Connexins: Key Players in the Control of Vascular Plasticity and Function.

Of the 21 members of the connexin family, 4 (Cx37, Cx40, Cx43, and Cx45) are expressed in the endothelium and/or smooth muscle of intact blood vessels to a variable and dynamically regulated degree. Full-length connexins oligomerize and form channel structures connecting the cytosol of adjacent cells (gap junctions) or the cytosol with the extracellular space (hemichannels). The different connexins vary mainly with regard to length and sequence of their cytosolic COOH-terminal tails. These COOH-terminal parts, which in the case of Cx43 are also translated as independent short isoforms, are involved in various cellular signaling cascades and regulate cell functions. This review focuses on channel-dependent and -independent effects of connexins in vascular cells. Channels play an essential role in coordinating and synchronizing endothelial and smooth muscle activity and in their interplay, in the control of vasomotor actions of blood vessels including endothelial cell reactivity to agonist stimulation, nitric oxide-dependent dilation, and endothelial-derived hyperpolarizing factor-type responses. Further channel-dependent and -independent roles of connexins in blood vessel function range from basic processes of vascular remodeling and angiogenesis to vascular permeability and interactions with leukocytes with the vessel wall. Together, these connexin functions constitute an often underestimated basis for the enormous plasticity of vascular morphology and function enabling the required dynamic adaptation of the vascular system to varying tissue demands.

Ubiquitination of VE-cadherin regulates inflammation-induced vascular permeability in vivo.

VE-cadherin is a major component of the cell adhesion machinery which provides integrity and plasticity of the barrier function of endothelial junctions. Here, we analyze whether ubiquitination of VE-cadherin is involved in the regulation of the endothelial barrier in inflammation in vivo. We show that histamine and thrombin stimulate ubiquitination of VE-cadherin in HUVEC, which is completely blocked if the two lysine residues K626 and K633 are replaced by arginine. Similarly, these mutations block histamine-induced endocytosis of VE-cadherin. We describe two knock-in mouse lines with endogenous VE-cadherin being replaced by either a VE-cadherin K626/633R or a VE-cadherin KallR mutant, where all seven lysine residues are mutated. Mutant mice are viable, healthy and fertile with normal expression levels of junctional VE-cadherin. Histamine- or LPS-induced vascular permeability in the skin or lung of both of these mutant mice are clearly and similarly reduced in comparison to WT mice. Additionally, we detect a role of K626/633 for lysosomal targeting. Collectively, our findings identify ubiquitination of VE-cadherin as important for the induction of vascular permeability in the inflamed skin and lung.

A Metabolic Basis for Endothelial-to-Mesenchymal Transition.

Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor ß (TGF-ß) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.

Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

Impaired high-density lipoprotein function and endothelial barrier stability in severe anaphylaxis.

BACKGROUND: Growing evidence demonstrates the importance of high- and low-density lipoprotein cholesterol in certain immune and allergy-mediated diseases. OBJECTIVE: This study aimed to evaluate levels of high- and low-density lipoprotein cholesterol and apolipoproteins A1 and B in sera from a cohort of patients presenting with hypersensitivity reactions. We further assessed the function of high-density lipoprotein particles as well as their involvement in the molecular mechanisms of anaphylaxis. METHODS: Lipid profile determination was performed in paired (acute and baseline) serum samples from 153 patients. Thirty-eight experienced a non-anaphylactic reaction and 115 had an anaphylactic reaction (88 moderate and 27 severe). Lecithin cholesterol acyl transferase activity was assessed in patient sera, and we also evaluated macrophage cholesterol efflux in response to the serum samples. Last, the effect of anaphylactic-derived high-density lipoprotein (HDL) particles on the endothelial barrier was studied. Detailed methods are provided in the Methods section in this article's Online Repository available at www.jacionline.org. RESULTS: Serum samples from severe anaphylactic reactions show statistically significant low levels of HDL cholesterol, low-density lipoprotein cholesterol, and apolipoproteins A1 and B, which points to their possible role as biomarkers. Specifically, HDL particles play a protective role in cardiovascular diseases. Using functional human serum cell assays, we observed impaired capacity of apolipoprotein B-depleted serum to induce macrophage cholesterol efflux in severe anaphylactic reactions. In addition, purified HDL particles from human anaphylactic sera failed to stabilize and maintain the endothelial barrier. CONCLUSION: These results encourage further research on HDL functions in severe anaphylaxis, which may lead to new diagnostic and therapeutic strategies.

Permissive role of vascular endothelium in fibrosis: focus on the kidney.

Fibrosis, the morphologic end-result of a plethora of chronic conditions and the scorch for organ function, has been thoroughly investigated. One aspect of its development and progression, namely the permissive role of vascular endothelium, has been overshadowed by studies into (myo)fibroblasts and TGF-ß; thus, it is the subject of the present review. It has been established that tensile forces of the extracellular matrix acting on cells are a prerequisite for mechanochemical coupling, leading to liberation of TGF-ß and formation of myofibroblasts. Increased tensile forces are prompted by elevated vascular permeability in response to diverse stressors, resulting in the exudation of fibronectin, fibrinogen/fibrin, and other proteins, all stiffening the extracellular matrix. These processes lead to the development of endothelial cells dysfunction, endothelial-to-mesenchymal transition, premature senescence of endothelial cells, perturbation of blood flow, and gradual obliteration of microvasculature, leaving behind "string" vessels. The resulting microvascular rarefaction is not only a constant companion of fibrosis but also an adjunct mechanism of its progression. The deepening knowledge of the above chain of pathogenetic events involving endothelial cells, namely increased permeability-stiffening of the matrix-endothelial dysfunction-microvascular rarefaction-tissue fibrosis, may provide a roadmap for therapeutic interventions deemed to curtail and reverse fibrosis.

The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability.

Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.

Obesity-induced blood-brain barrier dysfunction: phenotypes and mechanisms.

Obesity, a burgeoning global health issue, is increasingly recognized for its detrimental effects on the central nervous system, particularly concerning the integrity of the blood-brain barrier (BBB). This manuscript delves into the intricate relationship between obesity and BBB dysfunction, elucidating the underlying phenotypes and molecular mechanisms. We commence with an overview of the BBB's critical role in maintaining cerebral homeostasis and the pathological alterations induced by obesity. By employing a comprehensive literature review, we examine the structural and functional modifications of the BBB in the context of obesity, including increased permeability, altered transport mechanisms, and inflammatory responses. The manuscript highlights how obesity-induced systemic inflammation and metabolic dysregulation contribute to BBB disruption, thereby predisposing individuals to various neurological disorders. We further explore the potential pathways, such as oxidative stress and endothelial cell dysfunction, that mediate these changes. Our discussion culminates in the summary of current findings and the identification of knowledge gaps, paving the way for future research directions. This review underscores the significance of understanding BBB dysfunction in obesity, not only for its implications in neurodegenerative diseases but also for developing targeted therapeutic strategies to mitigate these effects.

Heterogeneity of endothelial VE-PTP downstream polarization, Tie2 activation, junctional claudin-5, and permeability in the aorta and vena cava.

Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous.

Importance of IgE-Induced Unique Plasma Leakage in the Skin for Urticaria-Like Symptoms in an Anaphylaxis-Dependent Spotted Distribution of Immune Complex in Skin (ASDIS) Mouse Model.

INTRODUCTION: Allergic diseases, such as anaphylaxis and urticaria, pose significant health concerns. The quest for improved prognostic outcomes in these diseases necessitates the exploration of novel therapeutic avenues. To address this need, we have developed a novel mouse model of anaphylaxis, denoted as anaphylaxis-dependent spotted distribution of immune complex in skin (ASDIS). ASDIS manifests as distinct dotted symptoms in the skin, detectable through in vivo imaging, resembling urticarial symptoms. In this study, we investigated the potential underlying mechanisms giving rise to these dotted symptoms, exploring the role of vascular permeability and characterizing the ASDIS model as a new urticaria model. METHODS: We employed haired and hairless HR mice (BALB/c background) and hairless HR-1 mice (a commercially available hairless strain with an unidentified genetic background). ASDIS was induced by the simultaneous intravenous injection of anti-ovalbumin IgE and fluorescein isothiocyanate (FITC)-ovalbumin, along with Evans blue - a recognized vascular permeability indicator. Anaphylaxis and scratching behavior were monitored through rectal temperature decrease and optical observation, respectively. Histamine, platelet-activating factor, and compound 48/80 were injected with or without FITC-ovalbumin for comparative analysis. The effects of an α1 adrenergic receptor agonist applied to the skin were also examined. RESULTS: In hairless mice, the simultaneous injection of histamine, compound 48/80, or IgE with FITC-ovalbumin induced comparable rectal temperature decreases and vascular permeability. However, only the combination of FITC-ovalbumin and IgE triggered ASDIS, specifically the dotted urticaria-like symptom. Evans blue visualization and optical observation of dotted swelling confirmed that the vascular permeability mediated the phenomenon. Hairless mice exhibited a more pronounced temperature decrease than their haired counterparts when exposed to histamine, platelet-activating factor, compound 48/80, and IgE with FITC-ovalbumin. The application of an α1 adrenergic receptor agonist to the skin attenuated the topical urticaria-like symptom. CONCLUSION: Our experiments revealed four findings. The first is that ASDIS mirrors urticaria-like symptoms resulting from increased vascular permeability, akin to human urticaria. The second finding is that the development of dotted symptoms involves an IgE-induced, yet unidentified, mechanism not triggered by histamine or compound 48/80 alone. The third finding highlights the heightened susceptibility of hairless mice to ASDIS induction. The fourth finding demonstrates that the inhibition of ASDIS by the topical application of an α1 adrenergic receptor agonist hints at a potential anti-urticarial application for this vasoconstrictor. Further elucidation of these unidentified IgE-dependent mechanisms and the specific generation of dotted symptoms by IgE-immune complexes could provide novel insights into allergic response processes and therapeutic interventions for these conditions.

CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE-cadherin.

Regulation of vascular permeability to plasma is essential for tissue and organ homeostasis and is mediated by endothelial cell-to-cell junctions that tightly regulate the trafficking of molecules between blood and tissue. The single-pass transmembrane glycoprotein CD93 is upregulated in endothelial cells during angiogenesis and controls cytoskeletal dynamics. However, its role in maintaining homeostasis by regulating endothelial barrier function has not been elucidated yet. Here, we demonstrate that CD93 interacts with vascular endothelial (VE)-cadherin and limits its phosphorylation and turnover. CD93 deficiency in vitro and in vivo induces phosphorylation of VE-cadherin under basal conditions, displacing it from endothelial cell-cell contacts. Consistent with this, endothelial junctions are defective in CD93-/- mice, and the blood-brain barrier permeability is enhanced. Mechanistically, CD93 regulates VE-cadherin phosphorylation and turnover at endothelial junctions through the Rho/Rho kinase-dependent pathway. In conclusion, our results identify CD93 as a key regulator of VE-cadherin stability at endothelial junctions, opening up possibilities for therapeutic strategies directed to control vascular permeability.

Potential Therapeutic Strategies and Drugs That Target Vascular Permeability in Severe Infectious Diseases.

Severe infection pathogenicity is induced by processes such as pathogen exposure, immune cell activation, inflammatory cytokine production, and vascular hyperpermeability. Highly effective drugs, such as antipathogenic agents, steroids, and antibodies that suppress cytokine function, have been developed to treat the first three processes. However, these drugs cannot completely suppress severe infectious diseases, such as coronavirus disease 2019 (COVID-19). Therefore, developing novel drugs that inhibit vascular hyperpermeability is crucial. This review summarizes the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced vascular hyperpermeability and identifies inhibitors that increase endothelial cell (EC) junction-related proteins and determines their efficacy in COVID-19 and endotoxemia models. Analyzing the effects of SARS-CoV-2 on vascular permeability revealed that SARS-CoV-2 suppresses Claudin-5 (CLDN5) expression, which is responsible for adhesion between ECs, thereby increasing vascular permeability. Inhibiting CLDN5 function in mice induced vascular hyperpermeability and pulmonary edema. In contrast, Enhancing CLDN5 expression suppressed SARS-CoV-2-induced endothelial hyperpermeability, suggesting that SARS-CoV-2-induced vascular hyperpermeability contributes to pathological progression, which can be suppressed by upregulating EC junction proteins. Based on these results, we focused on Roundabout4 (Robo4), another EC-specific protein that stabilizes EC junctions. EC-specific Robo4 overexpression suppressed vascular hyperpermeability and mortality in lipopolysaccharide-treated mice. An ALK1 inhibitor (a molecule that increases Robo4 expression), suppressed vascular hyperpermeability and mortality in lipopolysaccharide- and SARS-CoV-2-treated mice. These results indicate that Robo4 expression-increasing drugs suppress vascular permeability and pathological phenotype in COVID-19 and endotoxemia models.

Exploring the multifaceted effects of Ammi visnaga: subchronic toxicity, antioxidant capacity, immunomodulatory, and anti-inflammatory activities.

Ammi visnaga (A. visnaga) is an annual herb that has been used in traditional medicine to treat various ailments attributed to the presence of its bioactive compounds. The purpose of this study was to identify and examine the phytochemical properties of the hydroalcoholic extract of A. visnaga using in vitro and in vivo models. Our findings demonstrated that the extract contained a variety of beneficial components, including phenols, flavonoids, tannins, coumarins, saponins, khellin, and visnagin. The total polyphenolic content and total flavonoid content were 23.26 mg/GAE/g dry weight and 13.26 mg/GAE/g dry weight, respectively. In vitro tests demonstrated that the extract possessed antioxidant properties as evidenced by the ability to scavenge free radicals, including DPPH, ABTS, nitric oxide (NO), phosphomolybdate, and ferric-reducing antioxidant power (FRAP). Further, the extract was found to inhibit hydrogen peroxide (H2O2)-induced hemolysis. In a 90-d in vivo study, female Wistar rats were administered 1 g/kg of A. visnaga extract orally resulting in a significant increase in total white blood cell count. Although morphological changes were observed in the liver, no marked alterations were noted in kidneys and spleen. In a female Swiss albino mice model of acetic acid-induced vascular permeability, A. visnaga significantly inhibited extravasations of Evans blue at doses of 0.5 or 1 g/kg with inhibition percentages of 51 and 65%, respectively, blocking tissue necrosis. The extract also demonstrated potential immunomodulatory properties in mice by enhancing antibody production in response to antigens. In silico molecular docking studies demonstrated a strong affinity between khellin or visnagin and immunomodulatory proteins, NF-κB, p52, and TNF-α. These findings suggest that A. visnaga may be considered a beneficial antioxidant with immunomodulatory properties and might serve as a therapeutic agent to combat certain diseases.

Non-invasive evaluation of vascular permeability in formalin-induced orofacial pain model using infrared thermography.

Enhanced vascular permeability at the site of injury is a prominent feature in acute inflammatory pain models, commonly assessed through the Evans Blue test. However, this invasive test requires euthanasia, thereby precluding further investigations on the same animal. Due to these limitations, the integration of non-invasive tools such as IRT has been sought. Here, we aimed to evaluate the use of thermography in a common orofacial pain model that employs formalin as a chemical irritant to induce local orofacial inflammation. Male Hannover rats (290-300 g, N = 43) were used. In the first approach, radiometric images were taken before and after formalin administration, assessing temperature changes and extravasated Evans Blue. The second approach included capturing pre- and post-formalin test radiometric images, followed by cytokine measurements in excised vibrissae tissue. Rats were anesthetized for vibrissae tissue collection, allowing correlations between thermographic patterns, nocifensive behavior duration, and cytokine levels in this area. Our findings revealed a positive correlation between local temperature, measured via thermography, and vascular permeability in the contralateral (r2 = 0.3483) and ipsilateral (r2 = 0.4502) side, measured using spectrophotometry. The obtained data supports the notion that thermography-based temperature assessment can effectively evaluate vascular permeability in the orofacial region.

A ketogenic diet improves vascular hyperpermeability in type 2 diabetic mice by downregulating vascular pescadillo1 expression.

The role of pescadillo1 (PES1) in regulating vascular permeability has been unknown. This study probes the role of PES1 and its mediated molecular mechanism in modulating vascular hyperpermeability in diabetic mice. Male C57BL/6J and db/db mice were fed a standard diet and a ketogenic diet (KD). Meanwhile, mouse vascular endothelial cells (MVECs) were treated with ß-hydroxybutyric acid (ß-HB), Pes1 siRNA or a Pes1 overexpression plasmid. Additionally, knockout (KO) of Pes1 in mice was applied. After 12 weeks of feedings, enhanced vascular PES1 expression in diabetic mice was inhibited by the KD. The suppression of PES1 was also observed in ß-HB-treated MVECs. In mice with Pes1 KO, the levels of vascular VEGF and PES1 were attenuated, while the levels of vascular VE-cadherin, Ang-1 and Occludin were upregulated. Similar outcomes also occurred after the knockdown of Pes1 in cultured MVECs, which were opposite to the effects induced by PES1 overexpression in MVECs. In vitro and in vivo experiments showed that high glucose concentration-induced increases in vascular paracellular permeability declined after MVECs were treated by ß-HB or by knockdown of Pes1. In contrast, increases in vascular permeability were induced by overexpression of Pes1, which were suppressed by coadministration of ß-HB in cultured endothelial cells. Similarly declines in vascular permeability were found by Pes1 knockdown in diabetic mice. Mechanistically, ß-HB decreased PES1-facilitated ubiquitination of VE-cadherin. The KD suppressed the diabetes-induced increase in PES1, which may result in vascular hyperpermeability through ubiquitination of VE-cadherin in type 2 diabetic mice.

Targeting VEGF-A/VEGFR2 Y949 Signaling-Mediated Vascular Permeability Alleviates Hypoxic Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is associated with increased expression of VEGF-A (vascular endothelial growth factor A) and its receptor, VEGFR2 (vascular endothelial growth factor 2), but whether and how activation of VEGF-A signal participates in the pathogenesis of PH is unclear. METHODS: VEGF-A/VEGFR2 signal activation and VEGFR2 Y949-dependent vascular leak were investigated in lung samples from patients with PH and mice exposed to hypoxia. To study their mechanistic roles in hypoxic PH, we examined right ventricle systolic pressure, right ventricular hypertrophy, and pulmonary vasculopathy in mutant mice carrying knock-in of phenylalanine that replaced the tyrosine at residual 949 of VEGFR2 (Vefgr2Y949F) and mice with conditional endothelial deletion of Vegfr2 after chronic hypoxia exposure. RESULTS: We show that PH leads to excessive pulmonary vascular leak in both patients and hypoxic mice, and this is because of an overactivated VEGF-A/VEGFR2 Y949 signaling axis. In the context of hypoxic PH, activation of Yes1 and c-Src and subsequent VE-cadherin phosphorylation in endothelial cells are involved in VEGFR2 Y949-induced vascular permeability. Abolishing VEGFR2 Y949 signaling by Vefgr2Y949F point mutation was sufficient to prevent pulmonary vascular permeability and inhibit macrophage infiltration and Rac1 activation in smooth muscle cells under hypoxia exposure, thereby leading to alleviated PH manifestations, including muscularization of distal pulmonary arterioles, elevated right ventricle systolic pressure, and right ventricular hypertrophy. It is important that we found that VEGFR2 Y949 signaling in myeloid cells including macrophages was trivial and dispensable for hypoxia-induced vascular abnormalities and PH. In contrast with selective blockage of VEGFR2 Y949 signaling, disruption of the entire VEGFR2 signaling by conditional endothelial deletion of Vegfr2 promotes the development of PH. CONCLUSIONS: Our results support the notion that VEGF-A/VEGFR2 Y949-dependent vascular permeability is an important determinant in the pathogenesis of PH and might serve as an attractive therapeutic target pathway for this disease.

Biomarkers of alveolar epithelial injury and endothelial dysfunction are associated with scores of pulmonary edema in invasively ventilated patients.

Pulmonary edema is a central hallmark of acute respiratory distress syndrome (ARDS). Endothelial dysfunction and epithelial injury contribute to alveolar-capillary permeability but their differential contribution to pulmonary edema development remains understudied. Plasma levels of surfactant protein-D (SP-D), soluble receptor for advanced glycation end products (sRAGE), and angiopoietin-2 (Ang-2) were measured in a prospective, multicenter cohort of invasively ventilated patients. Pulmonary edema was quantified using the radiographic assessment of lung edema (RALE) and global lung ultrasound (LUS) score. Variables were collected within 48 h after intubation. Linear regression was used to examine the association of the biomarkers with pulmonary edema. In 362 patients, higher SP-D, sRAGE, and Ang-2 concentrations were significantly associated with higher RALE and global LUS scores. After stratification by ARDS subgroups (pulmonary, nonpulmonary, COVID, non-COVID), the positive association of SP-D levels with pulmonary edema remained, whereas sRAGE and Ang-2 showed less consistent associations throughout the subgroups. In a multivariable analysis, SP-D levels were most strongly associated with pulmonary edema when combined with sRAGE (RALE score: ßSP-D = 6.79 units/log10 pg/mL, ßsRAGE = 3.84 units/log10 pg/mL, R2 = 0.23; global LUS score: ßSP-D = 3.28 units/log10 pg/mL, ßsRAGE = 2.06 units/log10 pg/mL, R2 = 0.086), whereas Ang-2 did not further improve the model. Biomarkers of epithelial injury and endothelial dysfunction were associated with pulmonary edema in invasively ventilated patients. SP-D and sRAGE showed the strongest association, suggesting that epithelial injury may form a final common pathway in the alveolar-capillary barrier dysfunction underlying pulmonary edema.

Branched-chain keto-acid dehydrogenase kinase regulates vascular permeability and angiogenesis to facilitate tumor metastasis in renal cell carcinoma.

Branched-chain keto-acid dehydrogenase kinase (BCKDK) is the rate-limiting enzyme of branched-chain amino acid (BCAA) metabolism. In the last six years, BCKDK has been used as a kinase to promote tumor proliferation and metastasis. Renal cell carcinoma (RCC) is a highly vascularized tumor. A high degree of vascularization promotes tumor metastasis. Our objective is to explore the relationship between BCKDK and RCC metastasis and its specific mechanism. In our study, BCKDK is highly expressed in renal clear cell carcinoma and promotes the migration of clear cell renal cell carcinoma (ccRCC). Exosomes from ccRCC cells can promote vascular permeability and angiogenesis, especially when BCKDK is overexpressed in ccRCC cells. BCKDK can also augment the miR-125a-5p expression in ccRCC cells and derived exosomes, thereby decreasing the downstream target protein VE-cadherin level, weakening adhesion junction expression, increasing vascular permeability, and promoting angiogenesis in HUVECs. The novel BCKDK/Exosome-miR-125a-5p/VE-cadherin axis regulates intercellular communication between ccRCC cells and HUVECs. BCKDK plays a critical role in renal cancer metastasis, may be used as a molecular marker of metastatic ccRCC, and even may become a potential target of clinical anti-vascular therapy for ccRCC.

Spotted fever rickettsia-induced microvascular endothelial barrier dysfunction is delayed by the calcium channel blocker benidipine.

The tick-borne bacterium Rickettsia parkeri is an obligate intracellular pathogen that belongs to spotted fever group rickettsia (SFGR). The SFG pathogens are characterized by their ability to infect and rapidly proliferate inside host vascular endothelial cells that eventually result in impairment of vascular endothelium barrier functions. Benidipine, a wide range dihydropyridine calcium channel blocker, is used to prevent and treat cardiovascular diseases. In this study, we tested whether benidipine has protective effects against rickettsia-induced microvascular endothelial cell barrier dysfunction in vitro. We utilized an in vitro vascular model consisting of transformed human brain microvascular endothelial cells (tHBMECs) and continuously monitored transendothelial electric resistance (TEER) across the cell monolayer. We found that during the late stages of infection when we observed TEER decrease and when there was a gradual increase of the cytoplasmic [Ca2+], benidipine prevented these rickettsia-induced effects. In contrast, nifedipine, another cardiovascular dihydropyridine channel blocker specific for L-type Ca2+ channels, did not prevent R. parkeri-induced drop of TEER. Additionally, neither drug was bactericidal. These data suggest that growth of R. parkeri inside endothelial cells is associated with impairment of endothelial cell monolayer integrity due to Ca2+ flooding through specific, benidipine-sensitive T- or N/Q-type Ca2+ channels but not through nifedipine-sensitive L-type Ca2+ channels. Further study will be required to discern the exact nature of the Ca2+ channels and Ca2+ transporting system(s) involved, any contributions of the pathogen toward this process, as well as the suitability of benidipine and new dihydropyridine derivatives as complimentary therapeutic drugs against Rickettsia-induced vascular failure.

Hypoxic nasopharyngeal carcinoma-derived exosomal miR-455 increases vascular permeability by targeting ZO-1 to promote metastasis.

Nasopharyngeal carcinoma (NPC), the most frequent reason for treatment failure in head and neck tumors, has the greatest incidence of distant metastases. Increased vascular permeability facilitates metastasis. Exosomal microRNAs (miRNAs) have been implicated in the development of the premetastatic niche and are emerging as prospective biomarkers in cancer patients. We discovered that a higher level of miR-455 was connected to a larger propensity for NPC metastasis based on deep sequencing and RT-qPCR. We found that hypoxia promoted NPC exosomes release and increased miR-455 expression in a way that was hypoxia-inducible factor 1-alpha (HIF-1α) dependent. Exosomes from NPC cells with high levels of miR-455 were found to specifically target zonula occludens 1 (ZO-1), increasing the permeability of endothelial monolayers in vitro vascular permeability and transendothelial invasion experiments. Additional in vivo studies showed that zebrafish with sustained miR-455-overexpressing NPC cell xenografts displayed increased tumor cell mass throughout the body. In vivo, zebrafish vascular tight junction integrity was disrupted by exosomes produced by NPC cells with elevated miR-455 expression. Mice-bearing xenografts further supported the finding that exosomes containing miR-455 might reduce ZO-1 expression in addition to promote NPC cell growth. These findings suggest that in a hypoxic microenvironment, exosomal miR-455 released by NPC cells enhances vascular permeability and promotes metastasis by targeting ZO-1. The HIF-1α-miR-455-ZO-1 signaling pathway may be a promising predictor and potential therapeutic target for NPC with metastasis.