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Blood-CNS barriers protect the CNS from circulating immune cells and damaging molecules. It is thought barrier integrity becomes disrupted with aging, contributing to impaired CNS function. Using genome-wide and targeted molecular approaches, we found aging affected expression of predominantly immune invasion and pericyte-related genes in CNS regions investigated, especially after middle age, with spinal cord being most impacted. We did not find significant perturbation of endothelial cell junction genes or proteins, nor were vascular density or pericyte coverage affected by aging. We evaluated barrier paracellular permeability using small molecular weight tracers, serum protein extravasation, CNS water content, and iron labelling measures. We found no evidence for age-related increased barrier permeability in any of these tests. We conclude that blood-brain (BBB) and blood-spinal cord barrier (BSCB) paracellular permeability does not increase with normal aging in mouse. Whilst expression changes were not associated with increased permeability, they may represent an age-related primed state whereby additional insults cause increased leakiness., Competing Interests: Declarations. Ethics approval: All animal work was undertaken in strict accordance with the University of Newcastle Animal Ethics Committee (protocol numbers: A-2015–526, A-2021–137), and New South Wales and Australian animal research guidelines. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Background: Ischemia-reperfusion injury (IRI) stands as a major trigger for primary graft dysfunction (PGD) in lung transplantation (LTx). Especially in LTx from donation after cardiac death (DCD), effective control of IRI following warm ischemia (WIRI) is crucial to prevent PGD. This study aimed to identify the key factors affecting WIRI in LTx from DCD., Methods: Previously reported RNA-sequencing dataset of lung WIRI was reanalyzed to identify nuclear receptor subfamily 4 group A member 1 (NR4A1) as the immediate early gene for WIRI. Dynamics of NR4A1 expression were verified using a mouse hilar clamp model. To investigate the role of NR4A1 in WIRI, a mouse model of LTx from DCD was established using Nr4a1 knockout (Nr4a1 -/- ) mice., Results: NR4A1 was located around vascular cells, and its protein levels in the lungs increased rapidly and transiently during WIRI. LTｘ from Nr4a1 -/- donors significantly improved pulmonary graft function compared to wild-type donors. Histological analysis showed decreased microvascular endothelial cell death, neutrophil infiltration, and albumin leakage. Evans blue permeability assay demonstrated maintained pulmonary microvascular barrier integrity in grafts from Nr4a1 -/- donors, correlating with diminished pulmonary edema. However, NR4A1 did not significantly affect the inflammatory response during WIRI, and IRI was not suppressed when a wild-type donor lung was transplanted into the Nr4a1 -/- recipient., Conclusions: Donor NR4A1 plays a specialized role in the positive regulation of endothelial cell injury and microvascular hyperpermeability. These findings demonstrate the potential of targeting NR4A1 interventions to alleviate PGD and improve outcomes in LTx from DCD., Competing Interests: Disclosure statement The authors have no conflicts of interest to disclose. This study was supported by the Japan Society for the Promotion of Science KAKENHI (Grant no. 20KK0203 and 23K08320 to M.O.). We thank Tetsuo Kawakami and staff of the Department of Animal Resources, Advanced Science Research Center, Okayama University., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Angiogenesis, the formation of new blood microvessels, is a necessary physiological process for tissue generation and repair. Sufficient blood supply to the tissue is dependent on microvascular density, while the material exchange between the circulating blood and the surrounding tissue is controlled by microvascular permeability. We thus begin this article by reviewing the key signaling factors, particularly vascular endothelial growth factor (VEGF), which regulates both angiogenesis and microvascular permeability. We then review the role of angiogenesis in tissue growth (bone regeneration) and wound healing. Finally, we review angiogenesis as a pathological process in tumorigenesis, intraplaque hemorrhage, cerebral microhemorrhage, pulmonary fibrosis, and hepatic fibrosis. Since the glycocalyx is important for both angiogenesis and microvascular permeability, we highlight the role of the glycocalyx in regulating the interaction between tumor cells and endothelial cells (ECs) and VEGF-containing exosome release and uptake by tumor-associated ECs, all of which contribute to tumorigenesis and metastasis., (Copyright © 2025 Cold Spring Harbor Laboratory Press; all rights reserved.)

The activation of the bradykinin type 2 receptor is intricately involved in acute post-ischemic inflammatory responses. However, its precise role in different stages of ischemic injury, especially in the chronic phase, remains unclear. Following simultaneous cerebral and retinal ischemia, bradykinin type 2 receptor knockout mice and their controls were longitudinally monitored for 35 days via magnetic resonance imaging, fundus photography, fluorescein angiography, behavioral assessments, vascular permeability measurements, and immunohistochemistry, as well as glycemic status assessments. Without impacting the lesion size, bradykinin type 2 receptor deficiency reduced acute cerebral vascular permeability preventing the loss of pericytes and tight junctions. In the chronic phase of ischemia, however, it resulted in increased astrogliosis and cortical neuronal loss, as well as higher functional deficits. The retinal findings demonstrated a similar pattern. Bradykinin type 2 receptor deficiency delayed, but exacerbated the development of retinal necrosis, increased subacute vascular permeability, and promoted retinal ganglion cell loss in the chronic phase of ischemia. This investigation sheds light on the temporal dynamic of bradykinin type 2 receptor effects in ischemia, pointing to a therapeutic potential in the subacute and chronic phases of ischemic injury., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: Dynamic susceptibility contrast (DSC) MRI is commonly part of the clinical brain tumor imaging protocol. Usually, a preload of contrast agent is administered to minimize contrast-leakage T 1 effects. However, recent studies have indicated that with adaptation of scan parameters (in particular, low flip angle), a preload is not required. For advanced approaches, like MR vascular fingerprinting (MRVF), which is based on combining gradient-echo and spin-echo readouts, excitation flip angles cannot easily be reduced, but postprocessing might alleviate leakage effects. In this study, the aim was to verify whether DSC-MRVF can be performed without preload., Methods: A previously established simulation model was used to create a dictionary of DSC-MRVF signals resulting from a double-bolus injection with varying input values for permeability, vessel radius, and relative cerebral blood volume (rCBV). A sensitivity analysis based on a Cramer Rao Lower Bound approach was performed, from which the logarithmic ratio of standard deviations for parameter estimation was derived. A ratio above zero corresponded with a better determination without preload. Two brain tumor patients were included for validation., Results: The results show that the standard deviation for determining permeability was lower without preload, whereas for determining rCBV, the opposite result was found. For vessel radius, the standard deviation was similar with and without preload. The in vivo data reasonably agreed with the simulation results., Conclusion: This work has shown the potential of DSC-MRVF with a single bolus injection for determining vessel radius and permeability, although for rCBV, a preload is still advisable., (© 2024 The Author(s). Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Background: Cerebral blood flow normally places a limit on the magnitude of brain vascular permeability (P) that can be measured in vivo. At normal cerebral blood flow, this limit falls at the lower end of lipophilicity for most FDA-approved CNS drugs. In this study, we report on two methods that can be used to overcome this limitation and measure brain vascular permeability values that are up to ~1000 times higher using the in situ brain perfusion technique., Methods: Rat brain was perfused with physiological saline at increased flow rate and in the presence of various concentrations of plasma protein, serum albumin or alpha-acid glycoprotein. Plasma protein was added to the saline perfusion fluid to lower extraction into the measurable range using the Crone Renkin "diffusion-flow" equation to calculate brain P o S., Results: Cerebrovascular P o was determined for 125 solutes, of which 78 showed little or no evidence of active efflux transport. Fifty of the solutes were in the lipophilicity zone (Log P oct 1-5) of most FDA-approved CNS drugs. Care was taken to ensure the integrity of the brain vasculature during perfusion and to measure flow accurately using markers that had been verified for the flow rates. The results showed a linear relationship between Log P o and Log P oct over ~10 orders of magnitude with values for diazepam, estradiol, testosterone, and other agents that exceed prior published values by fivefold to 200-fold., Conclusions: The results show that brain vascular permeability can be measured directly in vivo for highly lipophilic solutes and the PS values obtained match reasonably with that predicted by the Crone-Renkin flow diffusion equation with care taken to validate the accuracy for the component measurements and with no need to invoke "enhanced" or "induced" dissociation., Competing Interests: Declarations. Ethics approval and consent to participate: All experiments were performed in accordance with guidelines and protocols approved by the Institutional Animal Care and Use Committee. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

We used spontaneously hypertensive rats (SHR) as a hypertensive cerebral small vessel disease (CSVD) model to quantify blood-brain barrier (BBB) disruption by 11.7TMR T1mapping and to investigate white matter lesions and microangiopathy in CSVD. Male SHR were used as a hypertensive CSVD animal model and normotensive Wistar-Kyoto rats (WKY) were used as a control model. After 18 weeks, the rats did the Morris water maze test were evaluated, blood-brain barrier (BBB) integrity were evaluated by using Bruker 11.7T MR T1 mapping. ITK-SNAP software was used to measure hippocampal volume. Then, pathological analysis was carried out on rats, myelin integrity, vascular permeability and microvessel density were assessed by immunohistochemistry. Our data showed that hypertensive CSVD model exhibited decreased memory function, BBB leakage could be detected differently in different brain regions, and T1 values of the hippocampus showed the greatest drop than other areas. Furthermore, the pathological changes in small vessels were more extensive, average optical density of myelin basic protein (MBP) in the white matter of SHR group was significantly reduced, moreover, VEGFR2 immunoreactivity scores (IRS) and CD34-assessed MVD in SHR group were significantly higher than WKY group. We find different parts of the brain tissues have different degrees of BBB leakage, hippocampal atrophy and hippocampal volume were decreased in hypertensive CSVD by using T1 Mapping. Loss of myelin integrity, vascular permeability increased and microangiopathy may contribute to hypertensive-related BBB functional deficits in CSVD model., Competing Interests: Declarations. Ethics approval: Animal studies were reported following ARRIVE guidelines. All the experiments were conducted according to the standards recommended by the United States National Institute of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee at Shanghai Pudong Hospital, Fudan University Pudong Medical Center (No.2020QKW-03). Consent for publication : All the authors agreed to publish the article. Consent to participate: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Contradictory results have been reported about the effects of liver diseases on the blood-brain barrier (BBB) permeability to markers. For instance, both an increase and no change in the BBB permeability to BBB markers sodium fluorescein and Evans blue have been reported in experimental cholestasis induced by bile duct ligation (BDL) in rats. These contradictory effects might be due to inherent limitations of these markers and/or methodological issues. Here, we investigated the time course of the impact of BDL in rats on BBB permeability using a recently developed stable isotope labeled marker [ 13 C]sucrose, which is expected to be devoid of limitations of other markers, such as sodium fluorescein. At various times (five days, two weeks, and four weeks) after BDL or sham surgery, the brain uptake clearance (K in ) of [ 13 C]sucrose was estimated using quantitation of the marker in plasma, blood, and brain by a specific LC-MS/MS analytical method. BDL caused substantial increases in the plasma concentrations of liver biochemical markers (bilirubin, total bile acids, ammonia, and cholesterol) and reduced liver cytochrome P450 content and metabolic activities. However, compared with the sham group, the plasma or blood AUC, brain concentrations, and K in of [ 13 C]sucrose in BDL animals remained unchanged at all the studied times. Additionally, we observed a negative correlation between the sucrose K in and plasma total bile acids concentrations in the BDL animals. It is concluded that cholestatic liver disease, induced by BDL surgery in rats, does not significantly affect the BBB permeability to sucrose up to 4 weeks after the surgery., Competing Interests: Declarations. Ethics approval: All the animal procedures used in this study were approved by Texas Tech University Health Sciences Center’s Institutional Animal Care and Use Committee and were consistent with the guidelines set by the National Research Council (USA) Committee for the Update of the Guide for the Care and Use of Laboratory Animals (8th edition, 2011). Consent to participate: Not applicable. Consent for publication: All authors have read and approved the manuscript. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).)

Background: HAdV-7 is a prevalent pathogen that can cause severe pneumonia in children. Previous studies have shown a significant increase in serum levels of vascular permeability factor (VPF/VEGF) and viral load in pediatric patients with fatal HAdV-7 infection, suggesting potential damage to the pulmonary vascular endothelium. Further research is necessary to elucidate the underlying mechanism., Methods: The human lung microvascular endothelial cell line-5a and human CD46 mice were used for in vitro and in vivo experiments, respectively. RNA-seq was employed for correlative omics analysis. Viral infection and copy status were examined using transmission electron microscopy to observe virus particles, immunofluorescence to detect the viral protein Hexon, and qPCR to assess HAdV-7 fiber gene copies. Various methods, including ELISAs for VEGF and other injury markers, the CCK8 assay for cell viability, and flow cytometry for endothelium numbers, were employed to evaluate endothelial damage. Acute lung injury severity was evaluated by scoring pathological inflammation and measuring pulmonary vascular permeability. Autophagy activation was assessed by observing autophagosomes and validating marker proteins., Results: GSEA analysis showed significant enrichment of gene sets related to endothelial functions (barrier, defense, and regeneration) and ALI in the HAdV-7-infected group. GO analysis indicated an enrichment of autophagy-related pathways linked to cell death. Subsequently, successful signs of HAdV-7 infection and replication were observed in the endothelium, including cytopathic effects, intracellular virions, and increased HAdV-7 fiber gene copies. Endothelial injury, including mitochondrial damage, decreased endothelium, and elevated levels of endothelial injury markers such as VEGF, sICAM-1, sVCAM-1, E-selectin, ESM1, MCP1, and IL1β were observed after HAdV-7 infection. Additionally, evidence of leaky lung blood vessels and ALI was observed, including progressive weight loss, elevated pulmonary vascular permeability, and severe lung consolidation. Furthermore, HAdV-7 infection induced autophagosome formation in the endothelium and triggered complete cell autophagy. Importantly, inhibiting autophagic flux reduced VEGF levels and other endothelial injury markers, decreased viral load, improved cell survival rate, alleviated pulmonary vessel leakage, and mitigated lung inflammation., Conclusions: HAdV-7 successfully infects pulmonary vascular endothelium and replicates effectively, causing injury to the endothelium, high VEGF expression and viral load in the serum, as well as ALI/ARDS. Autophagy inhibitors can alleviate endothelial injury, inhibit viral replication, relieve leakage from the vasculature, and reduce lung inflammation., Competing Interests: Declarations. Ethics approval and consent to participate: All procedures involving animals were approved by the Experimental Animal Committee of Chongqing Medical University [permits no. SYXK (Yu) 2022-0016]. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Dynamic monitoring of the blood-brain barrier (BBB) functional status in septic mice can help to explore the pathological mechanisms. Therefore, we proposed a new method for monitoring BBB permeability and applied it to the detection of sepsis models., Methods: The new method involves the construction of an optical cranial window and in vivo imaging. We performed dynamic monitoring of BBB permeability and cerebral blood flow (CBF) in cecal ligation puncture (CLP) and endotoxemia (lipopolysaccharide [LPS]) mice., Results: The sensitivity and accuracy of this method were higher than those of Evans blue evaluation. The increase of BBB permeability in the group of CLP mice was relatively mild and correlated with overall survival, and the damage was irreversible. Contrarily, BBB damage in the LPS group was more acute and severe, unrelated to overall survival, but recoverable. The CBF decreased significantly in both model mouse groups 24 h after modeling, but only the CBF proportion decrease in the LPS group was significantly correlated with an increase in BBB permeability. Within 24 h after both models were established, the decrease in blood flow in the digestive organs occurred earlier than in the brain and kidneys, and the decrease in small intestine blood flow in the LPS group progressed faster., Conclusions: We have successfully demonstrated the feasibility of our novel method to detect BBB permeability in mice. Our results revealed a significant difference in the BBB permeability change trend between the CLP and LPS model mice when survival curves were consistent. Notably, the CLP-model mice demonstrated a closer resemblance to clinical patients. Our findings suggest that early-stage brain tissue hypoperfusion has a greater impact on BBB function damage in endotoxemia mice, which is related to the faster progression of blood flow redistribution., Competing Interests: Conflict of interest: The authors declare that they have no conflict of interest. Human and Animal Rights: The Animal Ethics Committee of Central South University approved and monitored this experimental protocol (CSU-2023–00092023.01.03 review)., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society.)

Exposure to VEGF-A 165 a over several days leads to a persistent dysfunction of the very tight barrier formed by immortalized endothelial cells of the bovine retina (iBREC). Elevated permeability of the barrier is indicated by low cell index values determined by electric cell-substrate impedance measurements, by lower amounts of claudin-1, and by disruption of the homogenous and continuous staining of vascular endothelial cadherin at the plasma membrane. Because of findings that suggest modulation of VEGF-A's detrimental effects on the inner blood-retina barrier by the angiogenic growth factor angiopoietin-2, we investigated in more detail in vitro whether this growth factor indeed changes the stability of the barrier formed by retinal endothelial cells or modulates effects of VEGF-A. In view of the clinical relevance of anti-VEGF therapy, we also studied whether blocking VEGF-A-driven signaling is sufficient to prevent barrier dysfunction induced by a combination of both growth factors. Although angiopoietin-2 stimulated proliferation of iBREC, the formed barrier was not weakened at a concentration of 3 nM: Cell index values remained high and expression or subcellular localization of claudin-1 and vascular endothelial cadherin, respectively, were not affected. Angiopoietin-2 enhanced the changes induced by VEGF-A 165 a and this was more pronounced at lower concentrations of VEGF-A 165 a. Specific inhibition of the VEGF receptors with tivozanib as well as interfering with binding of VEGF-A to its receptors with bevacizumab prevented the detrimental effects of the growth factors; dual binding of angiopoietin-2 and VEGF-A by faricimab was marginally more efficient. Uptake of extracellular angiopoietin-2 by iBREC can be efficiently prevented by addition of faricimab which is also internalized by the cells. Exposure of the cells to faricimab over several days stabilized their barrier, confirming that inhibition of VEGF-A signaling is not harmful to this cell type. Taken together, our results confirm the dominant role of VEGF-A 165 a in processes resulting in increased permeability of retinal endothelial cells in which angiopoietin-2 might play a minor modulating role., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This investigation evaluated the microvascular permeability and ultrastructure of skeletal muscle capillaries in the skeletal muscle of diabetic (DIA) rats using two-photon laser scanning microscopy (TPLSM) and transmission electron microscopy (TEM). Microvascular permeability was assessed in the tibialis anterior muscle of control (CON) and DIA (streptozocin) male Wistar rats ( n = 20, 10-14 wk) by in vivo imaging using TPLSM after fluorescent dye intravenous infusion. Fluorescent dye leakage was quantified to determine microvascular permeability. The ultrastructure was imaged by TEM ex vivo to calculate the size and number of intercellular clefts between capillary endothelial cells and also intracellular vesicles. Compared with control, the volumetrically determined interstitial fluorescent dye leakage, the endothelial cell thickness, and the number of intercellular clefts per capillary perimeter were significantly higher, and the cleft width was significantly narrower in tibialis anterior (TA) of DIA (interstitial fluorescent dye leakage, 2.88 ± 1.40 vs. 10.95 ± 1.41 µm 3 × min × 10 6 ; endothelial thickness, 0.28 ± 0.02 vs. 0.45 ± 0.03 µm; number of intercellular clefts per capillary perimeter, 6.3 ± 0.80 vs. 13.6 ± 1.7/100 µm; cleft width, 11.92 ± 0.95 vs. 8.40 ± 1.03 nm, CON vs. DIA, respectively, all P < 0.05). The size of intracellular vesicles in the vascular endothelium showed an increased proportion of large vesicles in the DIA group compared with the CON group ( P < 0.05). Diabetes mellitus enhances the microvascular permeability of skeletal muscle microvessels due, in part, to a higher density and narrowing of the endothelial intercellular clefts, and larger intracellular vesicles. NEW & NOTEWORTHY Microvascular permeability in diabetic muscle was investigated using our original two-photon scanning laser microscopy method. Compared with controls, the leakage volume was increased in diabetic muscle, which was atrophic with smaller capillary diameter, endothelial cell thickening, and the appearance of more endothelial intercellular gaps or clefts, and large vesicles. Hyperpermeability was closely related to ultrafine structural changes of the capillary endothelial cell junctions.

Cells depend on precisely regulating barrier function within the vasculature to maintain physiological stability and facilitate essential substance transport. Endothelial cells achieve this through specialized adherens and tight junction protein complexes, which govern paracellular permeability across vascular beds. Adherens junctions, anchored by vascular endothelial (VE)-cadherin and associated catenins to the actin cytoskeleton, mediate homophilic adhesion crucial for barrier integrity. In contrast, tight junctions composed of occludin, claudin, and junctional adhesion molecule A interact with Zonula Occludens proteins, reinforcing intercellular connections essential for barrier selectivity. Endothelial cell-cell junctions exhibit dynamic conformations during development, maturation, and remodeling, regulated by local biochemical and mechanical cues. These structural adaptations play pivotal roles in disease contexts such as chronic inflammation, where junctional remodeling contributes to increased vascular permeability observed in conditions from cancer to cardiovascular diseases. Conversely, the brain microvasculature's specialized junctional arrangements pose challenges for therapeutic drug delivery due to their unique molecular compositions and tight organization. This commentary explores the molecular mechanisms underlying endothelial cell-cell junction conformations and their implications for vascular permeability. By highlighting recent advances in quantifying junctional changes and understanding mechanotransduction pathways, we elucidate how physical forces from cellular contacts and hemodynamic flow influence junctional dynamics.

Astrocytes in Alzheimer's disease (AD) exert a pivotal role in the maintenance of blood-brain barrier (BBB) integrity essentially through structural support and release of soluble factors. This study provides new insights into the vascular remodeling processes occurring in AD, and reveals, in vivo, a pathological profile of astrocytic secretion involving Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinases (MMP)-9, MMP-2 and Endothelin-1 (ET-1). Cerebrospinal fluid (CSF) levels of VEGF, MMP-2/-9 were lower in patients belonging to the AD continuum, compared to aged-matched controls. CSF levels of VEGF and ET-1 positively correlated with MMP-9 but negatively with MMP-2, suggesting a complex vascular remodeling process occurring in AD. Only MMP-2 levels were significantly associated with CSF AD biomarkers. Conversely, higher MMP-2 (β = 0.411, p < 0.001), ET-1 levels (β = 0.344, p < 0.001) and VEGF (β = 0.221, p = 0.022), were associated with higher BBB permeability. Astrocytic-derived vascular remodeling factors are altered in AD, disclosing the failure of important protective mechanisms which proceed independently alongside AD pathology., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Choroidal vascular hyperpermeability (CVH) on indocyanine green angiography (ICGA) is a hallmark feature of central serous chorioretinopathy (CSC). We identified three distinct CVH phenotypes in CSC: uni-focal indistinct signs of choroidal hyperpermeability (uni-FISH) with one focal area of CVH, multiple areas of focal CVH (multi-FISH), and diffuse hyperpermeability covering most of the posterior pole (DISH). This report investigates the distribution of these phenotypes and their association with signs of disease chronicity., Methods: The CERTAIN study is a monocentric, retrospective study on consecutive CSC patients referred to a large tertiary referral centre that underwent ultra-widefield (UWF) and 55° ICGA. Two independent graders assessed CVH patterns based on mid- to late-phase UWF and 55° ICGA with a third grader acting as referee., Results: Of the 167 eyes of 91 patients included in this study, 43 (26%) showed uni-FISH, 87 (52%) multi-FISH, and 34 (20%) showed DISH based on UWF ICGA. Median age (40 vs. 45 vs. 57; p < 0.001) and logMAR visual acuity (0 vs. 0 vs. 0.1, p < 0.001) differed significantly in-between groups, as did the occurrence of cystoid retinal degeneration (PCRD; 0% vs. 1% vs. 18%, p < 0.001) or diffuse atrophic RPE alterations (DARA; 0% vs. 17% vs. 29%, p < 0.001). The same was true when grading was based on 55° ICGA., Conclusions: The CVH patterns of uni-FISH, multi-FISH, and DISH are typical of CSC. These patterns correlate with established signs of CSC chronicity. Their predictive role in treatment response and prognosis remains to be evaluated., (© 2024 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

Oxaliplatin, a platinum-based anticancer drug, is associated with peripheral neuropathy (oxaliplatin-induced peripheral neuropathy, OIPN), which can lead to worsening of quality of life and treatment interruption. The endothelial glycocalyx, a fragile carbohydrate-rich layer covering the luminal surface of endothelial cells, acts as an endothelial gatekeeper and has been suggested to protect nerves, astrocytes, and other cells from toxins and substances released from the capillary vessels. Mechanisms underlying OIPN and the role of the glycocalyx remain unclear. This study aimed to define changes in the three-dimensional ultrastructure of capillary endothelial glycocalyx near nerve fibers in the hind paws of mice with OIPN. The mouse model of OPIN revealed disruption of the endothelial glycocalyx in the peripheral nerve compartment, accompanied by vascular permeability, edema, and damage to the peripheral nerves. To investigate the potential treatment interventions, nafamostat mesilate, a glycocalyx protective agent was used in tumor-bearing male mice. Nafamostat mesilate suppressed mechanical allodynia associated with neuropathy. It also prevented intra-epidermal nerve fiber loss and improved vascular permeability in the peripheral paws. The disruption of endothelial glycocalyx in the capillaries that lie within peripheral nerve bundles is a novel finding in OPIN. Furthermore, these findings point toward the potential of a new treatment strategy targeting endothelial glycocalyx to prevent vascular injury as an effective treatment of neuropathy as well as of many other diseases. PERSPECTIVE: OIPN damages the endothelial glycocalyx in the peripheral capillaries, increasing vascular permeability. In order to prevent OIPN, this work offers a novel therapy approach that targets endothelial glycocalyx., (Copyright © 2024 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

Nogo-A is a transmembrane protein with multiple functions in the central nervous system (CNS), including restriction of neurite growth and synaptic plasticity. Thus far, Nogo-A has been predominantly considered a cell contact-dependent ligand signaling via cell surface receptors. Here, we show that Nogo-A can be secreted by cultured cells of neuronal and glial origin in association with extracellular vesicles (EVs). Neuron- and oligodendrocyte-derived Nogo-A containing EVs inhibited fibroblast spreading, and this effect was partially reversed by Nogo-A receptor S1PR2 blockage. EVs purified from HEK cells only inhibited fibroblast spreading upon Nogo-A over-expression. Nogo-A-containing EVs were found in vivo in the blood of healthy mice and rats, as well as in human plasma. Blood Nogo-A concentrations were elevated after acute stroke lesions in mice and rats. Nogo-A active peptides decreased barrier integrity in an in vitro blood-brain barrier model. Stroked mice showed increased dye permeability in peripheral organs when tested 2 weeks after injury. In the Miles assay, an in vivo test to assess leakage of the skin vasculature, a Nogo-A active peptide increased dye permeability. These findings suggest that blood borne, possibly EV-associated Nogo-A could exert long-range regulatory actions on vascular permeability.

Background: This study aimed to explore the changes in blood-brain barrier (BBB) permeability and intracranial pressure (ICP) for the first 24 h after the return of spontaneous circulation (ROSC) and their association with injury severity of cardiac arrest., Methods: This prospective study analysed the BBB permeability assessed using the albumin quotient (Qa) and ICP every 2 h for the first 24 h after ROSC. The injury severity of cardiac arrest was assessed using Pittsburgh Cardiac Arrest Category (PCAC) scores. The primary outcome was the time course of changes in the BBB permeability and ICP for the first 24 h after ROSC and their association with injury severity (PCAC scores of 1-4)., Results: Qa and ICP were measured 274 and 197 times, respectively, in 32 enrolled patients. Overall, the BBB permeability increased progressively over time after ROSC, and then it increased significantly at 18 h after ROSC compared with the baseline. In contrast, the ICP revealed non-significant changes for the first 24 h after ROSC. The Qa in the PCAC 2 group was < 0.01, indicating normal or mild BBB disruption at all time points, whereas the PCAC 3 and 4 groups showed a significant increase in BBB permeability at 14 and 22 h, and 12 and 14 h after ROSC, respectively., Conclusion: BBB permeability increased progressively over time for the first 24 h after ROSC despite post-resuscitation care, whereas ICP did not change over time. BBB permeability has an individual pattern when stratified by injury severity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Purpose: The purpose of this study is to evaluate the feasibility of using 3-dimensional (3D) ultra-short echo time (UTE) radial imaging method for measurement of the permeability of the blood-brain barrier (BBB) to gadolinium-based contrast agent. In this study, we propose to use the golden-angle radial sparse parallel (GRASP) method with 3D center-out trajectories for UTE, hence named as 3D UTE-GRASP. We first examined the feasibility of using 3D UTE-GRASP dynamic contrast-enhanced (DCE)-magnetic resonance imaging (MRI) for differentiating subtle BBB disruptions induced by focused ultrasound (FUS). Then, we examined the BBB permeability changes in Alzheimer's disease (AD) pathology using Alzheimer's disease transgenic mice (5xFAD) at different ages., Methods: For FUS experiments, we used four Sprague Dawley rats at similar ages where we compared BBB permeability of each rat receiving the FUS sonication with different acoustic power (0.4-1.0 MPa). For AD transgenic mice experiments, we included three 5xFAD mice (6, 12, and 16 months old) and three wild-type mice (4, 8, and 12 months old)., Results: The result from FUS experiments showed a progressive increase in BBB permeability with increase of acoustic power (p < .05), demonstrating the sensitivity of DCE-MRI method for detecting subtle changes in BBB disruption. Our AD transgenic mice experiments suggest an early BBB disruption in 5xFAD mice, which is further impaired with aging., Conclusion: The results in this study substantiate the feasibility of using the proposed 3D UTE-GRASP method for detecting subtle BBB permeability changes expected in neurodegenerative diseases, such as AD., (© 2024 American Society of Neuroimaging.)

Background: In acute respiratory distress syndrome (ARDS), respiratory drive often differs among patients with similar clinical characteristics. Readily observable factors like acid-base state, oxygenation, mechanics, and sedation depth do not fully explain drive heterogeneity. This study evaluated the relationship of systemic inflammation and vascular permeability markers with respiratory drive and clinical outcomes in ARDS., Methods: ARDS patients enrolled in the multicenter EPVent-2 trial with requisite data and plasma biomarkers were included. Neuromuscular blockade recipients were excluded. Respiratory drive was measured as P ES 0.1, the change in esophageal pressure during the first 0.1 s of inspiratory effort. Plasma angiopoietin-2, interleukin-6, and interleukin-8 were measured concomitantly, and 60-day clinical outcomes evaluated., Results: 54.8% of 124 included patients had detectable respiratory drive (P ES 0.1 range of 0-5.1 cm H 2 O). Angiopoietin-2 and interleukin-8, but not interleukin-6, were associated with respiratory drive independently of acid-base, oxygenation, respiratory mechanics, and sedation depth. Sedation depth was not significantly associated with P ES 0.1 in an unadjusted model, or after adjusting for mechanics and chemoreceptor input. However, upon adding angiopoietin-2, interleukin-6, or interleukin-8 to models, lighter sedation was significantly associated with higher P ES 0.1. Risk of death was less with moderate drive (P ES 0.1 of 0.5-2.9 cm H 2 O) compared to either lower drive (hazard ratio 1.58, 95% CI 0.82-3.05) or higher drive (2.63, 95% CI 1.21-5.70) (p = 0.049)., Conclusions: Among patients with ARDS, systemic inflammatory and vascular permeability markers were independently associated with higher respiratory drive. The heterogeneous response of respiratory drive to varying sedation depth may be explained in part by differences in inflammation and vascular permeability., (© 2024. The Author(s).)

The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighboring cells to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as thrombin, along with mechanical properties of the cell and extracellular matrix are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modeling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behavior that VE-cadherin complexes are known to exhibit, we model the VE-cadherin homophilic bond as cohesive contact with damage following a traction-separation law. We explicitly model the actin cytoskeleton and substrate to study their role in permeability. Our studies show that mechanochemical coupling is necessary to simulate the influence of the mechanical properties of the substrate on permeability. Simulations show that shear between cells is responsible for the variation in permeability between bicellular and tricellular junctions, explaining the phenotypic differences observed in experiments. An increase in the magnitude of traction force due to disturbed flow that endothelial cells experience results in increased permeability, and it is found that the effect is higher on stiffer extracellular matrix. Finally, we show that the cylindrical monolayer exhibits higher permeability than the planar monolayer under unconstrained cases. Thus, we present a contact mechanics-based mechanochemical model to investigate the variation in the permeability of endothelial monolayer due to multiple loads acting simultaneously., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Increased endothelial permeability plays an important role in blood-brain barrier (BBB) dysfunction and is implicated in neuronal injury in many diseased conditions. BBB disruption is primarily determined by dysfunction of endothelial cell-cell junctions. Deprivation of oxygen supply or hypoxia, a common feature of a variety of human diseases, is a major risk factor for BBB disruption. The molecular regulatory mechanisms of hypoxia-induced BBB dysfunction remain incompletely understood. The mitochondrial enzyme, arginase type II (Arg-II), has been shown to promote endothelial dysfunction. However, its role in hypoxia-induced BBB dysfunction has not been explored. In the C57BL/6J mouse model, hypoxia (8% O 2 , 24 hours) augments vascular Arg-II in the hippocampus, decreases cell-cell junction protein levels of Zonula occludens-1 (ZO-1), occludin, and CD31 in endothelial cells, increases BBB leakage in the brain in old mice (20 to 24 months) but not in young animals (3 to 6 months). These effects of hypoxia in aging are suppressed in arg-ii -/- mice. Moreover, the age-associated vulnerability of endothelial integrity to hypoxia is demonstrated in senescent human brain microvascular endothelial cell (hCMEC/D3) culture model. Further results in the cell culture model show that hypoxia augments Arg-II, decreases ZO-1 and occludin levels, and increases endothelial permeability, which is prevented by arg-ii gene silencing or by inhibition of mitochondrial reactive oxygen species (mtROS) production. Our study demonstrates an essential role of Arg-II in increased endothelial permeability and BBB dysfunction by promoting mtROS generation, resulting in decreased endothelial cell-cell junction protein levels under hypoxic conditions particularly in aging.

The microvascular endothelium has a critical role in regulating the delivery of oxygen, nutrients, and water to the surrounding tissues. Under inflammatory conditions that accompany acute injury or disease, microvascular permeability becomes elevated. When microvascular hyperpermeability becomes uncontrolled or chronic, the excessive escape of plasma proteins into the surrounding tissue disrupts homeostasis and ultimately leads to organ dysfunction. Much remains to be learned about the mechanisms that control microvascular permeability. In addition to in vivo and isolated microvessel methods, the cultured endothelial cell monolayer protocol is an important tool that allows for understanding the specific, endothelial subcellular mechanisms that determine permeability of the endothelium to plasma proteins. In this chapter, two variations of the popular Transwell culture methodology to determine permeability to using fluorescently labeled tracers are presented. The strengths and weaknesses of this approach are also discussed., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

The integrity of the blood-brain barrier (BBB), the protective barrier of the brain, is key to maintaining normal microvascular permeability and brain homeostasis. Brain microvascular endothelial cells are primary components of the blood-brain barrier. Transendothelial electrical resistance (TEER) is the electrical resistance across a cellular monolayer such as the brain microvascular endothelial cell monolayers. Measurement of TEER is considered a sensitive, reliable, and noninvasive method for evaluating barrier integrity and permeability of an endothelial cell monolayer under in vitro conditions. Measurement of TEER is also helpful for studying various cellular and molecular changes and signaling events that regulate barrier functions in endothelial monolayers. One of the in vitro endothelial cell barrier models that have been commonly used for measuring TEER is the BBB model using human or rodent brain microvascular endothelial cells grown as a monolayer. In this protocol, we describe how TEER is measured in brain microvascular endothelial cell monolayers, to determine blood-brain barrier integrity under in vitro conditions., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

It is known that pulmonary vascular leakage, a key pathological feature of sepsis-induced lung injury, is largely regulated by perivascular cells. However, the underlying mechanisms have not been fully uncovered. In the present study, we aimed to evaluate the role of isthmin1, a secretory protein originating from alveolar epithelium, in the pulmonary vascular leakage during sepsis and to investigate the regulatory mechanisms of isthmin1 gene transcription. We observed an elevated isthmin1 gene expression in the pulmonary tissue of septic mice induced by cecal ligation and puncture (CLP), as well as in primary murine alveolar type II epithelial cells (ATII) exposed to lipopolysaccharide (LPS). Furthermore, we confirmed that isthmin1 derived from ATII contributes to pulmonary vascular leakage during sepsis. Specifically, adenovirus-mediated isthmin1 disruption in ATII led to a significant attenuation of the increased pulmonary microvascular endothelial cell (PMVEC) hyperpermeability in a PMVEC/ATII coculture system when exposed to LPS. In addition, adeno-associated virus 9 (AAV9)-mediated knockdown of isthmin1 in the alveolar epithelium of septic mice significantly attenuated pulmonary vascular leakage. Finally, mechanistic studies unveiled that nuclear transcription factor CCAAT/enhancer binding protein (C/EBP)β participates in isthmin1 gene activation by binding directly to the cis-regulatory element of isthmin1 locus and may contribute to isthmin1 upregulation during sepsis. Collectively, the present study highlighted the impact of the paracrine protein isthmin1, derived from ATII, on the exacerbation of pulmonary vascular permeability in sepsis and revealed a new regulatory mechanism for isthmin1 gene transcription. NEW & NOTEWORTHY This article addresses the role of the alveolar epithelial-secreted protein isthmin1 on the exacerbation of pulmonary vascular permeability in sepsis and identified nuclear factor CCAAT/enhancer binding protein (C/EBP)β as a new regulator of isthmin1 gene transcription. Targeting the C/EBPβ-isthmin1 regulatory axis on the alveolar side would be of great value in the treatment of pulmonary vascular leakage and lung injury induced by sepsis.

Inflammation in vascular structures due to external factors such as injury or infection inevitably leads to blood leakage. Therefore, measuring blood infiltrated into tissue may serve as an indication for the extent of an inflammatory reaction or injury. There are various methods of confirming vascular permeability in vivo and in vitro; for example, using a blood vessel permeable dye, the dye efflux can be quantitatively measured with a spectrophotometer. Although the aforementioned commonly used methods can measure leaked dye without difficulty, substantial limitations exist regarding the time points of blood leakage that can be measured. Here, we describe the details of a novel protocol to identify and analyze the real-time progression of blood leakage in vivo. This method, by combining existing methods with real-time imaging, is expected to immensely improve the visualization and evaluation of vascular permeability., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

This prospective, descriptive study focused on lung flukes ( Hematoloechus sp., H ) and their impact on systemic and individual capillary variables measured in pithed Rana pipiens , a long-standing model for studies of capillary physiology. Three groups were identified based on Hematoloechus attachment: no Hematoloechus (No H ), Hematoloechus not attached ( H Not Att), and Hematoloechus attached ( H Att). Among 38 descriptive, cardiovascular, and immunological variables, 18 changed significantly with H . Symptoms of H included weight loss, elevated immune cells, heart rate variability, faster coagulation, lower hematocrit, and fluid accumulation. Important capillary function discoveries included median baselines for hydraulic conductivity ( L p ) of 7.0 (No H ), 12.4 ( H Not Att), and 4.2 ( H Att) × 10 -7 cm·s -1 ·cmH 2 O -1 ( P < 0.0001) plus seasonal adaptation of sigma delta pi [σ(π c -π i ), P = 0.03]. Pro- and anti-inflammatory phases were revealed for L p and plasma nitrite/nitrate concentration ([NO x ]) in both H Not Att and H Att, whereas capillary wall tensile strength increased in the H Att. H attachment was advantageous for the host due to lower edema and for the parasite via a sustained food source illustrating an excellent example of natural symbiosis. However, H attachment also resulted in host weight loss: in time, a conundrum for the highly dependent parasite. The study increases overall knowledge of Rana pipiens by revealing intriguing effects of H and previously unknown, naturally occurring seasonal changes in many variables. The data improve Rana pipiens as a general scientific and capillary physiology model. Diseases of inflammation and stroke are among the clinical applications.

Bortezomib (BTZ), a chemotherapeutic drug used to treat multiple myeloma, induces life-threatening side effects, including severe pulmonary toxicity. However, the mechanisms underlying these effects remain unclear. The objectives of this study were to (1) investigate whether BTZ influences vascular permeability and (2) clarify the effect of BTZ on the expression of molecules associated with cell-cell junctions using human pulmonary microvascular endothelial cells in vitro. Clinically relevant concentrations of BTZ induced limited cytotoxicity and increased the permeability of human pulmonary microvascular endothelial cell monolayers. BTZ decreased the protein expression of claudin-5, occludin, and VE-cadherin but not that of ZO-1 and β-catenin. Additionally, BTZ decreased the mRNA expression of claudin-5, occludin, ZO-1, VE-cadherin, and β-catenin. Our results suggest that BTZ increases the vascular permeability of the pulmonary microvascular endothelium by downregulating cell-cell junction molecules, particularly claudin-5, occludin, and VE-cadherin.

Endothelial cells play a major part in the regulation of vascular permeability and angiogenesis. According to their duty to fit the needs of the underlying tissue, endothelial cells developed different subtypes with specific endothelial microdomains as caveolae, fenestrae and transendothelial channels which regulate nutrient exchange, leukocyte migration, and permeability. These microdomains can exhibit diaphragms that are formed by the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), the only known protein component of these diaphragms. Several studies displayed an involvement of PLVAP in diseases as cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Besides an upregulation of PLVAP expression within these diseases, pro-angiogenic or pro-inflammatory responses were observed. On the other hand, loss of PLVAP in knockout mice leads to premature mortality due to disrupted homeostasis. Generally, PLVAP is considered as a major factor influencing the permeability of endothelial cells and, finally, to be involved in the regulation of vascular permeability. Following these observations, PLVAP is debated as a novel therapeutic target with respect to the different vascular beds and tissues. In this review, we highlight the structure and functions of PLVAP in different endothelial types in health and disease., (© 2023. The Author(s).)

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 β-hydroxybutyric acid (β-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 β-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 β-HB or by knockdown of Pes1. In contrast, increases in vascular permeability were induced by overexpression of Pes1, which were suppressed by coadministration of β-HB in cultured endothelial cells. Similarly declines in vascular permeability were found by Pes1 knockdown in diabetic mice. Mechanistically, β-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., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Edema after spinal cord injury (SCI) is one of the first observations after the primary injury and lasts for few days after trauma. It has serious consequences on the affected tissue and can aggravate the initial devastating condition. To date, the mechanisms of the water content increase after SCI are not fully understood. Edema formation results in a combination of interdependent factors related to mechanical damage after the initial trauma progressing, along with the subacute and acute phases of the secondary lesion. These factors include mechanical disruption and subsequent inflammatory permeabilization of the blood spinal cord barrier, increase in the capillary permeability, deregulation in the hydrostatic pressure, electrolyte-imbalanced membranes and water uptake in the cells. Previous research has attempted to characterize edema formation by focusing mainly on brain swelling. The purpose of this review is to summarize the current understanding of the differences in edema formation in the spinal cord and brain, and to highlight the importance of elucidating the specific mechanisms of edema formation after SCI. Additionally, it outlines findings on the spatiotemporal evolution of edema after spinal cord lesion and provides a general overview of prospective treatment strategies by focusing on insights to prevent edema formation after SCI.

Recapitulating the normal physiology of the microvasculature is pivotal in the development of more complex in-vitro models and organ-on-chip designs. Pericytes are an important component of the vasculature, promoting vessel stability, inhibiting vascular permeability and maintaining the vascular hierarchical architecture. The use of such co-culture for the testing of therapeutics and nanoparticle safety is increasingly considered for the validation of therapeutic strategies. This report presents the use of a microfluidic model for such applications. Interactions between endothelial cells and pericytes are first explored. We identify basal conditions required to form stable and reproducible endothelial networks. We then investigate interactions between endothelial cells and pericytes via direct co-culture. In our system, pericytes prevented vessel hyperplasia and maintained vessel length in prolonged culture (> 10 days). In addition, these vessels displayed barrier function and expression of junction markers associated with vessel maturation, including VE-cadherin, β-catenin and ZO-1. Furthermore, pericytes maintained vessel integrity following stress (nutrient starvation) and prevented vessel regression, in contrast to the striking dissociation of networks in endothelial monocultures. This response was also observed when endothelial/pericyte co-cultures were exposed to high concentrations of moderately toxic cationic nanoparticles used for gene delivery. This study highlights the importance of pericytes in protecting vascular networks from stress and external agents and their importance to the design of advanced in-vitro models, including for the testing of nanotoxicity, to better recapitulate physiological response and avoid false positives., (© 2023. The Author(s).)

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., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Excessive vascular endothelial growth factor-A (VEGF-A) signaling induces vascular leakage and angiogenesis in diseases. VEGFR2 trafficking to the cell surface, mediated by kinesin-3 family protein KIF13B, is essential to respond to VEGF-A when inducing angiogenesis. However, the precise mechanism of how KIF13B regulates VEGF-induced signaling and its effects on endothelial permeability is largely unknown. Here we show that KIF13B-mediated recycling of internalized VEGFR2 through Rab11-positive recycling vesicle regulates endothelial permeability. Phosphorylated VEGFR2 at the cell-cell junction was internalized and associated with KIF13B in Rab5-positive early endosomes. KIF13B mediated VEGFR2 recycling through Rab11-positive recycling vesicle. Inhibition of the function of KIF13B attenuated phosphorylation of VEGFR2 at Y951, SRC at Y416, and VE-cadherin at Y685, which are necessary for endothelial permeability. Failure of VEGFR2 trafficking to the cell surface induced accumulation and degradation of VEGFR2 in lysosomes. Furthermore, in the animal model of the blinding eye disease wet age-related macular degeneration (AMD), inhibition of KIF13B-mediated VEGFR2 trafficking also mitigated vascular leakage. Thus, the present results identify the fundamental role of VEGFR2 recycling to the cell surface in mediating vascular permeability, which suggests a promising strategy for mitigating vascular leakage associated with inflammatory diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Vascular hyporeactivity and leakage are key pathophysiologic features that produce multi-organ damage upon sepsis. We hypothesized that pericytes, a group of pluripotent cells that maintain vascular integrity and tension, are protective against sepsis via regulating vascular reactivity and permeability., Methods: We conducted a series of in vivo experiments using wild-type (WT), platelet-derived growth factor receptor beta (PDGFR-β)-Cre + mT/mG transgenic mice and Tie2-Cre + Cx43 flox/flox mice to examine the relative contribution of pericytes in sepsis, either induced by cecal ligation and puncture (CLP) or lipopolysaccharide (LPS) challenge. In a separate set of experiments with Sprague-Dawley (SD) rats, pericytes were depleted using CP-673451, a selective PDGFR-β inhibitor, at a dosage of 40 mg/(kg·d) for 7 consecutive days. Cultured pericytes, vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were used for mechanistic investigations. The effects of pericytes and pericyte-derived microvesicles (PCMVs) and candidate miRNAs on vascular reactivity and barrier function were also examined., Results: CLP and LPS induced severe injury/loss of pericytes, vascular hyporeactivity and leakage (P < 0.05). Transplantation with exogenous pericytes protected vascular reactivity and barrier function via microvessel colonization (P < 0.05). Cx43 knockout in either pericytes or VECs reduced pericyte colonization in microvessels (P < 0.05). Additionally, PCMVs transferred miR-145 and miR-132 to VSMCs and VECs, respectively, exerting a protective effect on vascular reactivity and barrier function after sepsis (P < 0.05). miR-145 primarily improved the contractile response of VSMCs by activating the sphingosine kinase 2 (Sphk2)/sphingosine-1-phosphate receptor (S1PR)1/phosphorylation of myosin light chain 20 pathway, whereas miR-132 effectively improved the barrier function of VECs by activating the Sphk2/S1PR2/zonula occludens-1 and vascular endothelial-cadherin pathways., Conclusions: Pericytes are protective against sepsis through regulating vascular reactivity and barrier function. Possible mechanisms include both direct colonization of microvasculature and secretion of PCMVs., (© 2023. The Author(s).)

The endothelial cells (EC) of established blood vessels in adults remain extraordinarily quiescent in the sense that they are not actively proliferating, but they fulfill the necessary role to control the permeability of their monolayer that lines the interior of blood vessels. The cell-cell junctions between ECs in the endothelium comprise tight junctions and adherens homotypic junctions, which are ubiquitous along the vascular tree. Adherens junctions are adhesive intercellular contacts that are crucial for the organization of the EC monolayer and its maintenance and regulation of normal microvascular function. The molecular components and underlying signaling pathways that control the association of adherens junctions have been described in the last few years. In contrast, the role that dysfunction of these adherens junctions has in contributing to human vascular disease remains an important open issue. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator found at high concentrations in blood which has important roles in the control of the vascular permeability, cell recruitment, and clotting that follow inflammatory processes. This role of S1P is achieved through a signaling pathway mediated through a family of G protein-coupled receptors designated as S1PR1. This review highlights novel evidence for a direct linkage between S1PR1 signaling and the mediation of EC cohesive properties that are controlled by VE-cadherin.

Objective: Chronic subdural hematoma (CSDH) is a common neurological disease among elderly adults. The progression of CSDH is an angiogenic process, involving inflammatory mediators that affect vascular permeability, microvascular leakage, and hematoma thickness. The authors aimed to identify biomarkers associated with angiogenesis and vascular permeability that might influence midline shift and hematoma thickness., Methods: Medical records and laboratory data of consecutive patients who underwent surgery for CSDH were analyzed. Collected data were basic demographic data, CSDH classification, CSDH thickness, midline shift, heme oxygenase-1 (HO-1) levels in hematomas, and common laboratory markers. Linear regression analysis was used to evaluate the relationship of CSDH thickness with characteristic variables. The chick chorioallantoic membrane (CAM) assay was used to test the angiogenic potency of identified variables in ex ovo culture of chick embryos., Results: In total, 93 patients with CSDH (71.0% male) with a mean age of 71.0 years were included. The mean CSDH thickness and midline shift were 19.7 and 9.8 mm, respectively. The mean levels of HO-1, ferritin, total bilirubin, white blood cells, segmented neutrophils, lymphocytes, platelets, international normalized ratio, and partial thromboplastin time were 36 ng/mL, 14.8 μg/mL, 10.5 mg/dL, 10.3 × 103 cells/μL, 69%, 21.7%, 221.1 × 109 cells/μL, 1.0, and 27.8 seconds, respectively. Pearson correlation analysis revealed that CSDH thickness was positively correlated with midline shift distance (r = 0.218, p < 0.05) but negatively correlated with HO-1 concentration (r = -0.364, p < 0.01) and ferritin level (r = -0.222, p < 0.05). Multivariate linear regression analysis revealed that HO-1 was an independent predictor of CSDH thickness (β = -0.084, p = 0.006). The angiogenic potency of HO-1 in hematoma fluid was tested with the chick CAM assay; topical addition of CSDH fluid with low HO-1 levels promoted neovascularization and microvascular leakage. Addition of HO-1 in a rescue experiment inhibited CSDH fluid-mediated angiogenesis and microvascular leakage., Conclusions: HO-1 is an independent risk factor in CSDH hematomas and is negatively correlated with CSDH thickness. HO-1 may play a role in the pathophysiology and development of CSDH, possibly by preventing neovascularization and reducing capillary fragility and hyperpermeability.

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 ( Vefgr2 Y949F ) 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 Vefgr2 Y949F 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.

Background: Metastasis is the leading cause of death in patients with breast cancer (BC). Primary tumors create a premetastatic niche (PMN) in secondary organs for subsequent metastases. Cancer-associated fibroblasts (CAFs) are a predominant stromal component in the tumor microenvironment and serve as a major contributor to tumor metastasis. However, the function and mechanism of primary CAFs in the premetastatic niche of secondary organs remain unclear in BC. Methods: We investigated the expression profiles of lncRNAs in pairs of CAFs and NFs derived from breast tumor tissues using lncRNA microarray. The expression levels of lncSNHG5, ZNF281, IGF2BP2, CCL2 and CCL5 were assessed by qRT-PCR; the protein levels of related genes (e.g., ZNF281, IGF2BP2, CCL2, and CCL5) were analyzed using western blotting and/or ELISA in primary and immortalized CAFs and clinical samples. Tubule formation and three-dimensional sprouting assays and tissue fluorescence staining were conducted to investigate angiogenesis. In vitro permeability assays, trans-endothelial invasion assays, in vivo permeability assays and tissue fluorescence staining were conducted to examine vascular permeability. The regulatory mechanism of lncSNHG5 was investigated by RNA sequencing, fluorescent in situ hybridization, cellular fractionation assay, mass spectrometry, RNA pull-down, RNA immunoprecipitation, gene-specific m6A assay, chromatin immunoprecipitation, dual luciferase reporter assay and actinomycin D treatment in CAFs and NFs. Results: LncSNHG5 was highly expressed in breast CAFs and played an essential role in premetastatic niche formation by promoting angiogenesis and vascular leakiness through regulation of ZNF281 in CAFs. lncSNHG5 enhanced ZNF281 mRNA stability by binding with the m6A reader IGF2BP2. Enhanced ZNF281 transcriptionally regulated CCL2 and CCL5 expression to activate P38 MAPK signaling in endothelial cells. High CCL2 and CCL5 expression was associated with tumor metastasis and poor prognosis in BC patients. The inhibitors RS102895, marasviroc and cenicriviroc inhibited angiogenesis and vascular permeability in the PMN by blocking the binding of CCL2/CCR2 and CCL5/CCR5. The lncSNHG5-ZNF281-CCL2/CCL5 signaling axis plays an essential role in inducing premetastatic niche formation to promote BC metastasis. Conclusions: Our work demonstrates that lncSNHG5 and its downstream signaling ZNF281-CCL2/CCL5 in CAFs play a crucial role in premetastatic niche formation in breast cancer and may serve as potential targets for the diagnosis and treatment of BC metastasis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need., Methods: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) β2., Results: Global knock-out of PLCβ2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCβ2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCβ2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCβ2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls., Conclusions: The results implicate PLCβ2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCβ2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.

Several lines of evidence suggest that stress induces the neurovascular dysfunction associated with increased blood-brain barrier (BBB) permeability, which could be an important pathology linking stress and psychiatric disorders, including major depressive disorder (MDD). However, the detailed mechanism resulting in BBB dysfunction associated in the pathophysiology of MDD still remains unclear. Herein, we demonstrate the role of vascular endothelial growth factor (VEGF), a key mediator of vascular angiogenesis and BBB permeability, in stress-induced BBB dysfunction and depressive-like behavior development. We implemented an animal model of depression, chronic restraint stress (RS) in BALB/c mice, and found that the BBB permeability was significantly increased in chronically stressed mice. Immunohistochemical and electron microscopic observations revealed that increased BBB permeability was associated with both paracellular and transcellular barrier alterations in the brain endothelial cells. Pharmacological inhibition of VEGF receptor 2 (VEGFR2) using a specific monoclonal antibody (DC101) prevented chronic RS-induced BBB permeability and anhedonic behavior. Considered together, these results indicate that VEGF/VEGFR2 plays a crucial role in the pathogenesis of depression by increasing the BBB permeability, and suggest that VEGFR2 inhibition could be a potential therapeutic strategy for the MDD subtype associated with BBB dysfunction., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Protein kinase C (PKC) is a family of serine/threonine protein kinases, the activation of which plays an important role in the development of diabetic microvascular complications. The activation of PKC under high-glucose conditions stimulates redox reactions and leads to an accumulation of redox stress. As a result, various types of cells in the microvasculature are influenced, leading to changes in blood flow, microvascular permeability, extracellular matrix accumulation, basement thickening and angiogenesis. Structural and functional disorders further exacerbate diabetic microvascular complications. Here, we review the roles of PKC in the development of diabetic microvascular complications, presenting evidence from experiments and clinical trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pan, Xu and Guo.)

Background: The vascular endothelium maintains tissue-fluid homeostasis by controlling the passage of large molecules and fluid between the blood and interstitial space. The interaction of catenins and the actin cytoskeleton with VE-cadherin (vascular endothelial cadherin) is the primary mechanism for stabilizing AJs (adherens junctions), thereby preventing lung vascular barrier disruption. Members of the Rho (Ras homology) family of GTPases and conventional GEFs (guanine exchange factors) of these GTPases have been demonstrated to play important roles in regulating endothelial permeability. Here, we evaluated the role of DOCK4 (dedicator of cytokinesis 4)-an unconventional Rho family GTPase GEF in vascular function., Methods: We generated mice deficient in DOCK4' used DOCK4 silencing and reconstitution approaches in human pulmonary artery endothelial cells' used assays to evaluate protein localization, endothelial cell permeability, and small GTPase activation., Results: Our data show that DOCK4-deficient mice are viable. However, these mice have hemorrhage selectively in the lung, incomplete smooth muscle cell coverage in pulmonary vessels, increased basal microvascular permeability, and impaired response to S1P (sphingosine-1-phosphate)-induced reversal of thrombin-induced permeability. Consistent with this, DOCK4 rapidly translocates to the cell periphery and associates with the detergent-insoluble fraction following S1P treatment, and its absence prevents S1P-induced Rac-1 activation and enhancement of barrier function. Moreover, DOCK4-silenced pulmonary artery endothelial cells exhibit enhanced basal permeability in vitro that is associated with enhanced Rho GTPase activation., Conclusions: Our findings indicate that DOCK4 maintains AJs necessary for lung vascular barrier function by establishing the normal balance between RhoA (Ras homolog family member A) and Rac-1-mediated actin cytoskeleton remodeling, a previously unappreciated function for the atypical GEF family of molecules. Our studies also identify S1P as a potential upstream regulator of DOCK4 activity.

Objectives: Clinical and histological studies have found evidence that nerve ischemia is a major contributor to diabetic neuropathy (DN) in type 2 diabetes (T2D). The aim of this study was to investigate peripheral nerve microvascular permeability using dynamic contrast enhanced (DCE) magnetic resonance neurography (MRN) to analyze potential correlations with clinical, electrophysiological, and demographic data., Methods: Sixty-five patients (35/30 with/without DN) and 10 controls matched for age and body mass index (BMI) underwent DCE MRN of the distal sciatic nerve with an axial T1-weighted sequence. Microvascular permeability (K trans ), plasma volume fraction (v p ), and extravascular extracellular volume fraction (v e ) were determined with the extended Tofts model, and subsequently correlated with clinical data., Results: K trans and v e were lower in T2D patients with DN compared to patients without DN (0.037 min -1  ± 0.010 vs. 0.046 min -1  ± 0.014; p = 0.011, and 2.35% ± 3.87 vs. 5.11% ± 5.53; p = 0.003, respectively). In individuals with T2D, K trans correlated positively with tibial, peroneal, and sural NCVs (r = 0.42; 95%CI = 0.18 to 0.61, 0.50; 95%CI = 0.29 to 0.67, and 0.44; 95%CI = 0.19 to 0.63, respectively), with tibial and peroneal CMAPs (r = 0.27; 95%CI = 0.01 to 0.49 and r = 0.32; 95%CI = 0.07 to 0.53), and with the BMI (r = 0.47; 95%CI = 0.25 to 0.64). Negative correlations were found with the neuropathy deficit score (r = -0.40; 95%CI = -0.60 to -0.16) and age (r = -0.51; 95%CI = -0.67 to -0.31). No such correlations were found for v p ., Conclusion: This study is the first to find associations of MR nerve perfusion parameters with clinical and electrophysiological parameters related to DN in T2D. The results indicate that a decrease in microvascular permeability but not plasma volume may result in nerve ischemia that subsequently causes demyelination., (© 2022 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Central nervous system (CNS) dysfunction induced by sepsis and pathogenic microbial infections is reported to be closely associated with increased permeability of the blood-brain barrier (BBB), which is mainly mediated by the stimulation of lipopolysaccharide (LPS) on inflammatory signaling. Midazolam is a novel sedative acting on the benzodiazepine receptor, which is recently reported to exert a neuroprotective effect by inhibiting inflammation. The present study will explore the potential repair capacity of Midazolam on LPS-induced damage to the BBB. The in vivo mice model was established by intraperitoneal injection of LPS, while the in vitro model was constructed by stimulating endothelial cells utilizing LPS. We found that the increased malondialdehyde (MDA) level and reduced superoxide dismutase (SOD) activity in the brain cortices, promoted serum concentration of inflammatory factors, and elevated BBB permeability were found in the LPS group, all of which were dramatically reversed by 1 mg/kg and 2 mg/kg Midazolam. Interestingly, Midazolam increased the expression of the tight junction protein zonula occludens-1 (ZO-1). In LPS-challenged in vitro human brain microvascular endothelial cells (HBMECs), the increased concentration of inflammatory factors, reduced trans-endothelial electrical resistance (TEER) level, elevated relative value of trans-endothelial permeability, and downregulated ZO-1 were observed, all of which were pronouncedly alleviated by Midazolam, accompanied by the inhibition on the Ras homolog family member A/ Rho-kinase 2 (RhoA/ROCK-2) pathway. Furthermore, the regulatory effects of Midazolam on ZO-1 expression and the endothelial monolayer permeability in LPS-challenged HBMECs were abolished by the overexpression of RhoA. Collectively, our data imply that Midazolam ameliorated the impairment of the BBB against LPS by regulating the RhoA/ROCK2 pathway., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Fluid shear stress is an important mediator of vascular permeability, yet the molecular mechanisms underlying the effect of shear on the blood-brain barrier (BBB) have yet to be clarified in cerebral vasculature despite its importance for brain homeostasis. The goal of this study is to probe components of shear mechanotransduction within the BBB to gain a better understanding of pathologies associated with changes in cerebral perfusion including ischemic stroke. Interrogating the effects of shear stress in vivo is complicated by the complexity of factors in the brain parenchyma and the difficulty associated with modulating blood flow regimes. The in vitro model used in this study is compatible with real-time measurement of barrier function using a transendothelial electrical resistance as well as immunocytochemistry and dextran permeability assays. These experiments reveal that there is a threshold level of shear stress required for barrier formation and that the composition of the extracellular matrix, specifically the presence of high molecular weight hyaluronan, dictates the flow response. Gene editing to modulate the expression of CD44, a mechanosensitive receptor for hyaluronan, demonstrates that the receptor is required for the endothelial response to shear stress. Manipulation of small GTPase activity reveals CD44 activates Rac1 while inhibiting RhoA activation. Additionally, adducin-γ localizes to tight junctions in response to shear stress and RhoA inhibition and is required to maintain the barrier. This study identifies specific components of the mechanosensing complex associated with the BBB response to fluid shear stress and, therefore, illuminates potential targets for barrier manipulation in vivo., (© 2022 Federation of American Societies for Experimental Biology.)

Objective: To investigate the effect of interleukin-33 (IL-33) on lipopolysaccharide (LPS)-induced permeability of rat cardiac microvascular endothelial cells (RCMECs). Methods: RCMECs were cultured in vitro to be divided into control group, LPS group, IL-33 group and LPS+IL-33 group. The effect of IL-33 on the proliferation of RCMECs was detected by cell counting reagent (CCK8). Fluorescein isothiocyanate (FITC)-dextran assay was used to evaluate the permeability of RCMECs. The expression of vascular endothelial calmodulin, ras homologous gene family (Rho) member A (RhoA) and phosphorylated Rho-associated coiled-coil-containing protein kinase (p-ROCK2) proteins were tested by western blot. High-throughput sequencing and gene ontology (GO) were performed for gene expression in LPS and LPS+IL-33 groups. Results: No significant effect of IL-33 at 10-50 ng/ml on the proliferation of RCMECs was observed ( P> 0.05). Compared with the control group, the permeability of RCMECs (permeability coefficient ratio 1.404±0.029 vs. 1.000±0.200, P< 0.05) was significantly increased in LPS group and the expression of vascular endothelial calmodulin (relative gray value 0.429 5±0.012 9 vs. 0.594 9±0.014 2, P< 0.05) was down-regulated, while the permeability of monolayers (permeability coefficient ratio, 0.948±0.013, P< 0.01) was decreased in LPS+IL-33 group and the expression of vascular endothelial calmodulin (relative grayscale value 0.549 1±0.012 0, P< 0.005) was up-regulated compared with the LPS group. High-throughput sequencing data revealed that the differential genes downregulated in the LPS and LPS+IL-33 groups were associated with cytoskeleton and Rho signaling pathway. Compared with the control group, RhoA (relative gray value 0.211 4±0.009 9 vs. 0.135 0±0.007 6, P< 0.000 1) and p-ROCK (relative gray value 0.656 3±0.013 2 vs. 0.503 6±0.036 2, P< 0.000 1) protein expression was upregulated in the LPS group. When compared with LPS group, RhoA (relative gray value 0.157 7±0.010 7, P= 0.000 2), p-ROCK (relative gray value 0.427 7±0.003 8, P< 0.000 1) protein expression was decreased in LPS+IL-33 group. Conclusion: IL-33 may improve LPS-induced hyperpermeability of RCMECs by inhibiting RhoA and p-ROCK protein expression in Rho/Rho-associated coiled-coil-containing protein kinase signaling pathway.