Abstract 1679: Hypoxia and ECM Proteins Influences Angiogenesis and Lymphangiogenesis in Mouse Embryoid Bodies
Regulatory mechanisms for angiogenesis are relatively well established compared to lymphangiogenesis. Few early markers that distinguish lymphatic lineage committed endothelial cells (LECs) have been identified and include Lymphatic Endothelial Hyaluronan Receptor (LYVE1) and Prospero-related Homeobox 1 (Prox1). Hypoxia promotes angiogenesis while also upregulating VEGFR3 and VEGFR3 and its ligand VEGF-C induces LEC differentiation. This study tests if hypoxia in combination with prolonged VEGF-A/C treatment could induce lymphangiogenesis in addition to angiogenesis in mouse embryoid bodies (EBs). EBs were subjected to four different conditions and were maintained under normoxic (N) and hypoxic (H) conditions (21% and 2.6% O2, respectively) with and without VEGF-A/C. Double immunofluorescence stains with Meca32 and Prox1/LYVE1 was performed on E22.5. Hypoxia and VEGF-A/C treatment had the most potent effect on forming blood vessel and few lymphatic capillary-like structures (Figure A⇓). Furthermore we investigated the influences of extracellular matrix (ECM) proteins like laminin and collagen on angiogenesis and lymphangiogenesis in developing EBs. EBs were grown on coverslips with laminin or collagen-I coating and subjected to the four different conditions as described above. Double staining on E36.5 revealed that collagen-I promoted better attachment of LECs resulting in a dense network of lymphatic vessels as compared to laminin (Figure B⇓). Collectively, our data show that hypoxic microenvironments combined with growth factors and ECM proteins could induce early lymphangiogenesis processes in EBs.