properties of graphene oxide (GO) and reduced graphene oxide (rGO) have been demonstrated, observed through several in vitro and in vivo angiogenesis assays. The results here demonstrate that the intracellular formation of reactive oxygen species and reactive nitrogen species as well as activation of phospho-eNOS and phospho-Akt might be the plausible mechanisms for GO and rGO induced angiogenesis. The results altogether suggest the possibilities for the development of alternative angiogenic therapeutic approach for the treatment of cardiovascular related diseases where angiogenesis plays a significant role.

Endothelial cell-based angiogenesis requires activation of survival signals that generate resistance to external apoptotic stimuli, such as tumor necrosis factor-alpha (TNF-α), during pathobiologic settings. Mechanisms by which this is achieved are not fully defined. Here, we use a model in which the multifunctional cytokine nitric oxide counterbalances TNF-α-induced apoptosis, to define a role for membrane trafficking in the process of endothelial cell survival signaling. By perturbing dynamin GTPase function, we identify a key role of dynamin for ensuing downstream endothelial cell survival signals and vascular tube formation. Furthermore, nitric oxide is directly demonstrated to promote dynamin function through specific cysteine residue nitrosylation, which promotes endocytosis and endothelial cell survival signaling. Thus, these studies identify a novel role for dynamin as a survival factor in endothelial cells, through a mechanism by which dynamin S-nitrosylation regulates the counterbalances of TNF-α-induced apoptosis and nitric oxide-dependent survival signals, with implications highly relevant to angiogenesis.