Modulation of Cyclooxygenase in Endothelial Cells by Fibronectin: Relevance to Angiogenesis R.I. Viji, V.B. Sameer Kumar, M.S. Kiran, and P.R. Sudhakaran * Department of Biochemistry, University of Kerala, Thiruvananthapuram, Kerala 695 581, India ABSTRACT Cyclooxygenases (COX), which catalyze the formation of prostaglandins (PGs), have been implicated in angiogenesis. Adhesion of endothelial cells (ECs) to extracellular matrix (ECM) induces the expression of COX-2 and PG production. The present study was carried out to analyze the influence of the adhesive ECM protein, fibronectin (FN), in modulating COX expression and its implications to angiogenesis using in vitro cultures of human umbilical vein ECs. RT-PCR analysis showed that the level of COX-2 mRNA was significantly high while that of COX-1 decreased in ECs maintained on FN. On treatment with p38 MAPK inhibitor and anti-a 5 b 1 integrin antibody, FN dependent effect on COX expression was not observed. Analysis by ELISA and immunoblotting confirmed FN-dependent upregulation of COX-2 protein. The ratio of PG E 2 :PG D 2 was significantly high in cells maintained on FN and on treatment with p38 MAPK inhibitor, the relative level of PG D 2 increased and that of PG E 2 decreased. Concomitant with the modulation of COX-2 and changes in PGs, ECs maintained on FN showed angiogenic response in an a 5 b 1 integrin/p38 MAPK dependent manner as evidenced by the expression of angiogenic markers, CD 31 and E-selectin. These results suggest a FN-a 5 b 1 /FAK/p38 MAPK dependent upregulation of COX-2 causing a shift in the relative levels of PGs in HUVECs which contributes to the angiogenic effect of FN. J. Cell. Biochem. 105: 158–166, 2008. ß 2008 Wiley-Liss, Inc. KEY WORDS: HUVECs; FIBRONECTIN; COX-2; PROSTAGLANDINS; VEGF A ngiogenesis is the process by which new microvessels develop and grow from existing, quiescent vascular endothelium. Different cell types, extracellular matrix (ECM), growth factors and several small molecules interact in a complex manner and temporal sequence in the process of angiogenesis. Many molecules influencing angiogenesis have been identified [Yancopoulos et al., 2000], of which, inflammatory mediators are reported to contribute to the angiogenic process associated with pathological conditions such as metastatic cancer, rheumatoid arthritis, and diabetic retinopathy. Some of the inflammatory prostaglandins (PGs) are implicated in altered vascular permeability and angiogenesis [Ziche et al., 1982; Form and Auerbach, 1983]. PGs are synthesized from arachidonic acid (AA) by the action of cyclooxygenases (COX), which are the products of two distinct genes, referred to as COX-1 and COX-2. These isoforms catalyze the first two steps in prostanoid biosynthesis [Marnett and Kalgutkar, 1999]. They share about 60% homology at the amino acid level and have similar enzymatic activities, but have distinct biological functions [Tazawa et al., 1994; Williams and DuBois, 1996]. COX-1 expression is constitutive in many tissues whereas COX-2 expression is induced by inflammatory cytokines [Diaz et al., 1998]. The COX-2 of endothelial cells (ECs) is important in angiogenesis [Hull et al., 1999; Williams et al., 2000]. Over- expression of COX-2 appears to promote tumorigenesis, whereas non-steroidal anti-inflammatory drugs and COX-2 specific inhibi- tors suppress tumorigenesis and tumor progression [Dubois et al., 1998; Ruegg et al., 2003]. COX-2 is highly expressed in the tumor cells, activated stromal fibroblasts, infiltrating leukocytes, and angiogenic ECs. Hypoxia [Schmedtje et al., 1997], inflammatory cytokines [Nakagawa et al., 1999; Uracz et al., 2002], and angiogenic growth factors such as VEGF [Tamura et al., 2002] induce expression of COX-2 in ECs. ECM is a key component involved in regulating the process of angiogenesis. For ECs to enter avascular tissue they must first detach themselves from basement membrane through limited, focal proteolysis and during the later invasive and proliferative phases of angiogenesis, the ECs undergo extensive interactions with a fibronectin (FN) rich matrix [Risau and Lemmon, 1988]. ECs interact Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 105:158–166 (2008) 158 Grant sponsor: CSIR, Government of India; Grant sponsor: DST-FIST, Government of India; Grant sponsor: UGC-SAP, New Delhi. *Correspondence to: P.R. Sudhakaran, Department of Biochemistry, University of Kerala, Thiruvananthapuram, Kerala 695 581, India. E-mail: prsbn@md4.vsnl.net.in Received 18 October 2007; Accepted 7 April 2008  DOI 10.1002/jcb.21808  2008 Wiley-Liss, Inc. Published online 5 May 2008 in Wiley InterScience (www.interscience.wiley.com).