Peroxisome Proliferator-Activated Receptor Activation- Mediated Regulation of Endothelin-1 Production via Nitric Oxide and Protein Kinase C Signaling Pathways in Piglet Cerebral Microvascular Endothelial Cell Culture

Elevated endothelin (ET)-1 has been implicated in cerebrovascular complications following brain trauma characterized by dysregulation of endothelial nitric oxide synthase (eNOS), protein kinase C (PKC), and cerebral function. Recently, vascular expression of PPAR has been observed and suggested to improve vascular dysfunction. We speculate that activation of PPAR in cerebral microvessels can improve cerebral dysfunction following trauma, and we tested the hypothesis that activation of cerebral endothelial peroxisome proliferator-activated receptor (PPAR) will attenuate ET-1 production via a mechanism involving nitric oxide (NO) and PKC. Phorbol 12-myristate 13-acetate (PMA) (1 M), bradykinin (BK, 1 M), angiotensin II (AII, 1 M), or hemoglobin (Hem, 10 ) increased ET-1 levels by 24-, 11.4-, 3.6-, or 1.3-fold increasing ET-1 levels from 0.36 0.08 to 8.6 0.8, 4.1 0.7, 1.30 0.1, or 0.47 0.03 fmol/ g protein (p 0.05), respectively. Clofibrate (10 M) reduced basal ET-1 from 0.36 0.08 (control) to 0.03 0.01 and blunted vasoactive agent-induced increase to 0.12 0.07 (PMA), 0.6 0.04 (BK), 0.25 0.03 (AII), or 0.12 0.03 (Hem) fM/ g protein (p 0.05). L-Arginine methyl ester (100 M) inhibited clofibrate-induced reduction in basal ET-1 production. Clofibrate increased PPAR expression, accompanied by increased NO production and eNOS expression. PKC inhibition by calphostin C (10 M) blocked these effects, whereas activation by PMA reduced basal PPAR expression. Thus, PPAR activation attenuated ET-1 production by agents that mediate brain injury through mechanisms that probably result from PPAR -induced increase in eNOS expression/NO production and complex PKC signaling pathways. Therefore, PPAR activators can be appropriate therapeutic agents to alleviate cerebrovascular dysfunction following cerebral vasospasm. Pathogenesis of hemorrhage-induced cerebral dysfunction has been reported to result from potent and prolonged cerebral microvascular constriction. Cerebral arterial vasoconstriction following brain trauma has been associated with increased CSF concentration of blood derived vasoactive agents and endothelin (ET)-1 (Findlay et al., 1991; Yakubu and Leffler, 1996, 1999; Andaluz et al., 2002). ET-1, a 21amino acid peptide, has been implicated in brain injury and stroke-induced cerebral dysfunction (MacDonald and Weir, 1991; Yakubu and Leffler, 1996). The etiology of cerebral trauma such as subarachnoid hemorrhage (SAH)-induced alteration of cerebral microcirculation and vasospasm can result from hemolysis of blood clots leading to accumulation of ET-1 and other vasoactive agents such as leukotrienes, 5-hydroxytryptamine, thromboxane A2, lysophosphatidic acid, oxyhemoglobin, etc. (Findlay et al., 1991; Yakubu and Leffler, 1996; Yakubu et al., 1997). These vasoactive agents have been shown to stimulate ET-1 biosynthesis from vascular endothelial cells via activation of PKC and Ca (Yakubu and Leffler, 1999, 2002). In addition, diminished CSF levels of dilator prostanoids and nitric oxide (NO) have been observed following SAH (Findlay et al., 1991; Yakubu et al., 1994, 1995, 1997), and such reduction can result in high level of CSF ET-1 (Boulanger and Luscher, 1990; Yakubu and Leffler, 1997, 1999). Evidence for an important role for ET-1 This work was supported by the National Heart, Lung, and Blood Institute (Grants HL03674 and HL070669) and by the use of Texas Southern University Research Center for Minority Institute facilities. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.104992. ABBREVIATIONS: CSF, cerebrospinal fluid; ET, endothelin; SAH, subarachnoid hemorrhage; PKC, protein kinase C; NO, nitric oxide; PPAR, peroxisome proliferator-activated receptor; CMVEC, cerebral microvascular endothelial cell; L-NAME, L-arginine methyl ester; PMA, phorbol 12-myristate 13-acetate; eNOS, endothelial nitric-oxide synthase; FBS, fetal bovine serum; AII, angiotensin II; Hem, hemoglobin; BK, bradykinin; NFM/TBS, nonfat milk in Tris-buffered saline; ANOVA, analysis of variance. 0022-3565/07/3202-774–781$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 104992/3172307 JPET 320:774–781, 2007 Printed in U.S.A. 774 at A PE T Jornals on O cber 8, 2017 jpet.asjournals.org D ow nladed from in cerebral microcirculation is based on the demonstration that ET-1 antagonists and agents that interfere with its biosynthesis ameliorated the consequences of SAH-induced cerebral microvascular dysfunction (Yakubu and Leffler, 1996; Sobey and Faraci, 1998). Despite this important observation, therapeutic strategies to treat brain trauma-induced cerebral deficit are still lacking (Liu-Deryke and Rhoney, 2006; MacDonald, 2006). Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily that comprises three members, , , and / . All PPARs are widely distributed and activated by fatty acids to varying degrees. PPAR is widely expressed in tissues where fatty acid catabolism is important and regulates genes that are involved in lipid and lipoprotein metabolism (Ziouzenkova and Plutzky, 2004; Han et al., 2005). Of interest in this study is the PPAR that is highly expressed in endothelial and vascular smooth muscle cells (IsraelianKonaraki and Reaven, 2005) and activated by natural ligands, including polyunsaturated fatty acids, such as docosahexanoic acid and eicosapentaenoic acid, and lipolytic and synthetic ligands, including fibrates, such as fenofibrate, clofibrate, and gemfibrozil (Desvergne and Wahli, 1999). Activation of PPARs results in heterodimerization with another nuclear receptor partner retinoid X receptor, and the complex binds to specific PPAR-response elements in the promoter region of their target genes, thereby regulating gene function, through repression or activation of gene expression. PPARs can also repress gene expression in a DNA-bindingindependent fashion by interfering with other signaling pathways, such as PKC via a mechanism termed trans-repression (Blanquart et al., 2004). In addition to interfering with endothelial cell inflammatory mediators, PPAR has been reported to modulate endothelial NOS-induced NO production and NOS expression (Goya et al., 2004, Newaz et al., 2004), implying a possible vasculoprotective effect. PPAR activators have also been reported to reduce agonist-stimulated ET-1 expression and production (Irukayama-Tomobe et al., 2004) and to improve peripheral vascular function (Chinetti-Gbaguidi et al., 2005; Touyz and Schiffrin, 2006), but the mechanism(s) involved is not fully understood. However, studies suggest possible involvement of PKC and increased generation of endothelium-derived NO in the actions of PPAR (Blanquart et al., 2004; Goya et al., 2004). Although we are not aware of studies that demonstrate the expression of PPAR in cerebral microvasculature, there is a possibility that activation of cerebral microvascular endothelial cell PPAR could modulate cerebral function via stimulation of endothelial NO production and/or inhibition of ET-1-mediated vascular dysfunction through a mechanism that may involve PKC. PKC involvement in the modulation of NO and ET-1 production (Yakubu and Leffler, 1999; Ramzy et al., 2006) as well as in the actions of PPAR (Paumelle et al., 2006) has been reported. Therefore, we tested the hypothesis that activation of PPAR will attenuate ET-1 production from the cerebral microvascular endothelial cell (CMVEC) by a mechanism involving NO production and PKC activation. Materials and Methods Materials. Reagents used in the present study were obtained from the following companies: bradykinin, angiotensin II, clofibrate, hemoglobin, L-arginine methyl ester (L-NAME), endothelial growth supplement, Percoll, and Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO); calphostin C and PMA (Calbiochem, San Diego, CA), polyclonal antibodies for PPAR and endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Santa Cruz, CA); Endothelin-1 Kit (ALPCO Diagnostics, Winham, NH); Matrigel and cell culture plates (BD Biosciences, San Jose, CA); and fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA). Primary Cultures of Cerebral Microvascular Endothelial Cells. Primary cultures of cerebral microvascular endothelial cells from piglet brain were established as described previously (Yakubu and Leffler, 1999, 2005). In brief, cerebral cortical microvessels (60– 300 m) were isolated by differential filtration of cerebral cortex homogenate, first through a 300m and then through a 60m nylon mesh screen. The isolated microvessels were incubated in collagenase-Dispase solution (1 mg/ml) for 2 h at 37°C. At the end of the incubation, the dispersed microvascular endothelial cells were separated using Percoll density gradient centrifugation. Endothelial cells were resuspended in culture medium consisting of 20% FBS, 2 mg/ml sodium bicarbonate, 1 U/ml heparin, 30 mg/ml endothelial cell growth supplement, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin B. Endothelial cells were plated on 12-well Costar plates coated with Matrigel and maintained in a 5% CO2-95% air incubator at 37°C. The culture medium was changed every 2 to 3 days until cells attained confluence. Confluent cells were starved overnight with 1% FBS-conditioned media and used for the