The increased tubulogenesis of human LECs in response to LPS is demonstrated in Fig. 2B. Transfection of TLR4 siRNA

into human LECs significantly inhibited basal tubulogenesis in comparison with the transfection of a scrambled siRNA control (Fig. 2C,D; P < 0.05; the inset western blot depicts siRNA knockdown of the doublet TLR4 protein band as previously described27). Additionally, the reduction in tubulogenesis was not due to cell toxicity; this was INCB018424 solubility dmso assessed by the staining of cells in Matrigel with the cell viability dye calcein AM (Supporting Fig. 2A,B). Similar results were also obtained with a second TLR4 siRNA recognizing a distinctly different region of human TLR4 mRNA (Fig. 2C). In these and ensuing in vitro AZD2014 molecular weight experiments of tubulogenesis conducted on Matrigel,

we observed prominent effects of experimental interventions on basal responses in the absence of LPS, and these were likely due to endogenous TLR4 ligands present within matrix-rich environments such as Matrigel.28-30 Therefore, the data are depicted as basal responses to Matrigel rather than the addition of exogenous LPS. These complementary genetic and molecular approaches provide evidence that TLR4 promotes angiogenesis in LECs in vitro. TLR4 signaling in response to LPS may occur by an MyD88-dependent or MyD88-independent, TRAM-dependent pathway.6 To identify the pathway that mediates the angiogenic signals of TLR4, we overexpressed MyD88 with a retroviral construct in human LECs. MyD88 overexpression in human LECs significantly enhanced tubulogenesis in comparison with cells transduced with a control retrovirus (Fig. 3A). To confirm specificity, we transfected human LECs with MyD88 siRNA or control siRNA. Basal tubulogenesis was reduced in LECs transfected with MyD88 siRNA

in comparison with control siRNA (P < 0.05; Fig. 3B) in the absence of siRNA-induced cell toxicity (Supporting Fig. 2C,D). To further confirm whether TLR4-dependent angiogenesis occurs through MyD88 function, BCKDHA we blocked MyD88 homodimerization with the peptide IMG-2005-1 and thus blocked MyD88 function.19 The MyD88 inhibitory peptide attenuated tubulogenesis in human LECs in comparison with a vehicle control peptide (Fig. 3C). Furthermore, overexpression of a dominant-negative, N-terminal truncated form of MyD88 also significantly reduced tubulogenesis (Fig. 3D). To further link TLR4 signals through MyD88, we silenced MyD88 in human LECs and returned to the LPS stimulation model. Indeed, silencing of MyD88 reduced LPS-mediated tube formation in comparison with control siRNA, and this suggested that angiogenic signaling in these cells requires MyD88 activation downstream of TLR4 (Fig. 3E). Conversely, a small and not statistically significant difference in tubulogenesis was observed through the silencing of TRAM with siRNA (Supporting Fig. 3). In all, these studies using multiple complementary approaches indicate that TLR4-dependent tubulogenesis is mediated through MyD88.