Endostatin, the Proteolytic Fragment of Collagen XVIII, Induces Vasorelaxation

Abstract

Abstract-Collagen XVIII is an important component of the extracellular matrix and is expressed in basement membranes.Its degradation results in the generation of endostatin claimed to possess antiangiogenic activity. To date, only limited knowledge exists with regard to the cellular signaling of this molecule. We show in single-cell measurements using the Ca 2ϩ indicator fura-2 acetoxy methylester (fura-2 AM) and the nitric oxide (NO) indicator 4,5-diaminofluorescein diacetate that application of endostatin (ES) (5 pmol/L, 100 ng/mL) induced Ca 2ϩ spikes and an increase of NO production in human and murine endothelial cells. The NO response was independent of an increase in cytosolic Ca 2ϩ and blocked by the endothelial NO synthase (eNOS) inhibitor N G -nitro-L-arginine methyl ester and by incubation with pertussis toxin known to inhibit G i/o proteins. The physiological relevance of this novel signaling pathway of ES was assessed with isometric force measurements in large and small arteries of mouse. Physiological concentrations of ES were found to decrease vascular tone in an endothelium-dependent manner. This occurred via an Arg-Gly-Asp (RGD) peptide-independent pathway through activation of G i/o proteins, phosphatidylinositol 3-kinase, Akt, and eNOS. We conclude that the proteolytic matrix fragment ES is a prominent vasorelaxing agent. Because ES is constantly released into the blood, it is a novel regulator of blood pressure and, therefore, represents an interesting pharmacological target. Key Words: vascular tone Ⅲ extracellular matrix Ⅲ endostatin Ⅲ nitric oxide Ⅲ G proteins T he identification of novel endogenous and exogenous regulators of vascular tone is of great interest. Advances in this field may lead to a better understanding of existing pathophysiological conditions and reveal novel therapeutic targets. Besides the canonical agonists such as noradrenaline, acetylcholine, or histamine, components of the extracellular matrix (ECM) have been proposed to potentially play an important role in vasoregulation. 1 Collagen XVIII is an ECM protein that is diffusely expressed in basement membranes and best known by its degradation product endostatin (ES). ES, a 20-kDa C-terminal fragment, is generated by cleavage of the ECM involving matrix metalloproteases, cathepsins, 2 and elastases. 3 ES is a potent angiogenesis inhibitor preventing neovascularization in vitro as well as in vivo. This effect is mediated by the inhibition of proliferation and migration and the enhancement of apoptosis in endothelial cells. 4,5 In vivo, ES was reported to prevent tumor growth and metastasis, 6 suggesting a promising therapeutic potential as anticancer agent. The biological effects of ES are mainly attributed to its antagonism to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) signaling. 7 ES was found to bind to the VEGF receptor kinase-inserted domaincontaining receptor (KDR), thereby blocking tyrosine phosphorylation and the activation of mitogen-activated protein kinase (MAPK), wh...

Endostatin lowers blood pressure via nitric oxide and prevents hypertension associated with VEGF inhibition

“…These cell types regulate vascular tone and BP. This finding is consistent with previous studies showing that ES induces NO release by cultured endothelial cells and relaxation of ex vivo vascular rings (29,30). Further, we show that circulating nitrate levels were higher in ES-treated mice than in controls (Fig.…”

“…This result is consistent with other studies showing that short-term exposure of bovine aortic endothelial cells to ES induced events associated with eNOS activation (30). Using single-cell measurements, Wenzel et al (29) showed that ES induced increases in NO production by human and murine endothelial cells that were blocked by an eNOS inhibitor. Ex vivo studies showing vessel relaxation suggested that this mechanism might be physiologically relevant.…”

“…Endogenous and Exogenous ES Increases NO. Physiologic concentrations of ES have been shown to increase NO production in endothelial cells and to decrease vascular tone in ex vivo studies (29). We found that murine Fc-ES significantly increased nitrite production in microvascular (MS1) endothelial cells (P = 0.005) ( Fig.…”

“…Identifying an agent that can mitigate the hypertension and augment the antiangiogenic effects of VEGF inhibitors would address this pressing clinical need. ES may be uniquely suited for this purpose, being an angiogenesis inhibitor shown to increase NO bioavailability in vitro (27)(28)(29)(30).…”

“…Although the molecular pathways are not fully defined, major effects of ES signaling include inhibition of endothelial cell migration and survival and angiogenesis. In addition, ES induces NO release by cultured endothelial cells and relaxation of ex vivo vascular rings (29,30). Down syndrome patients have an extra copy of chromosome 21 and a negligible incidence of solid tumors (31).…”