Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers

© 2018 Hilfenhaus et al.Through multiple cell–cell and cell–matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability.Deutsche Forschungsgemeinschaft (STE 2045/1-1) Fundación Ramón Areces (NO AWARD) Ministerio de Economía y Competitividad (NO AWARD) National Institutes of Health (P40OD018537) Worldwide Cancer Research (13-0170

Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers

[EN]Through multiple cell-cell and cell-matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability. © 2018 Hilfenhaus et al

Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers.

Through multiple cell-cell and cell-matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability

A High-Content Screen Identifies Drugs That Restrict Tumor Cell Extravasation across the Endothelial Barrier

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg

Factors Associated With Humeral Avulsion of Glenohumeral Ligament Lesions in Patients With Anterior Shoulder Instability: An Analysis of the MOON Shoulder Instability Cohort.

BACKGROUND: Humeral avulsion of the glenohumeral ligament (HAGL) lesions are an uncommon cause of anterior glenohumeral instability and may occur in isolation or combination with other pathologies. As HAGL lesions are difficult to detect via magnetic resonance imaging (MRI) and arthroscopy, they can remain unrecognized and result in continued glenohumeral instability. PURPOSE: To compare patients with anterior shoulder instability from a large multicenter cohort with and without a diagnosis of a HAGL lesion and identify preoperative physical examination findings, patient-reported outcomes, imaging findings, and surgical management trends associated with HAGL lesions. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: Patients with anterior glenohumeral instability who underwent surgical management between 2012 and 2020 at 11 orthopaedic centers were enrolled. Patients with HAGL lesions identified intraoperatively were compared with patients without HAGL lesions. Preoperative characteristics, physical examinations, imaging findings, intraoperative findings, and surgical procedures were collected. The Student t test, Kruskal-Wallis H test, Fisher exact test, and chi-square test were used to compare groups. RESULTS: A total of 21 HAGL lesions were identified in 915 (2.3%) patients; approximately one-third (28.6%) of all lesions were visualized intraoperatively but not identified on preoperative MRI. Baseline characteristics did not differ between study cohorts. Compared with non-HAGL patients, HAGL patients were less likely to have a Hill-Sachs lesion (54.7% vs 28.6%; P = .03) or an anterior labral tear (87.2% vs 66.7%; P = .01) on preoperative MRI and demonstrated increased external rotation when their affected arm was positioned at 90° of abduction (85° vs 90°; P = .03). Additionally, HAGL lesions were independently associated with an increased risk of undergoing an open stabilization surgery (odds ratio, 74.6 [95% CI, 25.2-221.1]; P < .001). CONCLUSION: Approximately one-third of HAGL lesions were missed on preoperative MRI. HAGL patients were less likely to exhibit preoperative imaging findings associated with anterior shoulder instability, such as Hill-Sachs lesions or anterior labral pathology. These patients underwent open procedures more frequently than patients without HAGL lesions

1‑O‑Octadecyl-2‑O‑benzyl-sn-glyceryl-3-phospho-GS-441524 (V2043). Evaluation of Oral V2043 in a Mouse Model of SARS-CoV‑2 Infection and Synthesis and Antiviral Evaluation of Additional Phospholipid Esters with Enhanced Anti-SARS-CoV‑2 Activity

Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn-2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections

Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers.

Through multiple cell-cell and cell-matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability