We therefore created a retroviral transduction system to stably overexpress AQP-1 in vitro (pMMP-AQP1). IF (Fig. 3A) and western blotting (Fig. 3B) demonstrated robust overexpression of AQP-1 after treatment with pMMP-AQP1 compared with pMMP-LacZ controls, providing a mechanistic

in vitro model in which to study the biological effects of AQP-1. Based on our a priori hypothesis that AQP-1 promotes angiogenesis, we speculated that AQP-1 up-regulation would increase LEC motility. We therefore tested the effects of AQP-1 overexpression on LEC chemotaxis using modified Boyden chamber chemotaxis assays. However, contrary to our initial hypothesis, we found that after AQP-1 overexpression with pMMP-AQP1, traditional chemotaxis in LEC was actually reduced compared with LacZ controls, Etoposide order both in the basal state and in response to FGF (Fig. 4A). Similar results were Selleck Selumetinib observed using human hepatic sinusoidal endothelial cells, various chemotactic

agents, and both adenoviral and retroviral overexpression (Supporting Fig. 2A). Using AQP-1-specific siRNA or scrambled siRNA, we found, again, that AQP-1 expression was inversely correlated with HSEC chemotaxis in primary cells (Supporting Fig. 2B). Attempts to modulate AQP-1 function with chemical inhibitors, such as mercuric chloride, resulted in endothelial cell toxicity selleck chemicals llc and therefore were not pursued in greater depth (Supporting Fig. 3). Recent studies have revealed that, in the context of desmoplasia, cells frequently modify their migration pattern from a traditional, actin-based, mesenchymal mechanism, to a membrane deformation mechanism that has been referred to as ameboid motility.36 This invasive form of motility, although slower, is nonetheless, more adaptable in circumstances requiring cell shape deformation and dynamic membrane blebbing events to squeeze through confined areas. We hypothesized that the dense fibrotic ECM of the cirrhotic microenvironment requires invasion

and that LECs undergo mode-switching to a more primitive form of amoeboid motility in this setting. We therefore tested the effects of AQP-1 overexpression on FGF-induced endothelial cell invasion capacity using three-dimensional collagen invasion assays. In striking contrast to our chemotaxis results, we observed that AQP-1 overexpression in TSEC significantly increased both basal and FGF-induced invasion (Fig. 4B), suggesting that bleb-based amoeboid motility occurs in this setting. Because dynamic membrane blebbing is the hallmark of amoeboid motility,15 we used phase-contrast, time-lapse video microscopy to examine blebbing behavior in TSEC. We hypothesized that altered blebbing dynamics may contribute to amoeboid motility and explain the increased invasion capacity conferred by AQP-1. Phase contrast and SEM (Fig.