ELSEVIER Neuroscience Letters 197 (1995) 219-222 NEUROSCIEHC[ LETTERS Evidence for the role of protein kinase C in astrocyte-induced proliferation of rat cerebromicrovascular endothelial cells Danica B. Stanimirovic*, Rita Ball, Jon P. Durkin Cellular Neurobiology Group, Institute for Biological Sciences, National Research Council of Canada, Montreal Road Campus, Bldg. M-54, Ottawa, ONT, KIA OR6, Canada Received 9 June 1995; revisedversion received4 August 1995; accepted9 August 1995 Abstract The proliferation of cerebral endothelial cells is a crucial step in neural angiogenesis and is a process responsive to changes in the surrounding environment. Serum-free medium conditioned by rat cortical astrocytes was found to accelerate DNA synthesis, induce transient activation of protein kinase C (PKC), and increase the endogenous phosphorylation of the PKC-specific substrate, the 85 kDa MARCKS protein, in rat cerebromicrovascular endothelial cells (RCEC). The stimulatory factor(s) in astrocyte conditioned media (ACM) were heat- and trypsin-sensitive and found to have an apparent molecular weight greater than 10 kDa. The potent PKC activa- tor, 12-O-tetradecanoyl phorbol 13-acetate (TPA), also stimulated RCEC proliferation, whereas the inhibition of PKC by staurosporine caused a concomitant loss in ACM-induced PKC translocation, MARCKS protein phosphorylation and DNA synthesis. These findings implicate PKC activation as a critical early event in cerebral endothelial cell proliferation triggered by astrocyte-derived mitogen(s). Keywords: Cerebromicrovascular; Endothelium; Protein kinase C; DNA synthesis; MARCKS protein; Astrocyte; Rat Neural capillary angiogenesis is a crucial process in the microvascular response to development, ischemia, trauma or neoplasia of the brain. During angiogenesis, endothelial cells undergo distinct phenotypic changes which cause them to proliferate, to become highly 'in- vasive', and to interact fastidiously with other cells and the surrounding extracellular matrix [2]. Cerebral endothelial cells lining microvessels are en- sheathed by abluminally localized astrocytic processes. The extent to which astrocytes influence angiogenic transformation of cerebral endothelial cells in vivo re- mains unknown. However, astrocytes have been shown to induce and maintain critical aspects of the blood-brain barrier (BBB) phenotype of cultured cerebral endothelial cells [3] and express and release various trophic and an- giogenic factors such as fibroblast growth factor [7]. It has also been demonstrated that the formation of capil- lary-like tubular structures by cerebral endothelial cells in culture, an in vitro model of the angiogenic process in vivo, can be induced under the combined influence of extracellular matrix and, still unidentified, astrocyte- derived factor(s) [9]. * Corresponding author, Tel.: +1 613 9933730; Fax: +1 613 9414475; E-mail: danica@biologym54.1an.nrc.ca. Elsevier Science IrelandLtd. SSDI 0304-3940(95)11941-L To more clearly understand the influence of astrocytes on endothelial cell function, it is important to elucidate the effects astrocytes have on endothelial cell signaling. Protein kinase C (PKC) is a key signaling enzyme which regulates a myriad of cellular processes including cell cycle progression and proliferation [5]. Previous studies have demonstrated that the activation of PKC in cerebral endothelial cells stimulates fluid-phase endocytosis [8], secretion of vasoactive prostaglandins [14], and phospho- rylation of endothelial cytoskeletal proteins (i.e. F-actin, caldesmon, and vimentin) (reviewed in Ref. [11]). In this study we provide evidence that astrocyte-derived factor(s) stimulate DNA synthesis in rat cerebromicrovascular en- dothelial cells (RCEC) by a mechanism that likely in- volves the early activation of PKC and the PKC-mediated phosphorylation of endothelial cell proteins. Cerebral capillaries and microvessels were isolated from the brains of 2-4 days old Sprague-Dawley rats by separation on 20% dextran and sequential filtration through 350, 112, and 20/lm Nitex meshes [6] and cul- tured in rat-tail collagen-coated tissue culture dishes in complete medium (M199, Gibco BRL, supplemented with 1% basal media Eagle's (BME) amino acids, 1% BME vitamins, 1% glucose, 0.05% peptone) containing 20% FBS. Cultures of highly purified RCEC were ob-