Biomedical subjects

C D Kontos

Publications and source records attributed to C D Kontos.

7 recordsLinked to original sources

PubMed2001

Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide.

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.

Cell Division↗

PubMed1999

HCPTPA, a protein tyrosine phosphatase that regulates vascular endothelial growth factor receptor-mediated signal transduction and biological activity.

Angiogenesis is a tightly controlled process in which signaling by the receptors for vascular endothelial growth factor (VEGF) plays a key role. In order to define signaling pathways downstream of VEGF receptors (VEGFR), the kinase domain of VEGFR2 (Flk-1) was used as a bait to screen a human fetal heart library in the yeast two-hybrid system. One of the signaling molecules identified in this effort was HCPTPA, a low molecular weight, cytoplasmic protein tyrosine phosphatase. Although HCPTPA possesses no identifiable phosphotyrosine binding domains (i.e. SH2 or phosphotyrosine binding domains), it bound specifically to active, autophosphorylated VEGFR2 but not to a mutated, kinase-inactive VEGFR2. Recombinant VEGFR2 and endogenous VEGFR2 were substrates for recombinant HCPTPA, and HCPTPA was co-expressed with VEGFR2 in endothelial cell lines, suggesting that HCPTPA may be a negative regulator of VEGFR2 signal transduction. To pursue this possibility, an adenovirus directing the expression of HCPTPA was constructed. When used to infect cultured endothelial cells, this adenovirus directed high level expression of HCPTPA that resulted in impairment of VEGF-mediated VEGFR2 autophosphorylation and mitogen-activated protein kinase activation. Adenovirus-mediated overexpression of HCPTPA also inhibited VEGF-induced cellular responses (endothelial cell migration and proliferation) and inhibited angiogenesis in the rat aortic ring assay. Taken together, these findings indicate that HCPTPA may be an important regulator of VEGF-mediated signaling and biological activity. Potential interactions with other signaling pathways and possible therapeutic implications are discussed.

Animals↗

PubMed1999

Angiogenesis.

Angiogenesis, the growth and proliferation of blood vessels from existing vascular structures, is tightly regulated in adult tissues, and abnormalities in angiogenesis are associated with a number of pathologic states. Strategies designed to promote angiogenesis to treat disorders of inadequate tissue perfusion, such as occurs in coronary artery and peripheral vascular disease, have led to the area of therapeutic angiogenesis. Approaches to block angiogenesis are actively being explored to treat diseases that range from arthritis to cancer. This article will review some of the basic concepts of vascular development and the mechanisms involved in angiogenesis. Particular attention will be paid to the growth factors and receptors that are known to mediate angiogenesis, and a description of some of the cell signaling mechanisms that are involved in the regulation of angiogenesis will be described. Finally, potential targets that may provide opportunities to enhance or block angiogenesis will be discussed.

Angiogenesis Inhibitors↗

PubMed1999

Allelic and locus heterogeneity in inherited venous malformations.

Venous malformations are low-flow vascular lesions consisting of disorganized thin-walled vascular channels. These can occur sporadically but also as an autosomal dominant condition termed venous malformations, cutaneous and mucosal (VMCM; OMIM 600195). In two large unrelated kindreds mapping to chromosome 9, the identical R849W missense mutation was identified in the first kinase domain of Tie2, an endothelial cell-specific receptor tyrosine kinase. We report here the identification of four new kindreds with inherited venous malformations. Unlike the initial two families described, these four families demonstrate allelic and locus heterogeneity. In one of these families, the R849W mutation co-segregates with the disease phenotype. Three other families with venous malformations lack this mutation. One of these families is linked to markers near TIE2 on chromosome 9. In this family, we identified a novel mutation within the first kinase domain of Tie2 resulting in a Y897S change. Results from COS-1 cell transfections using expression constructs containing either the R849W or the Y897S mutation suggest that the receptors containing either mutation show ligand-independent hyperphosphorylation. These results suggest a gain-of-function mechanism for development of venous malformations in these families. Of the two remaining families, one excludes linkage to the TIE2 locus, establishing the existence of at least one additional locus for dominantly inherited venous malformations.

Amino Acid Sequence↗

PubMed1998

Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumour angiogenesis.

Endothelial receptor tyrosine kinases may play important roles in pathological vascular growth, particularly in tumours. In this study, immunohistochemistry was used to evaluate the expression of a novel endothelial receptor tyrosine kinase, Tie2/Tek, in the endothelium of vascular 'hotspots' in normal breast tissue (n = 10), benign breast lesions (n = 10) and in breast tumours (n = 123). Tie2 expression was detected in the endothelium of all breast tissues examined. However, the strongest expression of Tie-2 was seen in vascular 'hot spots' within the inflammatory infiltrate at the periphery of invasive tumours. Moreover, the proportion of Tie2-positive vessels (Tie2 counts/CD31 counts) was significantly higher in breast tumours than the proportion of Tie2-positive vessels in either normal breast tissue or benign breast lesions (P = 0.004 and 0.0001 respectively). These data are consistent with a role for Tie2 in tumour angiogenesis and demonstrate the potential use of Tie2 expression as a novel marker of the tumour vasculature.

Antibodies, Monoclonal↗

PubMed1998

Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt.

Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3, 4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

3T3 Cells↗

PubMed1992

Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo.

We used a cytochemical technique for the detection of superoxide in cerebral inflammation and ischemia-reperfusion in anesthetized cats. The technique is based on the oxidation of Mn2+ to Mn3+ by superoxide; Mn3+, in turn, oxidizes diaminobenzidine. The oxidized diaminobenzidine forms an osmiophilic electron-dense product that is detected by electron microscopy. The reagents, manganese chloride (2 mM) and diaminobenzidine (2 mg/ml), were placed topically on the brain surface of anesthetized cats equipped with cranial windows. Inflammation was induced by topical carrageenan with or without phorbol 12-myristate 13-acetate to activate leukocytes. In inflammation, superoxide was detected in the plasma membrane and in the phagocytic vacuoles of leukocytes. In ischemia-reperfusion, superoxide was identified in the meninges in association with blood vessels. It was located primarily in the extracellular space and occasionally in endothelial and vascular smooth muscle cells. In both inflammation and ischemia, the reaction product was eliminated by superoxide dismutase or by the omission of either manganese or diaminobenzidine. It was unaffected by sodium azide, which inhibits peroxidases. No superoxide was detected in the brain parenchyma. The findings confirm the generation of superoxide is cerebral ischemia-reperfusion and show that it is produced in cerebral vessels.

Animals↗