Abstract
Abstract 3265
An important role for Endothelin-1 (ET-1) has been proposed in patients with sickle cell disease (SCD). We, and others, have reported that endothelin-1 (ET-1) receptor antagonists in vivo ameliorate numerous complications observed in sickle transgenic mice through mechanisms that remain unclear. ET-1 is a vasoconstrictor and important regulator of endothelial cells migration that promotes structural changes in blood vessels in response to increases in vasoactive hormones, growth factors, hypoxia, shear stress and free radicals; events commonly observed in patients with SCD. Endothelial cell activation is characterized in part by increases of cytokines and growth factors that are important in vascular maintenance and fibrogenesis such as connective tissue growth factor (CTGF). CTGF plays a role in blood vessel remodeling and fibrogenesis. Indeed, there is in vitro evidence that ET-1 stimulates the production of CTGF in smooth muscle cells. We hypothesized that increasing ET-1 would stimulate CTGF production and lead to endothelial cell activation. We studied the effects of ET-1 on the human endothelial cell line, EA.hy926 (EA), as well as in primary cultures of mouse aortic endothelial cells (MAEC). We analyzed the effects ET-1 on cytosolic Ca2+ levels by spectrofluorimetry of FURA-2AM loaded EA cells and noted that, as observed in fibroblasts, incubation with 10−8 M ET-1 led to increases in cytosolic Ca2+ levels that were blocked by pre-incubation with the ET-1 receptor B antagonist, BQ-788. We performed CTGF gene expression time course experiments (0, 2, 4, 8, 16, 24 Hr) on EA cells following incubation with 100 nM ET-1 using quantitative RT-PCR with TaqMan detection probes and GAPDH and beta-actin as endogenous controls. We observed increases of CTGF and VEGF expression between 4 and 8 hr for CTGF (1.74 fold increase vs time 0, n=6, P<0.03) and 4 hr for VEGF (2.14 fold increase vs time 0, n=3, P<0.04). Additional experiments on EA cells showed that incubation with 100 nM ET-1 for 4 hr in the presence of BQ123 and BQ788, two inhibitors of ET-1 type A and B receptors, respectively, blocked the ET-1 stimulated rises in CTGF and VEGF mRNA expression. We then performed western blot analyses and showed increases in cell-associated CTGF protein levels following incubation of EA cells with 10 nM ET-1 for 24 hr that were blocked by both BQ123 and BQ788. We then studied the effect of ET-1 on CTGF production in cell supernatants at various dose and time points by sandwich ELISA immunoassay. We observed increased levels of CTGF in cellular supernatants after 48 h of exposure that peaked at 10 nM ET-1 as compared to 24 h, 12 h and vehicle treated cells (P<0.001). To study whether the effects of ET-1 were unique to EA cells, we also analyzed the effects of ET-1 on early cultures of MAEC. Consistent with our observations in human EA cells, incubation of MAEC with 100 nM ET-1 for 4 hr was associated with increases of CTGF (1.86 fold vs vehicle, n=3, P<0.03) and was blocked by pre-incubation with BQ123 and BQ788. As CTGF has been shown to be chemotactic as well, we studied the effect of supernatants isolated from EA cells following stimulation with 10 nM ET-1 for 48 hrs on ex vivo human mononuclear cells (MNC) isolated from otherwise healthy subjects. We determined MNC transmigration by Cyquant fluorescence and observed enhanced migration of cells exposed to supernatants from ET-1-stimulated cells when compared to vehicle treatment that was significantly blunted by BQ123 and BQ788 pre-incubation. Further western blot analyses of the MNC that migrated following incubation with ET-1-stimulated supernatants showed the presence of CTGF in the MNC that otherwise do not express CTGF. Thus, we conclude that ET-1 leads to activation of endothelial cells and increases in CTGF from endothelial cells and suggest that therapies designed to block ET-1 receptors will also reduce endothelial cell activation in part by reducing CTGF production leading to alterations in cellular and tissue architecture.
Disclosures:
No relevant conflicts of interest to declare.
Fisp12/Mouse Connective Tissue Growth Factor Mediates Endothelial Cell Adhesion and Migration through Integrin αvβ3, Promotes Endothelial Cell Survival, and Induces Angiogenesis In Vivo
