Acute respiratory distress syndrome (ARDS) is a severe pulmonary disease characterized by acute, noncardiogenic pulmonary edema and hypoxemia leading to respiratory failure. It is induced by a diverse array of etiologies, including recent SARS-CoV-2 infection. The current standard of care for ARDS remains predominantly supportive, underscoring the urgent need for targeted pharmacological interventions. To address this critical gap, we developed an inhibitor of the microtubule accessory factor end-binding protein 3 (EB3), a key mediator of pathological calcium signaling in endothelial cells. During injury, EB3 facilitates inositol 1,4,5-trisphosphate receptor 3 (IP3R3) clustering on the endoplasmic reticulum membrane, activating widespread calcium release from intracellular stores and leading to endothelial barrier disruption. Using nuclear magnetic resonance (NMR)-guided approaches, we designed and optimized a synthetic EB3 inhibitor, termed vascular therapeutics (VT)-109, with enhanced physicochemical properties. We evaluated the therapeutic potential of VT-109 across a wide range of preclinical models in which pathogenic insults target epithelial or endothelial barriers. Treatment with VT-109 promptly restored the tissue‒fluid balance in the injured lung by inducing the reannealing of VE-cadherin junctions and restoring the endothelial barrier. In addition to vascular protection, VT-109 improved lung architecture and function, normalized immune responses, and significantly reduced both morbidity and mortality in ARDS models. At the molecular level, VT-109 blocks inflammatory NFAT and NFκB signaling while concurrently activating FOXM1-dependent endothelial regeneration. These findings support EB3 inhibition as a promising therapeutic strategy for ARDS and highlight VT-109 as a versatile drug candidate capable of addressing the multifaceted pathophysiology of this disease.

Acute respiratory distress syndrome (ARDS) is the acute onset of non-cardiogenic pulmonary edema, hypoxemia, and respiratory failure. ARDS is frequently caused by bacterial and viral lung infections including the recent SARS-CoV-2. Development of therapeutic approaches to combat ARDS have become an urgent unmet medical need. We address this medical problem by developing a pharmacological treatment for pulmonary vascular leakage, a culprit of alveolar damage and lung inflammation. Our novel therapeutic target is the microtubule accessory factor End Binding protein 3 (EB3), which contributes to pulmonary vascular leakage by amplifying pathological calcium signaling in endothelial cells. Using a structure-activity-relationship approach by Nuclear Magnetic Resonance, we have identified a drug candidate Vascular Therapeutics (VT)-109, an allosteric EB3 inhibitor with optimized physicochemical and biochemical properties. The VT-109 shows marked therapeutic benefits in treating lung injury and inflammation in various models of ARDS including high-volume mechanical ventilation, polymicrobial and SARS-CoV-2 infections. Transcriptome analysis of lung endothelial cells in mice challenged with endotoxin shows that VT-109 upregulates FOXM1 target genes suggesting that the treatment not only mitigates vascular injury but also promotes endothelial cell regeneration by activating the FOXM1 pathway. Our work demonstrates that VT-109 is a promising drug candidate for future clinical studies. Funding for this work is provided by the Congressionally Directed Medical Research Programs through the Peer Reviewed Medical Research Program (PRMRP) under award No. W81XWH-21-1-0639. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Acute respiratory distress syndrome (ARDS) is the acute onset of non-cardiogenic pulmonary edema, hypoxemia, and respiratory failure, conditions associated with a high mortality rate. ARDS is frequently caused by lung infections including SARS-CoV-2. In light of the COVID-19 pandemic, development of therapeutic approaches to combat ARDS have become an urgent unmet medical need. As increased vascular leakage is a culprit of alveolar damage and lung inflammation, we hypothesize that an effective therapy against vascular leakage should improve the clinical outcome of ARDS patients. We have identified a drug candidate Vascular Therapeutics (VT)-109, an allosteric inhibitor of End Binding protein 3, which contributes to vascular leakage by amplifying pathological calcium signaling in endothelial cells. Treatment of mice with VT-109 reduced the leakage of proteinous fluids in lungs to 55.4% (n=7, p<0.05) after endotoxin challenge, 77.7% (n=6, p<0.01) after exposure to high volume mechanical ventilation, and 42.5% (n=7, p<0.05) in the two-hit model, a combination of polymicrobial sepsis with normal tidal volume mechanical ventilation. VT-109 reduced the mortality of both septic (n=10, p<0.01) and endotoxin-challenged (n=10, p<0.05) mice. Furthermore, treatment with VT-109 in mice expressing human ACE2 receptor blocked diffuse alveolar damage (n=5, p<0.0001) caused by SARS-CoV-2 infection. In this COVID-19 model, the therapeutic benefits of VT-109 were observed when treatment was initiated at either day 1 or day 3 post-viral infection. Transcriptome analysis of lung endothelial cells in mice challenged with endotoxin showed that VT-109 upregulated 23 FOXM1 target genes (>2-fold) suggesting that it promotes endothelial cell regeneration by activating the FOXM1 pathway. Our data demonstrate that VT-109 successfully treats vascular leakage and is a promising drug candidate for future clinical studies. Funding for this work is provided by the Congressionally Directed Medical Research Programs through the Peer Reviewed Medical Research Program (PRMRP) under award No. W81XWH-21-1-0639. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.

In our previous studies, we described a critical role of microtubule-associated end binding protein 3 (EB3) in regulating pathological calcium release from endoplasmic reticulum (ER) stores through interaction with inositol 1,4,5-trisphosphate (IP3R3) within endothelial cells. The short cognate peptide we designed based on this interaction blocked VEGF-evoked ER calcium release and decreased laser-induced choroidal neovascularization (CNV) in both mouse and non-human primate models of age-related macular degeneration. Here we analyzed the global effects of this peptide on choroidal and retinal cells using single nuclei-RNA (snRNA) and single nuclei-assay for transposase-accessible chromatin (snATAC) sequencing of non-human primate eye tissue. Within the snRNA-seq transcriptome data, we detected 18 different cell types residing in the retina and choroid using well-established markers. For the characterization of snATAC-seq clusters, we performed Pearson correlation of the genes expressed in each cluster of our snRNA-seq data and the peaks found in each cluster of our snATAC-seq data. Our snATAC-seq analysis demonstrated a widespread increase in chromatin accessibility at the promoter region across different cell types. These changes in chromatin accessibility were associated with increased acetylation in lysine 27 of histone 3 (H3K27ac) but not lysine 8 of histone 4 (H4K8ac). Concurrent analysis between our snATAC-seq and snRNA-seq data further revealed increased chromatin opening of MEIS2 and PAX6 within our metabolic-active endothelial cells of neovascularized lesions, leading to activation of downstream transcription factors such as NCOR2, NR2C1, SREBF2, TCF4, TCF12, TEF, and THRA. The activation of this transcriptional program promoted neovascular regeneration and healing of ablated regions within CNV lesion of non-human primate eye in the treatment group. Taken together, we concluded that EB3 inhibition prevented aberrant angiogenesis through epigenetic reprogramming of injured endothelial cells and through activation of MEIS2 and PAX6's transcriptional program.

Vascular endothelial (VE) protein tyrosine phosphatase (PTP) is an endothelial-specific phosphatase that stabilizes VE-cadherin junctions. Although studies have focused on the role of VE-PTP in dephosphorylating VE-cadherin in the activated endothelium, little is known of VE-PTP's role in the quiescent endothelial monolayer. Here, we used the photoconvertible fluorescent protein VE-cadherin-Dendra2 to monitor VE-cadherin dynamics at adherens junctions (AJs) in confluent endothelial monolayers. We discovered that VE-PTP stabilizes VE-cadherin junctions by reducing the rate of VE-cadherin internalization independently of its phosphatase activity. VE-PTP serves as an adaptor protein that through binding and inhibiting the RhoGEF GEF-H1 modulates RhoA activity and tension across VE-cadherin junctions. Overexpression of the VE-PTP cytosolic domain mutant interacting with GEF-H1 in VE-PTP-depleted endothelial cells reduced GEF-H1 activity and restored VE-cadherin dynamics at AJs. Thus, VE-PTP stabilizes VE-cadherin junctions and restricts endothelial permeability by inhibiting GEF-H1, thereby limiting RhoA signaling at AJs and reducing the VE-cadherin internalization rate.

The PU.1 transcription factor plays a critical role in the regulation of T cell development, so a report that it is dispensable for fetal thymopoiesis is puzzling. To understand this paradox, we examined the requirement for PU.1, encoded by Spi1, during fetal, neonatal, and adult thymopoiesis in a PU.1 hypomorphic mouse generated by deletion of the Spi1 14-kb upstream regulatory element and by analysis of patterns of gene expression in fetal and adult T cell progenitors. Our data demonstrate that the initiation of thymopoiesis during early gestation is less dependent on PU.1 compared with T cell differentiation in adults and that fetal T cell progenitors express lower levels of Spi1 compared with their adult counterparts. We also show that expression of the core network of T lineage transcription factors regulated by PU.1 differs in fetal and adult T cell progenitors. In particular, PU.1-regulated genes that promote T cell differentiation are differentially expressed in fetal versus adult early T lineage progenitors. These results indicate that the transcriptional differences between the fetal and adult T cell developmental programs are driven in part by differential levels of PU.1 expression and that this likely underlies the differences in the properties of fetal and adult T cell progenitors.