Quantification of the gelatinolytic areas was measured with ImageJ (National Institutes of Health, Bethesda, MD). Frozen liver sections from TLR4-WT and TLR4-MT mice were incubated in 1% agarose fortified with fluorescent gelatin (Molecular Probes, Invitrogen). The sections were then incubated at 37°C in a substrate

development buffer, and ethylene diamine tetraacetic acid was used as a negative control as previously described.25 Primary LECs were cultured for 24 and 48 hours and subsequently incubated with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent (Promega, Madison, WI). The absorbance of the plate was read colorimetrically at an optical density of 490 nm. ABT-199 manufacturer Standardization and other steps were performed according to the manufacturer’s instructions. Data are expressed as means and standard errors of the mean (SEMs) of at least three independent NVP-LDE225 experiments. Groups were compared by a two-tailed Student t test. A P value less than 0.05 was considered statistically significant. As a first step in exploring a role for TLR4 in liver fibrosis–associated angiogenesis, we determined

the expression of TLR4 in LECs from both humans and mice. Confirming prior studies,26 quantitative RT-PCR analysis detected TLR4 mRNA levels in both human and murine LECs; levels were substantially elevated in comparison with other systemic human endothelial cells such as human umbilical vein endothelial cells (HUVECs), although they were less elevated than the levels of lymphocyte-positive control Raji cells (Fig. 1A). This observation was substantiated by detection of a specific immunofluorescence signal for TLR4 in isolated mouse and human LECs (Fig. 1B); this 上海皓元医药股份有限公司 indicated that TLR4 was expressed in both

murine and human LECs. Although other TLR molecules were also expressed within LECs (data not shown), they were not pursued in further detail in this work. Instead, the present study was focused in a hypothesis-based manner on the recognition of TLR4 by LPS and its potential relevance to liver injury, fibrosis, and vascular integrity due to the proposed links of LPS with these processes. Reorganization of endothelial cells into tubelike vascular structures in Matrigel, which is called tubulogenesis, provides an in vitro estimation of the angiogenic capacity of vascular cells because a number of steps required for angiogenesis in vivo are required for tubulogenesis in vitro.15 To test TLR4 functional relevance in angiogenesis, we isolated LECs from TLR4-MT or TLR4-WT mice and measured tubulogenesis. As shown in Fig. 2A,B, although LPS prominently stimulated tubulogenesis in WT mice, both basal tubulogenesis and LPS-stimulated tubulogenesis were markedly attenuated in TLR4-MT mice. The antibiotic polymyxin-B inhibited tubulogenesis in all groups, and this further supports the role of basal LPS and TLR4 in this process.