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Tipping off endothelial tubes: nitric oxide drives tip cells.

ABSTRACT: Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called "tip cell," followed by vessel elongation by "stalk cells." Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (NO) is an important modulator of angiogenesis, its role in angiogenic sprouting and specifically in tip cell formation is poorly understood. The present study tested the role of endothelial nitric oxide synthase (eNOS)/NO/cyclic GMP (cGMP) signaling in tip cell formation. In primary endothelial cell culture, about 40% of the tip cells showed characteristic sub-cellular localization of eNOS toward the anterior progressive end of the tip cells, and eNOS became phosphorylated at serine 1177. Loss of eNOS suppressed tip cell formation. Live cell NO imaging demonstrated approximately 35% more NO in tip cells compared with stalk cells. Tip cells showed increased level of cGMP relative to stalk cells. Further, the dissection of NO downstream signaling using pharmacological inhibitors and inducers indicates that NO uses the sGC/cGMP pathway in tip cells to lead angiogenesis. Taken together, the present study confirms that eNOS/NO/cGMP signaling defines the direction of tip cell migration and thereby initiates new blood vessel formation.

SUBMITTER: Priya MK

PROVIDER: S-EPMC5957492 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Tipping off endothelial tubes: nitric oxide drives tip cells.

Priya Mani Krishna MK Sahu Giriraj G Soto-Pantoja David R DR Goldy Naga N Sundaresan Abaya Meenakshi AM Jadhav Vivek V Barathkumar T R TR Saran Uttara U Jaffar Ali B M BM Roberts David D DD Bera Amal Kanti AK Chatterjee Suvro S

Angiogenesis 20141216 2

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called "tip cell," followed by vessel elongation by "stalk cells." Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (N ...[more]

PMID: 25510468

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Project description:Previous studies have demonstrated that G protein-coupled receptor kinase interacting-1 protein (GIT1) associates with endothelial nitric oxide synthase (eNOS) to regulate nitric oxide production in sinusoidal endothelial cells (SECs). Here, we hypothesized that GIT1's tightly associated binding partner, β-PIX (p21-activated kinase-interacting exchange factor β, ARHGEF7) is specifically important in the regulation of eNOS activity. We examined β-PIX expression in normal rat liver by immunohistochemistry and explored β-PIX protein-protein interactions using immunoprecipitation and immunoblotting. The role of β-PIX in regulating eNOS enzymatic activity was studied in GIT1-deficient SECs. Finally, structural analysis of interaction sites in GIT1 and β-PIX required to regulate eNOS activity were mapped. β-PIX was expressed primarily in SECs in normal liver and was either absent or expressed at extremely low levels in other liver cells (stellate cells, Kupffer cells, and hepatocytes). β-PIX interacted with GIT1 and eNOS to form a trimolecular signaling module in normal SECs and was important in stimulating eNOS activity. Of note, GIT1-β-PIX interaction led to synergistic enhancement of eNOS activity, and β-PIX-driven increase in eNOS activity was GIT1 dependent. Disruption of β-PIX or GIT1 in normal SECs using β-PIX siRNA or GIT1-deficient SECs led to reduced eNOS activity. Finally, specific GIT1 domains [Spa2 homology domain (SHD) and synaptic localization domain (SLD), aa 331-596] and the β-PIX COOH terminal (aa 496-555) appeared to be critical in the regulation eNOS activity. The data indicate that β-PIX regulates eNOS phosphorylation and function in normal SECs and highlight the importance of the GIT1/β-PIX/eNOS trimolecular complex in normal liver SEC function.NEW & NOTEWORTHY β-PIX is a multidomain protein known to be a GIT1 binding partner. We report here that in the normal liver, the distribution and cellular localization of β-PIX are restricted largely to sinusoidal endothelial cells. Furthermore, β-PIX interacts with eNOS and GIT1 promotes eNOS activity and NO production and therefore exerts a novel posttranslational regulatory function on eNOS activity in sinusoidal endothelial cells. We also have identified specific molecular domains important in GIT1 and β-PIX's interaction with eNOS, which may represent novel therapeutic targets in the control of sinusoidal blood flow and intrahepatic resistance.

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Tipping off endothelial tubes: nitric oxide drives tip cells.

Publications

Tipping off endothelial tubes: nitric oxide drives tip cells.

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