Dissecting VEGF-Induced Acute Versus Chronic Vascular Hyperpermeability: Essential Roles of Dimethylarginine Dimethylaminohydrolase-1 (preprint)

Abnormal vascular leakiness plays crucial roles in several pathogenesis including cancer, macular degeneration, cardiovascular diseases and even in COVID-19-related pulmonary edema. Vascular endothelial cell growth factor (VEGF), also well-known as vascular permeability factor (VPF), activates VEGF receptor 2 (VEGFR2) and its downstream signaling leading to both acute and chronic vascular permeability. While many key downstream components are identified in cultured cells and murine models upon acute VEGF administration, the key molecular mediators of VEGF-induced chronic permeability remain unclear due to lack of appropriate in experimental model. Here we demonstrated that chronic VEGF exposure induces unique pattern of signaling as compared to that of acute exposure, including phosphorylation of VEGFR2, c-Src, and steady increases of nitric oxide (NO). Utilizing heat-inducible VEGF transgenic zebrafish (Danio rerio) and establishing an algorithm incorporating segmentation techniques to precisely quantify the extravasated dye, we monitored acute and chronic VEGF-induced vascular hyperpermeability in real time. Importantly, knockdown of dimethylarginine dimethylaminohydrolase-1 (DDAH1), an enzyme essential for NO generation, significantly altered downstream signaling pathways due to both acute and chronic VEGF exposure in human endothelial cells and the VEGF-inducible zebrafish permeability model. Finally, the effect of DDAH1 on VEGF-induced hyperpermeability was confirmed using DDAH1 transgenic mice as well as DDAH1 knockout mice by Miles assays, which exhibited increased and decreased leakage of Evan’s blue dye, respectively. Taken together, our data reveal novel divergent signaling of VEGF-induced acute versus chronic hyperpermeability and identify DDAH1 as a critical player and potentially a therapeutic target of vascular hyperpermeability-mediated pathogenesis.

Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability (preprint)

ABSTRACT Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression, and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, such as cancer, ischemic stroke, cardiovascular disease, retinal conditions, and COVID-19-associated pulmonary edema and sepsis, which often leads to acute lung injury, including acute respiratory distress syndrome. However, after initially stimulating permeability, VEGF subsequently mediates angiogenesis to repair damaged tissue. Consequently, understanding temporal molecular regulation of VEGF-induced vascular permeability will facilitate developing therapeutics that achieve the delicate balance of inhibiting vascular permeability while preserving tissue repair. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. Specifically, we show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9 generated genomic STAT3 knockout zebrafish. Importantly, STAT3 deficiency does not impair vascular development and function in vivo. We identify intercellular adhesion molecule 1 (ICAM-1) as a STAT3-dependent transcriptional regulator and show VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved anti-microbial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse, and human endothelium. Indeed, pharmacologically targeting STAT3 increases vascular barrier integrity using two additional compounds, atovaquone and C188-9. Collectively, our findings suggest that the VEGF, VEGFR-2, JAK2, and STAT3 signaling cascade regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability in vertebrate models. Key Points Genetic STAT3 deficiency in VEGF-inducible zebrafish and mice reveals that VEGF signals through STAT3 to promote vascular permeability Pyrimethamine, a clinically available agent that inhibits STAT3 activity, reduces VEGF-induced vascular permeability in preclinical models