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...

COX-2 (cyclooxygenase-2) is a pivotal player in inflammatory processes, and ultraviolet radiation is a known stimulus for COX-2 expression in skin cells. Here, an induction of COX-2 expression in HaCaT human keratinocytes was observed only upon exposure of cells to UVB (280 -320 nm) but not to UVA radiation (320 -400 nm), as demonstrated by reverse transcription-PCR and Western blotting. Prostaglandin E 2 levels were elevated in cell culture supernatants of HaCaT cells exposed to UVB. COX-2 mRNA stability was dramatically increased by UVB irradiation. Both the stabilization of COX-2 mRNA and the enhancement of COX-2 steady-state mRNA and protein levels caused by UVB were prevented both by inhibition and small interfering RNA-induced depletion of p38 MAPK , a kinase strongly activated upon exposure to UVB, suggesting p38 MAPK -dependent mRNA stabilization as a mechanism of UVB-induced COX-2 expression. A dramatic decrease in COX-2 expression induced by UVB was elicited by small interfering RNA-based depletion of a stress-responsive mRNA stabilizing protein regulated by p38 MAPK , i.e. HuR; UVB-induced elevation of COX-2 mRNA and protein levels coincided with an accumulation of HuR in the cytoplasm and was attenuated in cells depleted of HuR. Moreover, UVB-induced generation of prostaglandin E 2 by HaCaT cells was blunted by HuR depletion, suggesting that stress kinases (such as p38 MAPK ) as well as HuR are excellent targets for approaches aiming at interfering with induction of COX-2 expression by UVB.Cyclooxygenases catalyze the rate-limiting step in prostaglandin biosynthesis, i.e. the conversion of arachidonic acid to prostaglandin (PG) 3 H 2 , which in turn is converted by various synthases to different prostaglandins or thromboxane A 2 , important mediators in inflammatory processes. Two genes coding for isoforms of cyclooxygenase (COX-1 and COX-2) are known (1). Although COX-1 and a COX-1 variant, termed COX-3 (2), are constitutively expressed, expression of COX-2 is strongly inducible by growth factors, cytokines, and other stimuli, resulting in the production of prostaglandins during inflammatory processes. One such potent stimulus for COX-2 induction is UV radiation. Both UVB (280 -320 nm) (3) and UVA (320 -400 nm) (4) were reported previously to enhance the expression of COX-2 in human keratinocytes, followed by an increased production of the inflammatory mediator PGE 2 , a major prostaglandin in skin. Analysis of the relative contributions of UV ranges to the effects of solar light on COX-2 levels demonstrated that UVB is a far more efficient inducer of COX-2 expression; for example, UVB and UVA-2 (320 -350 nm) but not UVA-1 (350 -400 nm) contributed to COX-2 induction by simulated solar light in artificial human epidermis (5). Several lines of evidence link COX-2 and PGE 2 to the development of UV-induced skin cancer, such as the findings that COX-2 and PGE 2 levels are elevated in skin cancer versus normal tissue, that PGE 2 is a promoting factor in skin carcinogenesis, and that depletion or inhib...

Abstract:Thalidomide as an effective treatment for multiple myeloma and leprosy has also caused birth defects in thousands of children five decades ago particularly in Europe. Thus its use in humans remains limited. The rapid and fatal approval of thalidomide at that time ultimately was a consequence of the sole use of thalidomide-insensitive species in animal toxicity tests. Here, we aimed at elucidating the molecular basis for the resistance of mice to thalidomide teratogenicity. By using hydroethidine staining we demonstrate that thalidomide induces the formation of superoxide in embryonic fibroblasts of thalidomide-sensitive species but not in those of mice. As determined by trypan blue staining, scavenging of superoxide prevents thalidomide-induced apoptosis, a marker for thalidomide teratogenicity. Mouse embryonic fibroblasts are found to have higher glutathione levels than those of sensitive species and can be sensitized for thalidomide by glutathione depletion with diethyl maleate or diamide. Accordingly, experimental increase of glutathione levels in human embryonic fibroblasts by adding N-acetyl cysteine or glutathione ethyl ester to the culture medium counteracts thalidomideinduced apoptosis. Finally, we show that thalidomide-induced molecular pathology downstream of superoxide is essentially identical in human and sensitized mouse embryonic fibroblasts. In conclusion, thalidomide-resistance is based on the capacity of the glutathione-dependent antioxidant defense. We provide a basis to pharmacologically overcome the limitations of thalidomide use at humans and describe substantial differences between human and mouse embryonic cells regarding the protection against oxidative stress.