Abstract A07: Knockdown laminin-511 expression blocked endlthelial cell function in vitro and angiogenesis in vivo knockout skin model

Laminin-511 (composed of alpha5, beta1 and gamma1 chains) is a major extracellular matrix component in the basement membrane of human skin dermal microvascular endothelial cells (HDMECs). In this study the functions of laminin-511 in angiogenesis were investigated using in vitro knockdown and in vivo knockout skin model. In the in vitro studies, normal primary HDMECs and RNA interfering technique (siRNA) to silence the gene expression strategy were used. A lentiviral-based delivery system was developed that expresses laminin alpha5 chain specific siRNA (pL1) or control LUC siRNA (pLUC). HDMECs transduced with Laminin alpha5 siRNA (HDMEC/pL1) or control (HDMEC/pLUC) were analyzed in cell attachment, migration and a tubule formation assays. The mRNA expressions of laminin alpha5 and integrins were examined by real-time RT-PCR. Focal adhesion kinase (FAK) activation was studied by Western blot. In the in vivo study, a knockout mouse skin angiogenesis model was used. Since laminin alpha5 homozygous knockout (-/-) is embryonic lethal in mouse, skin from laminin alpha5 (-/-) embryos was transplanted onto the back of nude mouse, excisional wounds were created and wound angiogenesis was analyzed. Our results demonstrated that knockdown laminin alpha5 expression significantly inhibited the ability of endothelial cells in attachment, migration and tubule formation in vitro and blood vessel growth in vivo. Cell adhesive ability of HDMEC/pL1 was reduced 60% during a 60 minute attachment assay. Cell mobility of HDMEC/pL1 was found remarkably decreased 83% and 71% in a 12-hour and 48-hour chamber migration assay respectively; and in consistence, cell migration rates in a scratch migration assay were 56% and 53% reduced in HDMEC/pL1 cells at 48 and 72 hours respectively. Moreover, depletion of laminin alpha5 prevented HDMEC from forming enclosed capillary-like tubular structure at later stage in a three-dimensional tubule formation assay. While, in vivo, homozygous laminin alpha5 knockout skin was associated with significantly reduced growth of new blood vessels. A simultaneous and significant down regulation of integrins alphaV and beta3 was found correlated with the laminin alpha5 knockdown and reduced rate of cell migration. Further, cells with laminin alpha5 knockdown failed FAK activation, FAK phosphorylation on tyrosine 397 changed little in HDMEC/pL1 cells while there was markedly increase in control cells. In conclusion, our data suggested a possible involvement of laminin-511 in integrin alphaV and beta3-dependent angiogenesis and blood vessel maturation. Our works revealed the important roles of laminin-511 in endothelial cell activities, which proved its significance for angiogenesis. Citation Format: Jie Li, Tengjiao Cui. Knockdown laminin-511 expression blocked endlthelial cell function in vitro and angiogenesis in vivo knockout skin model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A07.