Blockade of Angiogenesis by Small Molecule Antagonists to Protease-Activated Receptor-1: Association with Endothelial Cell Growth Suppression and Induction of Apoptosis

Abstract

Many studies support the notion that protease-activated receptor (PAR)-1 plays a pivotal role in angiogenesis. However, direct evidence and understanding the molecular mechanisms involved were limited because PAR-1-specific antagonists have been developed only recently. In the present study, we evaluated the effects of two well characterized PAR-1 antagonists,, in the angiogenic cascade. These antagonists suppressed both the basic angiogenesis and that stimulated by thrombin in the chick chorioallantoic membrane model in vivo. PAR-1 antagonists also abrogated tube formation in the in vitro Matrigel system. These inhibitory effects were dose-dependent and well correlated with the inhibitory effects of SCH79797 and RWJ56110 on primary endothelial cell proliferation and on the initiation of apoptosis. PAR-1 blockage resulted in inhibition of endothelial cell growth by increasing the sub-G 0 /G 1 fraction and reducing the percentage of cells in the S phase. Consistent with this, PAR-1 antagonists reduced incorporation of [ 3 H]thymidine in endothelial cells and blocked the phosphorylation of extracellular signal-regulated kinases in a fashion depending specifically on PAR-1 activation. Analysis by annexin V/propidium iodide staining and poly(ADP-ribose) polymerase cleavage revealed that PAR-1 blockage increased apoptotic cell death by a mechanism involving caspases. These results provide further evidence that PAR-1 is a key receptor that mediates angiogenesis and suggest PAR-1 as target for developing antiangiogenic agents with potential therapeutic application in cancer and other angiogenesis-related diseases.

Protease-Activated Receptor-1 (hPar1), A Survival Factor Eliciting Tumor Progression

“…Overall, the withdrawal of hPar1 initiates apoptosis in immature blood vessels and subsequently in tumor epithelia. Similarly, it has been recently shown that blockade of PAR1 reduced the incorporation of 3 H-thymidine in endothelial cells and increased PARP cleavage, indicative of the apoptotic pathway (38).…”

Parstatin, the Cleaved Peptide on Proteinase-Activated Receptor 1 Activation, Is a Potent Inhibitor of Angiogenesis

“…The Matrigel tube formation assay was used as described previously (Zania et al, 2006). In brief, Matrigel (BD Biosciences Pharmingen, San Diego, CA) was used to coat 24-well plates (0.25 ml/well).…”

“…In addition, signaling through PAR1 has been pro-posed to influence a wide range of pathophysiological responses, including platelet activation, intimal hyperplasia, inflammation, and tumor growth and metastasis (Tsopanoglou and Maragoudakis, 2007). In this context, PAR1 presents a novel target for developing new therapeutic agents for treating these conditions and angiogenesis-related diseases (Zania et al, 2006).…”

Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer

“…In addition, PAR1 activates ␣v␤5 integrin in melanoma to promote migration and invasion (Even-Ram et al, 2001). PAR1 may also contribute to tumor cell survival since it can prevent apoptosis of certain cell types (Guo et al, 2004;Zania et al, 2006). However, the role of PAR1 in tumor cell apoptosis has not been firmly established.…”