Endothelial Cell Disruption Research Articles

Rational formulation of saponin and dexamethasone for the synergistic treatment of rheumatoid arthritis in vivo

Rheumatoid arthritis (RA) is a common autoimmune disease, and the abnormal proliferation of fibroblast-like synoviocytes (FLSs) in inflamed joints plays a key role in the pathogenesis of RA, which has become an important therapeutic target for RA treatment. This work reported a facilely formulated nanoparticle from saponin (Sap) and dexamethasone (Dex) in precise ratio, affording the resultant Dex@Sap nanoparticles. Benefiting from the enlargement of vascular endothelial cell gap at the inflammation site and intra-articular injection, Dex@Sap could accumulate at the inflamed joints to inhibit excessive proliferation of FLSs and mediate cell apoptosis, achieving symptomatic relief and efficient RA treatment, while reducing the amount of drug needed as well as the occurrence of adverse reactions. Typical AKT (also known as protein kinase B, PKB)/mTOR (mammalian target of rapamycin) pathway was observed to be inhibited, and the mitochondria-mediated intracellular reactive oxygen species (ROS) level was also upregulated by Dex@Sap. Further in vivo evaluations demonstrated that Dex@Sap could significantly alleviate RA-induced inflammatory response and the expression of pro-inflammatory cytokines to cure joint damage in collagen-induced arthritis mice and rats. This work provides a clinically promising nanomedicine to synergistically treat RA.

Simulation of Conducted Responses in Microvascular Networks: Role of Current Rectification by Endothelial Cell Gap Junctions

Neuronal activity in the brain stimulates local increases in blood flow. Such flow increases require dilation of feeding arterioles, which can be initiated by upstream conducted responses in the form of electrical currents transmitted along the endothelial lining of the vessels. One potential trigger for this response is hyperpolarization of capillary endothelial cells in active regions resulting from local elevation of extracellular K+ concentration. The conducted hyperpolarization causes arteriolar dilation. For this mechanism to be effective in delivering increased flow to a specific location, the conducted response must travel in the upstream direction only, without reentry into adjacent parallel flow pathways. Such specificity in the response to activation has been observed in skeletal muscle, and may result from partial rectification of current in gap junctions connecting adjacent endothelial cells. The goal of this work is to simulate the spread of hyperpolarizing currents along vessel walls in microvascular networks of the cerebral circulation, including the effects of rectification in gap junctions. A theoretical model was developed for the spread of electrical current in the endothelial cells of a network of microvessels in response to local K+- induced hyperpolarization. Transmembrane currents are represented in the model by a KIR-channel conductance, a non-voltage-dependent K+ conductance, and a background conductance. Currents along vessel walls are represented assuming asymmetric gap junction conductance between adjacent endothelial cells, with higher conductance for downstream currents (causing upstream hyperpolarization) than for upstream currents. The model was applied to a synthetic network of 45 segments, assuming 20 nS conductance for upstream propagation, while a range of values was used for downstream propagation. The model shows effective upstream propagation of conducted responses when the gap junction conductance for downstream propagation is 1 nS or less. This result was confirmed in a simulation of a reconstructed murine cerebral cortex network with 4881 segments. In conclusion, partial current rectification by endothelial gap junctions can result in preferential upstream propagation of conducted responses, consistent with experimental observations of local flow regulation. Supported by NIH grant U01 HL133362. This is the full abstract presented at the American Physiology Summit 2024 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.

Ultrasound-Based Radiation Enhancement: Concepts, Mechanisms and Therapeutic Applications.

Microbubbles have emerged as versatile carriers used both for cancer diagnosis and therapy. Microbubbles in the presence of ultrasound waves undergo cavitation, generating bioeffects near the cell's vicinity. Studies have shown ultrasound-stimulated microbubbles (USMB) to cause mechanical perturbation of endothelial cells, resulting in acid sphingomyelinase (ASMase)-induced ceramide production. Disruption of endothelial cells further causes vascular deterioration, leading to secondary tumor cell death. These effects are known to be synergistically higher when USMB is combined with radiation. This paper provides insight into the use of USMB as a potential radioenhancer. The possible underlying mechanism and therapeutic effects of combining USMB and radiation therapy are also presented.

Human Placental Mesenchymal Stem Cells-Exosomes Alleviate Endothelial Barrier Dysfunction via Cytoskeletal Remodeling through hsa-miR-148a-3p/ROCK1 Pathway.

Endothelial barrier disruption of human pulmonary vascular endothelial cells (HPVECs) is an important pathogenic factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Mesenchymal stem cells-exosome (MSCs-Exo) represents an ideal carrier for cell-free therapy. The therapeutic implication and underlying mechanism of human placental MSCs-Exo (HPMSCs-Exo) in ALI/ARDS need to be further explored. HPMSCs-Exo was extracted from HPMSCs and characterized. Then, the therapeutic effects of exosomes were evaluated in ALI mice and HPVECs. RNA-sequencing was applied to reveal the miRNA profile of HPMSCs-Exo and differentially expressed genes (DEGs) in HPMSCs-Exo-pretreated HPVECs. The targets of miRNAs were predicted by bioinformatics methods and correlated to DEGs. Finally, the role of hsa-miR-148a-3p/ROCK1 pathway in HPVECs has been further discussed. The results showed that HPMSCs-Exo could downregulate Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), upregulate the expression of zonula occludens-1 (ZO-1) and F-actin, promote HPVECs migration and tube formation, reduce cytoskeletal disorders and cell permeability, and thus improve ALI/ARDS. RNA-sequencing revealed the DEGs were mainly enriched in cell junction, angiogenesis, inflammation, and energy metabolism. HPMSCs-Exo contains multiple miRNAs which are associated with cytoskeletal function; the expression abundance of hsa-miR-148a-3p is the highest. Bioinformatic analysis identified ROCK1 as a target of hsa-miR-148a-3p. The overexpression of hsa-miR-148a-3p in HPMSCs-Exo promoted the migration and tube formation of HPVECs and reduced ROCK1 expression. However, the overexpression of ROCK1 on HPVECs reduced the therapeutic effect of HPMSCs-Exo. HPMSCs-Exo represents a protective regimen against endothelial barrier disruption of HPVECs in ALI/ARDS, and the hsa-miR-148a-3p/ROCK1 pathway plays an important role in this therapeutics implication.

Single nuclei transcriptomics in diabetic mice reveals altered brain hippocampal endothelial cell function, permeability, and behavior

Type 2 diabetes mellitus (T2DM) is a metabolic disorder with cerebrovascular and cardiovascular sequelae. Yet, a clear pattern of gene dysregulation by T2DM in dementia has yet to be defined. We used single nuclei RNA sequencing technology to profile the transcriptome of endothelial cells (EC) from anatomically defined hippocampus of db/db mice to identify differentially expressed (DE) genes, gene pathways and networks, predicted regulating transcription factors, and targets of DE long noncoding RNAs. We also applied gadolinium (Gd) enhanced magnetic resonance imaging (MRI) to assess blood brain barrier (BBB) permeability, and functionally assessed cognitive behavior. The murine gene expression profiles were then integrated with those of persons with Alzheimer's disease (AD) and vascular dementia (VaD).We reveal that the transcriptome of the diabetic hippocampal murine brain endothelium differs substantially from control wild types with molecular changes characterized by differential RNA coding and noncoding pathways enriched for EC signaling and for endothelial functions for neuroinflammation, endothelial barrier disruption, and neurodegeneration. Gd enhanced structural brain MRI linked endothelial molecular alterations to BBB dysfunction by neuroimaging. Integrated multiomics of hippocampal endothelial gene dysregulation associated with impairments in cognitive adaptive capacity. In addition, the diabetic transcriptome significantly and positively correlated with that of persons with AD and VaD. Taken together, our results from comprehensive, multilevel, integrated, single nuclei transcriptomics support the hypothesis of T2DM-mediated neuroinflammation and endothelial cell and barrier disruption as key mechanisms in cognitive decline in T2DM, thereby suggesting potential endothelial-specific molecular therapeutic targets.

Cortistatin deficiency reveals a dysfunctional brain endothelium with impaired gene pathways, exacerbated immune activation, and disrupted barrier integrity

BackgroundBrain activity governing cognition and behaviour depends on the fine-tuned microenvironment provided by a tightly controlled blood–brain barrier (BBB). Brain endothelium dysfunction is a hallmark of BBB breakdown in most neurodegenerative/neuroinflammatory disorders. Therefore, the identification of new endogenous molecules involved in endothelial cell disruption is essential to better understand BBB dynamics. Cortistatin is a neuroimmune mediator with anti-inflammatory and neuroprotective properties that exerts beneficial effects on the peripheral endothelium. However, its role in the healthy and injured brain endothelium remains to be evaluated. Herein, this study aimed to investigate the potential function of endogenous and therapeutic cortistatin in regulating brain endothelium dysfunction in a neuroinflammatory/neurodegenerative environment.MethodsWild-type and cortistatin-deficient murine brain endothelium and human cells were used for an in vitro barrier model, where a simulated ischemia-like environment was mimicked. Endothelial permeability, junction integrity, and immune response in the presence and absence of cortistatin were evaluated using different size tracers, immunofluorescence labelling, qPCR, and ELISA. Cortistatin molecular mechanisms underlying brain endothelium dynamics were assessed by RNA-sequencing analysis. Cortistatin role in BBB leakage was evaluated in adult mice injected with LPS.ResultsThe endogenous lack of cortistatin predisposes endothelium weakening with increased permeability, tight-junctions breakdown, and dysregulated immune activity. We demonstrated that both damaged and uninjured brain endothelial cells isolated from cortistatin-deficient mice, present a dysregulated and/or deactivated genetic programming. These pathways, related to basic physiology but also crucial for the repair after damage (e.g., extracellular matrix remodelling, angiogenesis, response to oxygen, signalling, and metabolites transport), are dysfunctional and make brain endothelial barrier lacking cortistatin non-responsive to any further injury. Treatment with cortistatin reversed in vitro hyperpermeability, tight-junctions disruption, inflammatory response, and reduced in vivo BBB leakage.ConclusionsThe neuropeptide cortistatin has a key role in the physiology of the cerebral microvasculature and its presence is crucial to develop a canonical balanced response to damage. The reparative effects of cortistatin in the brain endothelium were accompanied by the modulation of the immune function and the rescue of barrier integrity. Cortistatin-based therapies could emerge as a novel pleiotropic strategy to ameliorate neuroinflammatory/neurodegenerative disorders with disrupted BBB.

Design of in vitro biomimetic experimental system and simulation analysis for transvascular transport of nano-preparation

Recently, the enhanced penetration and retention (EPR) effect of nano-preparations has been questioned. Whether the vascular endothelial cell gap (VECG) is the main transport pathway of nano-preparations has become a hot issue at present. Therefore, we propose an in vitro biomimetic experimental system that demonstrates the transvascular transport of nano-preparation. Based on the tumor growth process, the experimental system was used to simulate the change process of abnormal factors (vascular endothelial cell gap and interstitial fluid pressure (IFP)) in the tumor microenvironment. The influence of change in the abnormal factors on the enhanced penetration and retention effect of nano-preparation was explored, and simulation verification was performed. The results show that when the interstitial fluid pressure is close to the vascular fluid pressure (VFP), the transport of nano-preparation is obstructed, resulting in the disappearance of enhanced penetration and retention effect of the nano-preparation. This indicates that the pressure gradient between vascular fluid pressure and interstitial fluid pressure determines whether the enhanced penetration and retention effect of nano-preparations can exist.

SARS-CoV-2–Induced Vasculitic Skin Lesions Are Associated with Massive Spike Protein Depositions in Autophagosomes

In patients infected with severe acute respiratory syndrome coronavirus 2, vasculopathic changes of the skin are associated with a severe prognosis. However, the pathogenesis of this vasculopathy is not conclusively clarified. In this study, 25 prospectively collected skin samples from patients with COVID-19-related skin lesions were examined for vasculopathic changes and, in case of vasculitis, were further analyzed with electron microscopy and immunohistochemistry. Vasculopathy was observed in 76% of all COVID-19-related inflammatory skin lesions. Visual endothelial changes without manifest leukocytoclastic vasculitis were found in 60% of the COVID-19-related skin lesions, whereas leukocytoclastic vasculitis was diagnosed in 16%. In the cases of vasculitis, there were extensive spike protein depositions in microvascular endothelial cells that colocalized with the autophagosome proteins LC3B and LC3C. The autophagy protein complex LC3-associated endocytosis in microvascular endothelial cells seems to be an important pathogenic factor for severe acute respiratory syndrome coronavirus 2-related vasculitis in the skin. On ultrastructural morphology, the vasculitic process was dominated by intracellular vesicle formation and endothelial cell disruption. Direct presence of severe acute respiratory syndrome coronavirus 2 particles in the skin was not observed. Therefore, our results suggest that instead of direct viral infection, dermal vasculitic lesions in COVID-19 are caused by severe acute respiratory syndrome coronavirus 2 spike protein deposition followed by endothelial damage with activation of autophagy.

Dl-3-n-butylphthalide improves intestinal microcirculation disorders in septic rats by regulating the PI3K/AKT signaling pathway and autophagy.

Sepsis has complex pathophysiological mechanisms that bring new challenges in the treatment of sepsis at a time when the intestinal microcirculation in sepsis is receiving increasing attention. Dl-3-n-butylphthalide (NBP), which is a drug that can improve multiorgan ischemic diseases, is also worth examining to improve the intestinal microcirculation in sepsis. In this study, male Sprague-Dawley rats were divided into the sham group (n=6), CLP group (n=6), NBP group (n=6) and NBP+LY294002 group (n=6). The rat model of severe sepsis was established by cecal ligation and puncture (CLP). Abdominal wall incisions and sutures were performed in the first group, and CLP was performed in the latter three groups. Normal saline/NBP/NBP+LY294002 solution was injected intraperitoneally 2h or 1h before modeling. Hemodynamic data (blood pressure and heart rate) were recorded at 0, 2, 4 and 6h. Sidestream dark field (SDF) imaging and the Medsoft System were used to observe the intestinal microcirculation of rats and obtain data at 0, 2, 4, and 6h. Six hours after the model was established, the serum levels of TNF-α and IL-6 were measured to evaluate the level of systemic inflammation. Pathological damage to the small intestine was evaluated by electron microscopy and histological analysis. The expression levels of P-PI3K, PI3K, P-AKT, AKT, LC3 and p62 in the small intestine were analyzed by Western blotting. The expressions of P-PI3K, P-AKT, LC3 and P62 in small intestine were detected by immunohistochemical staining. NBP improved intestinal microcirculation disturbances in septic rats, alleviated the systemic inflammatory response, reduced the destruction of the small intestinal mucosa and the disruption of microvascular endothelial cells, and alleviated autophagy in vascular endothelial cells. NBP increased the ratio of P-PI3K/total PI3K, P-AKT/total AKT, and P62/β-actin and decreased the ratio of LC3 II/LC3 I. NBP ameliorated intestinal microcirculation disturbances and the destruction of small intestinal vascular endothelial cells in septic rats by activating the PI3K/Akt signaling pathway and regulating autophagy.

Abstract 132: Mechanism By Which Coagulation Factor XI Regulates Endothelial Cell Permeability And Barrier Function In Vitro And In Vivo

Introduction: Proteolysis of vascular endothelial (VE)-cadherin is a biomarker for disruption of endothelial cell (EC) barrier function, with elevated levels of soluble VE (sVE)-cadherin associated with atherosclerosis and sepsis. Factor XI (FXI) plays a regulatory role in inflammation, and while a relationship between FXI activity and VE-cadherin expression levels has been observed, the mechanism by which this process is mediated is unknown. Hypothesis: Activated FXI (FXIa) induces VE-cadherin proteolysis. Methods: Human umbilical vein ECs were stimulated with FXIa (30 and 5 nM) for six hours. Western blot analysis was used to study VE-cadherin proteolysis, disintegrin and metalloproteinase 10 (ADAM10) expression, FXIa-PAI-1 or -very low density lipoprotein receptor (VLDLR) interactions, and cell signaling events. SVE-cadherin in baboons challenged with Staphylococcus aureus was detected by ELISA. Results: FXIa (5 nM) generated a VE-cadherin fragment and increased ECs permeability; the serine protease inhibitor, PPACK, and an inhibitor of ADAM10 reversed these processes. FXIa induced the expression of active ADAM10 on ECs. The receptor-associated protein (RAP) blocked FXIa-induced VE-cadherin proteolysis by preventing FXIa interaction with PAI-1 and VLDLR on ECs. FXIa-VLDLR interaction induced Dab1 and Src kinases phosphorylation. FXIa activated the VEGFR2-PLCgamma1-ERK1/2 signaling pathway. The ERK1/2 inhibitor, LY3214996, and the Src kinases inhibitor, PP2, inhibited VE-cadherin proteolysis by FXIa. PLCgamma1 phosphorylation was also prevented with PP2. The VEGFR2 kinase inhibitor, SU1498, prevented the cleavage of VE-cadherin by FXIa; in contrast, a VEGF blocking antibody had no effect on FXIa-induced VE-cadherin cleavage. Finally, the inhibition of FXI activation in a baboon model of sepsis significantly decreased the levels of sVE-cadherin. Conclusion: The complex formation of FXIa with PAI-1 and VLDLR promoted Src kinase phosphorylation. Src activation triggered the VEGFR2-PLCgamma1-ERK1/2 signaling pathway, which induced the trafficking of active ADAM10 to the EC surfaces inducing the cleavage of VE-cadherin. FXI may be a druggable target to protect EC barrier function in inflammatory diseases.

Liver alterations and detection of SARS-CoV-2 RNA and proteins in COVID-19 autopsies

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

Activating NO-sGC crosstalk in the mouse vascular niche promotes vascular integrity and mitigates acute lung injury.

Disruption of endothelial cell (ECs) and pericytes interactions results in vascular leakage in acute lung injury (ALI). However, molecular signals mediating EC-pericyte crosstalk have not been systemically investigated, and whether targeting such crosstalk could be adopted to combat ALI remains elusive. Using comparative genome-wide EC-pericyte crosstalk analysis of healthy and LPS-challenged lungs, we discovered that crosstalk between endothelial nitric oxide and pericyte soluble guanylate cyclase (NO-sGC) is impaired in ALI. Indeed, stimulating the NO-sGC pathway promotes vascular integrity and reduces lung edema and inflammation-induced lung injury, while pericyte-specific sGC knockout abolishes this protective effect. Mechanistically, sGC activation suppresses cytoskeleton rearrangement in pericytes through inhibiting VASP-dependent F-actin formation and MRTFA/SRF-dependent de novo synthesis of genes associated with cytoskeleton rearrangement, thereby leading to the stabilization of EC-pericyte interactions. Collectively, our data demonstrate that impaired NO-sGC crosstalk in the vascular niche results in elevated vascular permeability, and pharmacological activation of this crosstalk represents a promising translational therapy for ALI.

Differential Effects of Vascular Endothelial Growth Factor on Glycocalyx of Endothelial and Tumor Cells Uncovered by Super‐resolution Optical Microscopy and Potential Targets for Tumor Metastasis

On the surface of every mammalian cell, there is a matrix‐like glycocalyx (GCX) consisting of proteoglycans and glycosaminoglycans (GAGs). Disruption of endothelial cell (EC) GCX by vascular endothelial growth factor (VEGF, VEGF‐A165), a tumor secretion, was found to be an early event in tumor cell (TC) metastasis across vascular barriers. But how the TC secretion VEGF affects its own GCX is unknown although bulkier GCX of tumor cells was found to enhance its metastasis. To investigate the VEGF effect on TC GCX and to elucidate the ultrastructural organization of EC and TC GCX and their alteration by VEGF, we employed super‐resolution stochastic optical reconstruction microscopy (STORM) to observe the spatio‐chemical organizations of the heparan sulfate (HS) and hyaluronic acid (HA), two representative GAGs of GCX, on human cerebral microvascular endothelial cells (hCMEC) and malignant breast cancer cells MDA‐MB‐231 (MB231). We found that HS and HA have distinct organizations on hCMEC and MB231. Only HS of hCMEC is perpendicular to the cell surface, while HA of hCMEC, as well as HS and HA of MB231 all lie in the same plane as the cell surface where they appear to weave into a 2D network covering the cell. We also found that VEGF significantly reduces the length and coverage of HS on hCMEC but does not change the thickness and coverage of HA on hCMEC. On the contrary, VEGF significantly enhances the coverage of HS and HA on MB231 although it does not alter the thickness. The differential effects of VEGF on the GCX of TC and that of EC may favor TC adhesion and transmigration across EC barriers for their metastasis.

Abstract 133: Coagulation FXI Regulates Endothelial Cell Barrier Function By Inducing VE-cadherin Proteolysis In An ADAM10-dependent Manner

Introduction: Proteolysis of vascular endothelial (VE)-cadherin is a biomarker for disruption of endothelial cell (EC) barrier function, with elevated levels of soluble VE-cadherin associated with atherosclerosis and sepsis. Factor XI (FXI), known for its role in the coagulation cascade, also plays a regulatory role in inflammation. While a relationship between FXI activity, VE-cadherin expression levels, and endothelial barrier function has been observed, the mechanisms by which this process is mediated are unknown. Hypothesis: Activated FXI (FXIa) regulates EC barrier function by inducing VE-cadherin proteolysis. Methods: Human umbilical vein endothelial cells were stimulated with FXIa (30 and 5 nM) for six hours. VE-cadherin proteolysis and expression levels were analyzed by Western blot and immunofluorescence. ECs permeability was quantified by measuring the flux of Evans blue-albumin across the ECs. Immunoprecipitation and Western blot analysis were used to study FXIa-PAI-1 or -very low density lipoprotein receptor (VLDLR) interactions. Results: FXIa (5 nM) generated a VE-cadherin C-terminal fragment and decreased VE-cadherin expression on ECs; this process was reversed by the serine protease inhibitor, PPACK. Thrombin, kallikrein, or FXIIa did not induce VE-cadherin proteolysis. FXIa increased ECs permeability, yet did not induce the expression of VCAM-1. In contrast, while VCAM-1 expression was upregulated by tumor necrosis factor alpha or vascular endothelial growth factor, neither of these induced VE-cadherin proteolysis. A pharmacological inhibitor of a disintegrin and metalloproteinase 10 (ADAM10) abrogated the effects of FXIa on ECs, including proteolysis of VE-cadherin. Similarly, the low density lipoprotein antagonist, receptor-associated protein (RAP), inhibited FXIa-induced VE-cadherin proteolysis by preventing FXIa complex formation with PAI-1 and VLDLR on ECs. Conclusion: These results suggest that FXIa disrupts VE-cadherin-mediated EC permeability by increasing ADAM10 activity through complex formation and internalization by PAI-1 and VLDLR. It remains to be seen whether this pathway represents a druggable target to protect EC barrier function in inflammatory diseases by inhibiting FXIa.

Implications of fibrotic extracellular matrix in diabetic retinopathy.

Fibrosis is an accumulation of extracellular matrix (ECM) proteins and fibers in a disordered fashion, which compromises cell and tissue functions. High glucose-induced fibrosis, a major pathophysiological change of diabetic retinopathy (DR), severely affects vision by compromising the retinal vasculature and ultimately disrupting retinal tissue organization. The retina is a highly vascularized, stratified tissue with multiple cell types organized into distinct layers. Chronically high blood glucose stimulates certain retinal cells to increase production and assembly of ECM proteins resulting in excess ECM deposition primarily in the capillary walls on the basal side of the endothelium. This subendothelial fibrosis of the capillaries is the earliest histological change in the diabetic retina and has been linked to the vascular dysfunction that underlies DR. Proteins that are not normally abundant in the capillary basement membrane (BM) matrix, such as the ECM protein fibronectin, are assembled in significant quantities, disrupting the architecture of the BM and altering its properties. Cell culture models have identified multiple mechanisms through which elevated glucose can stimulate fibronectin matrix assembly, including intracellular signaling pathways, alternative splicing, and non-enzymatic glycation of the ECM. The fibrotic subendothelial matrix alters cell adhesion and supports further accumulation of other ECM proteins leading to disruption of endothelial cell-cell junctions. We review evidence supporting the notion that these molecular changes in the ECM contribute to the pathogenesis of DR, including vascular leakage, loss of endothelial cells and pericytes, changes in blood flow, and neovascularization. We propose that the accumulation of ECM, especially fibronectin matrix, first around the vasculature and later in extravascular locations, plays a critical role in DR and vision loss. Strategies for DR prevention and treatment should consider the ECM a potential therapeutic target.

Differential effects of vascular endothelial growth factor on glycocalyx of endothelial and tumor cells and potential targets for tumor metastasis

On the surface of every mammalian cell, there is a matrix-like glycocalyx (GCX) consisting of proteoglycans and glycosaminoglycans (GAGs). Disruption of endothelial cell (EC) GCX by a vascular endothelial growth factor (VEGF, VEGF-A165), a tumor secretion, was found to be an early event in tumor cell (TC) metastasis across vascular barriers. However, how the TC secretion VEGF affects its own GCX is unknown. To investigate the VEGF effect on TC GCX and to elucidate the ultrastructural organization of EC and TC GCX and their alteration by VEGF, we employed super-resolution stochastic optical reconstruction microscopy to observe the spatio-chemical organizations of the heparan sulfate (HS) and hyaluronic acid (HA), two representative GAGs of GCX, on human cerebral microvascular endothelial cells (hCMEC) and malignant breast cancer cells MDA-MB-231 (MB231). We found that HS and HA have distinct organizations on hCMEC and MB231. Only HS of hCMEC is perpendicular to the cell surface, while HA of hCMEC as well as HS and HA of MB231 all lie in the same plane as the cell surface where they appear to weave into a 2D network covering the cell. We also found that VEGF significantly reduces the length and coverage of HS on hCMEC but does not change the thickness and coverage of HA on hCMEC. On the contrary, VEGF significantly enhances the coverage of HS and HA on MB231 although it does not alter the thickness. The differential effects of VEGF on the GCX of TC and that of EC may favor TC adhesion and transmigration across EC barriers for their metastasis.

Ebola Virus GP Activates Endothelial Cells via Host Cytoskeletal Signaling Factors.

Ebola virus disease (EVD) is a lethal disease caused by the highly pathogenic Ebola virus (EBOV), and its major symptoms in severe cases include vascular leakage and hemorrhage. These symptoms are caused by abnormal activation and disruption of endothelial cells (ECs) whose mediators include EBOV glycoprotein (GP) without the need for viral replication. However, the detailed molecular mechanisms underlying virus–host interactions remain largely unknown. Here, we show that EBOV-like particles (VLPs) formed by GP, VP40, and NP activate ECs in a GP-dependent manner, as demonstrated by the upregulation of intercellular adhesion molecules-1 (ICAM-1) expression. VLPs-mediated ECs activation showed a different kinetic pattern from that of TNF-α-mediated activation and was associated with apoptotic ECs disruption. In contrast to TNF-α, VLPs induced ICAM-1 overexpression at late time points. Furthermore, screening of host cytoskeletal signaling inhibitors revealed that focal adhesion kinase inhibitors were found to be potent inhibitors of ICAM-1 expression mediated by both TNF-α and VLPs. Our results suggest that EBOV GP stimulates ECs to induce endothelial activation and dysfunction with the involvement of host cytoskeletal signaling factors, which represent potential therapeutic targets for EVD.

Soluble vascular cell adhesion molecule-1 levels and severity of dengue hemorrhagic fever in children

Background The clinical manifestations of dengue infection vary widely, ranging from asymptomatic to severe forms that can cause death. In severe infections, the expression of soluble vascular cell adhesion molecule-1 (sVCAM-1) in endothelial cells is reportedly excessive, causing endothelial cell gaps through VE-cadherin and plasma leakage, which is the basic mechanism for shock in dengue hemorrhagic fever (DHF).
 Objective To determine the association between sVCAM-1 levels and severity of dengue hemorrhagic fever in children.
 Methods This cross-sectional study was done in children with DHF at Dr. M. Djamil Hospital, Padang, West Sumatera. Subjects were diagnosed according to the 2011 WHO criteria and selected by consecutive sampling. They were grouped as DHF with or without shock. Examination of sVCAM-1 levels was done by ELISA method. Mann-Whitney test with a significance of P<0.05 was used for statistical analysis.
 Results A total of 66 patients were collected from January 2018 to December 2019, but 2 patients were excluded. The 64 subjects who met the inclusion criteria consisted of 32 (50%) DHF without shock and 32 (50%) DHF with shock. Median sVCAM-1 was significantly higher in the DHF with shock group (840 ng/mL) than in DHF without shock group (598 ng/mL) (P<0.05).
 Conclusion There was a significant association between higher sVCAM-1 levels and greater severity of dengue hemorrhagic fever in children.

Critical Role of Caveolin-1 Loss/Dysfunction in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a rare disease with a high morbidity and mortality rate. A number of systemic diseases and genetic mutations are known to lead to PH. The main features of PH are altered vascular relaxation responses and the activation of proliferative and anti-apoptotic pathways, resulting in pulmonary vascular remodeling, elevated pulmonary artery pressure, and right ventricular hypertrophy, ultimately leading to right heart failure and premature death. Important advances have been made in the field of pulmonary pathobiology, and several deregulated signaling pathways have been shown to be associated with PH. Clinical and experimental studies suggest that, irrespective of the underlying disease, endothelial cell disruption and/or dysfunction play a key role in the pathogenesis of PH. Endothelial caveolin-1, a cell membrane protein, interacts with and regulates several transcription factors and maintains homeostasis. Disruption of endothelial cells leads to the loss or dysfunction of endothelial caveolin-1, resulting in reciprocal activation of proliferative and inflammatory pathways, leading to cell proliferation, medial hypertrophy, and PH, which initiates PH and facilitates its progression. The disruption of endothelial cells, accompanied by the loss of endothelial caveolin-1, is accompanied by enhanced expression of caveolin-1 in smooth muscle cells (SMCs) that leads to pro-proliferative and pro-migratory responses, subsequently leading to neointima formation. The neointimal cells have low caveolin-1 and normal eNOS expression that may be responsible for promoting nitrosative and oxidative stress, furthering cell proliferation and metabolic alterations. These changes have been observed in human PH lungs and in experimental models of PH. In hypoxia-induced PH, there is no endothelial disruption, loss of endothelial caveolin-1, or enhanced expression of caveolin-1 in SMCs. Hypoxia induces alterations in membrane composition without caveolin-1 or any other membrane protein loss. However, caveolin-1 is dysfunctional, resulting in cell proliferation, medial hypertrophy, and PH. These alterations are reversible upon removal of hypoxia, provided there is no associated EC disruption. This review examined the role of caveolin-1 disruption and dysfunction in PH.

SARS-CoV-2 Infection in Hereditary Hemorrhagic Telangiectasia Patients Suggests Less Clinical Impact Than in the General Population.

At the moment of writing this communication, the health crisis derived from the COVID-19 pandemic has affected more than 120 million cases, with 40 million corresponding to Europe. In total, the number of deaths is almost 3 million, but continuously rising. Although COVID-19 is primarily a respiratory disease, SARS-CoV-2 infects also endothelial cells in the pulmonary capillaries. This affects the integrity of the endothelium and increases vascular permeability. In addition, there are serious indirect consequences, like disruption of endothelial cells’ junctions leading to micro-bleeds and uncontrolled blood clotting. The impact of COVID-19 in people with rare chronic cardiovascular diseases is unknown so far, and interesting to assess, because the virus may cause additional complications in these patients. The aim of the present work was to study the COVID-19 infection among the patients with Hereditary Hemorrhagic Telangiectasia (HHT). A retrospective study was carried out in a 138 HHT patients’ sample attending an Ear Nose and Throat (ENT) reference consult. The evaluation of the COVID-19 infection in them reveals milder symptoms; among the 25 HHT patients who were infected, only 3 cases were hospitalized, and none of them required ICU or ventilation assistance. The results are discussed in the light of macrophage immune response.