The BMP/TGFβ-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.

Noncovalent and electrostatic interactions facilitate the formation of complex networks through molecular self-assembly in biomolecules such as proteins and glycosaminoglycans. Self-assembling peptide amphiphiles (PA) are a group of molecules that can form nanofibrous structures and may contain bioactive epitopes to interact specifically with target molecules. Here, we report the presentation of cationic peptide sequences on supramolecular nanofibers formed by self-assembling peptide amphiphiles for cooperative enhanced antibacterial activity. Antibacterial properties of self-assembled peptide nanofibers were significantly higher than soluble peptide molecules with identical amino acid sequences, suggesting that the tandem presentation of bioactive epitopes is important for designing new materials for bactericidal activity. In addition, bacteria were observed to accumulate more rapidly on peptide nanofibers compared to soluble peptides, which may further enhance antibacterial activity by increasing the number of peptide molecules interacting with the bacterial membrane. The cationic peptide amphiphile nanofibers were observed to attach to bacterial membranes and disrupt their integrity. These results demonstrate that short cationic peptides show a significant improvement in antibacterial activity when presented in the nanofiber form.

Super-enhancers are clusters of enhancers associated with cell lineage. They can be powerful gene-regulators and may be useful in cell-type specific viral-vector development. Here, we have screened for endothelial super-enhancers and identified an enhancer from within a cluster that conferred 5–70-fold increase in transgene expression. Importantly, CRISPR/Cas9 deletion of enhancers demonstrated regulation of ADAMTS18, corresponding to evidence of chromatin contacts between these genomic regions. Cell division-related pathways were primarily affected by the enhancer deletions, which correlated with significant reduction in cell proliferation. Furthermore, we observed changes in angiogenesis-related genes consistent with the endothelial specificity of this SE. Indeed, deletion of the enhancers affected tube formation, resulting in reduced or shortened sprouts. The super-enhancer angiogenic role is at least partly due to its regulation of ADAMTS18, as siRNA knockdown of ADAMTS18 resulted in significantly shortened endothelial sprouts. Hence, functional characterization of a novel endothelial super-enhancer has revealed substantial downstream effects from single enhancer deletions and led to the discovery of the cis-target gene ADAMTS18 and its role in endothelial function.

Objective: Previous studies have demonstrated that the expression of several lysine (K)-specific demethylases (KDMs) is induced by hypoxia. Here, we sought to investigate the exact mechanisms underlying this regulation and its functional implications for endothelial cell function, such as angiogenesis. Approach and Results: We analyzed the expression changes of KDMs under hypoxia and modulation of HIF (hypoxia-inducible factor) expression using GRO-Seq and RNA-Seq in endothelial cells. We provide evidence that the majority of the KDMs are induced at the level of nascent transcription mediated by the action of HIF-1α and HIF-2α. Importantly, we show that transcriptional changes at the level of initiation represent the major mechanism of gene activation. To delineate the epigenetic effects of hypoxia and HIF activation in normoxia, we analyzed the genome-wide changes of H3K27me3 using chromosome immunoprecipitation-Seq. We discovered a redistribution of H3K27me3 at ≈2000 to 3000 transcriptionally active loci nearby genes implicated in angiogenesis. Among these, we demonstrate that vascular endothelial growth factor A ( VEGFA ) expression is partly induced by KDM4B- and KDM6B-mediated demethylation of nearby regions. Knockdown of KDM4B and KDM6B decreased cell proliferation, tube formation, and endothelial sprouting while affecting hundreds of genes associated with angiogenesis. These findings provide novel insights into the regulation of KDMs by hypoxia and the epigenetic regulation of VEGFA-mediated angiogenesis. Conclusions: Our study describes an additional level of epigenetic regulation where hypoxia induces redistribution of H3K27me3 around genes implicated in proliferation and angiogenesis. More specifically, we demonstrate that KDM4B and KDM6B play a key role in modulating the expression of the major angiogenic driver VEGFA.