Endothelial Barrier Function Research Articles

Heparan sulfate acts in synergy with tight junction through STAT3 signaling to maintain the endothelial barrier and prevent lung injury development.

Damage to glycocalyx and tight junction are key determinants of endothelial permeability, which is the main pathological feature of acute respiratory distress syndrome (ARDS). However, the effect of glycocalyx heparan sulfate (HS) on tight junction proteins occludin and ZO-1 has not been revealed. In this study, the mice exposed to LPS results showed that FITC-albumin infiltration, HS shedding, and tight junction protein impairment were most severe at 6h of LPS treatment compared with those in other treatment times. The in vitro and vivo experiments revealed that tight junction damage, FITC-albumin infiltration, and pathological injury induced by LPS were significantly alleviated via protection of glycocalyx HS shedding. mRNA sequencing analysis demonstrated that the STAT signaling pathways played a crucial role in the inhibition of LPS-induced HS shedding in mice. Supplementation of exogenous HS in human umbilical vein endothelial cells (HUVECs) and mice ameliorated LPS-induced the tight junction barrier defect by inhibiting STAT3 phosphorylation. Further analysis uncovered that intervention of STAT3 signaling significantly alleviated LPS-induced tight junction proteins damage and vascular permeability in HUVECs and mice. Mechanistically, HS modulated tight junction proteins by STAT3 signaling, which might directly bind to the promoter regions of occludin and ZO-1. In conclusion, glycocalyx HS played an important role in protecting endothelial barrier function and preventing injury development, in synergy with tight junction through STAT3 signaling, which further alleviated pulmonary edema.

Protection of liver sinusoidal endothelial cells using different preservation solutions.

Donor liver preservation methods and solutions have evolved over the last years. Liver sinusoidal endothelial cell (LSEC) barrier function and integrity during preservation is crucial for outcomes of liver transplantation. Therefore, the present study aimed to determine optimal preservation of LSEC barrier function and integrity, using different preservation solutions. Human Umbilical Vein Endothelial Cells (HUVEC) and LSEC were incubated in either University of Wisconsin machine perfusion solution (UW-MPS), histidine-tryptophan-ketoglutarate (HTK), or endothelial cell growth medium 2 (EGM2) (as a golden standard for cell culturing). Endothelial integrity was assessed by measurement of cellular morphology and expression of membrane proteins ; PECAM-1, ICAM-1, and Fc-gamma receptor CD32b (FcΥRCD32b). Endothelial barrier function was measured by electric cell-substrate impedance sensing (ECIS). Cellular respondence to inflammatory stimuli with tumor necrosis factor-alpha (TNF-α), was tested by studying trans-endothelial migration (TEM) under flow conditions. Differences in these parameters were analyzed between the different preservation solutions. PECAM-1 expression was high for all preservation solutions in HUVEC and LSEC. ICAM-1 expression was increased in both LSEC and HUVEC in all preservation solutions plus TNF-α. UW reduced PECAM-1 expression, whereas EGM2 medium promoted barrier function in LSEC and HUVEC, and monolayer recovery after wounding was best achieved in cells incubated in EGM2. LSEC and HUVEC incubated with EGM2 plus TNF-α both supported neutrophil adhesion and TEM, but much less to none when incubated in UW plus TNF-α. Overall, EGM2 showed the best results in preserving endothelial barrier function for both HUVEC and LSEC.

Impacts of APOE-ε4 and exercise training on brain microvascular endothelial cell barrier function and metabolism

The APOE-ε4 genotype is the highest genetic risk factor for Alzheimer's disease (AD), and exercise training can reduce the risk of AD. Two early pathologies of AD are degradation of tight junctions between brain microvascular endothelial cells (BMEC) and brain glucose hypometabolism. Therefore, the objective of this work was to determine how the APOE-ε4 genotype and serum from exercise trained individuals impacts BMEC barrier function and metabolism. iPSC homozygous for the APOE-ε3 and APOE-ε4 alleles were differentiated to BMEC-like cells and used to measure barrier function and metabolism. To investigate exercise effects, serum was collected from older adults pre- and post- 6 months of exercise training (n=9 participants per genotype). APOE-ε3 and APOE-ε4 BMEC were treated with genotype-matched serum, and then barrier function and metabolism were measured. APOE-ε4 genotype impaired BMEC barrier function and metabolism by reducing sirtuin 1 (SIRT1) levels by 27% (p=0.0188) and baseline insulin signalling by 37% (p=0.0186) compared to APOE-ε3 BMEC. Exercise-trained serum increased SIRT1 by 33% (p=0.0043) in APOE-ε3 BMEC but decreased SIRT1 by 22% (p=0.0004) in APOE ε4 BMEC. APOE-ε4 directly impairs glucose metabolism and barrier function. Serum from exercise trained individuals alters SIRT1 in a genotype-dependent manner but may require additional cues from exercise to decrease AD pathologies. Brain and Behaviour Initiative at the University of Maryland through the Seed Grant Program, NSF-GRFP DGE 1840340, Fischell Fellowship in Biomedical Engineering, NSF CBET-2211966 and DGE-1632976, National Niemann-Pick Disease Foundation, University of Maryland ASPIRE Program, NIH R01HL165193, R01HL140239-01, and R01AG057552.

Rnf40 Exacerbates Hypertension-Induced Cerebrovascular Endothelial Barrier Dysfunction by Ubiquitination and Degradation of Parkin.

We aimed to investigate the role of Rnf40 in hypertension-induced cerebrovascular endothelial barrier dysfunction and cognitive impairment. We employed microarray data analysis and integrated bioinformatics databases to identify a novel E3 ligase, Rnf40, that targets Parkin. To understand the role of RNF40 in hypertension-induced cerebrovascular endothelial cell damage, we used pAAV-hFLT1-MCS-EGFP-3×Flag-mir30shRnf40 to establish an Rnf40-deficient model in spontaneously hypertensive rats (SHRs). We also evaluated the cerebrovascular endothelial barrier function, cerebral blood flow, and cognitive performance. We observed reduced mitophagy in cerebrovascular endothelial cells of SHRs compared with that in Wistar-Kyoto rats. Rnf40 facilitated K48-linked polyubiquitination and degradation of Parkin, thereby inhibiting mitophagy. In the Rnf40-deficient SHR model, knocking down Rnf40 restored mitophagy in cerebrovascular endothelial cells. Additionally, levels of tight junction proteins and cerebrovascular endothelial barrier function improved following Rnf40 downregulation. Rnf40 depletion also improved global cognitive performance and restored cerebral blood flow in SHRs. Our findings suggest that increased Rnf40 levels exacerbate hypertension-induced cerebrovascular endothelial barrier dysfunction by ubiquitinating Parkin.

Expression of Yes-associated protein in endothelial cells of human corneas before and after storage in organ culture

The cornea, the anterior meniscus-shaped transparent and refractive structure of the eyeball, is the first mechanical barrier of the eye. Its functionality heavily relies on the health of its endothelium, its most posterior layer. The treatment of corneal endothelial cells (CECs) deficiency is allogeneic corneal graft using stored donor corneas. One of the main goals of eye banks is to maintain endothelial cell density (ECD) and endothelial barrier function, critical parameters influencing transplantation outcomes. Unlike in vivo, the stored cornea is not subjected to physiological mechanical stimuli, such as the hydrokinetic pressure of the aqueous humor and intraocular pressure (IOP). YAP (Yes-Associated Protein), a pivotal transcriptional coactivator, is recognized for its ability to sense diverse biomechanical cues and transduce them into specific biological signals, varying for each cell type and mechanical forces. The biomechanical cues that might regulate YAP in human corneal endothelium remain unidentified. Therefore, we investigated the expression and subcellular localization of YAP in the endothelium of corneas stored in organ culture (OC). Our findings demonstrated that CEC morphology, ECD and cell–cell interactions are distinctly and differentially associated with modifications in the expression, subcellular localization and phosphorylation of YAP. Notably, this phosphorylation occurs in the basal region of the primary cilium, which may play central cellular roles in sensing mechanical stimuli. The sustained recruitment of YAP in cellular junctions, nucleus, and cilium under long-term OC conditions strongly indicates its specific role in maintaining CEC homeostasis. Understanding these biophysical influences could aid in identifying molecules that promote homeostasis and enhance the functionality of CECs.

Talin1 dysfunction is genetically linked to systemic capillary leak syndrome.

Systemic capillary leak syndrome (SCLS) is a rare life-threatening disorder due to profound vascular leak. The trigger and the cause of the disease are currently unknown and there is no specific treatment. Here, we identified a rare heterozygous splice-site variant in the TLN1 gene in a familial SCLS case, suggestive of autosomal dominant inheritance with incomplete penetrance. Talin1 has a key role in cell adhesion by activating and linking integrins to the actin cytoskeleton. This variant causes in-frame skipping of exon 54 and is predicted to affect talin's C-terminal actin-binding site (ABS3). Modeling the SCLS-TLN1 variant in TLN1-heterozygous endothelial cells (ECs) disturbed the endothelial barrier function. Similarly, mimicking the predicted actin-binding disruption in TLN1-heterozygous ECs resulted in disorganized endothelial adherens junctions. Mechanistically, we established that the SCLS-TLN1 variant, through the disruption of talin's ABS3, sequestrates talin's interacting partner, vinculin, at cell-extracellular matrix adhesions, leading to destabilization of the endothelial barrier. We propose that pathogenic variants in TLN1 underlie SCLS, providing insight into the molecular mechanism of the disease that can be explored for future therapeutic interventions.

Shear stress-induced restoration of pulmonary microvascular endothelial barrier function following ischaemia reperfusion injury requires VEGFR2 signalling.

Normal shear stress produced by blood flow is sensed by the vascular endothelium and required for maintenance of the homeostatic functions of the endothelium in systemic conduit and resistance vessels. Many critical illnesses are characterised by periods of abnormally reduced or absent shear stress in the lung (e.g. haemorrhagic shock, embolism, ischaemia reperfusion injury and lung transplantation) and are complicated by pulmonary oedema following reperfusion due to microvascular leak. The role of shear stress in regulating the pulmonary microvascular endothelial barrier in the intact vascular bed has not been previously examined. We tested the hypothesis that, in lungs injured by a period of ischaemia and reperfusion (IRI), reduced shear stress contributes to increased pulmonary microvascular endothelial barrier permeability and oedema formation. Furthermore, we examined the role of VEGFR2 as a mechanosensor mediating the endothelial response to this altered shear stress. Following IRI, we perfused isolated ventilated mouse lungs with a low viscosity solution (LVS) or a higher, physiological viscosity solution (PVS) at constant flow to produce differing endothelial shear stresses in of the intact microcirculation. Lungs perfused with LVS developed pulmonary oedema due to increased endothelial permeability whereas those perfused with PVS were protected from oedema formation by reduced endothelial permeability. This effect of PVS required normal VEGFR2 mechanoreceptor function. These data show for the first time that shear stress has an important role in restoring endothelial barrier function in the intact pulmonary microcirculation following injury and have important implications for the treatment of pulmonary oedema in critically ill patients.

In vitro examination of Piezo1-TRPV4 dynamics: implications for placental endothelial function in normal and preeclamptic pregnancies.

Mechanosensation is essential for endothelial cell (EC) function, which is compromised in early-onset preeclampsia (EPE), impacting offspring health. The ion channels Piezo-type mechanosensitive ion channel component 1 (Piezo1) and transient receptor potential cation channel subfamily V member 4 (TRPV4) are coregulated mechanosensors in ECs. Current evidence suggests that both channels could mediate aberrant placental endothelial function in EPE. Using isolated fetoplacental ECs (fpECs) from early control (EC) and EPE pregnancies, we show functional coexpression of both channels and that Ca2+ influx and membrane depolarization in response to chemical channel activation is reduced in EPE fpECs. Downstream of channel activation, Piezo1 alone can induce phosphorylation of endothelial nitric oxide synthase (eNOS) in fpECs, while combined activation of Piezo1 and TRPV4 only affects eNOS phosphorylation in EPE fpECs. Additionally, combined activation reduces the barrier integrity of fpECs and has a stronger effect on EPE fpECs. This implies altered Piezo1-TRPV4 coregulation in EPE. Mechanistically, we suggest this to be driven by changes in the arachidonic acid metabolism in EPE fpECs as identified by RNA sequencing. Targeting of Piezo1 and TRPV4 might hold potential for EPE treatment options in the future.NEW & NOTEWORTHY This study shows Piezo-type mechanosensitive ion channel component 1 (Piezo1) and transient receptor potential cation channel subfamily V member 4 (TRPV4) coexpression and functionality within primary human fetoplacental endothelial cells (fpECs), mediating nitric oxide (NO) production and barrier integrity. In early-onset preeclampsia (EPE), fpEC channel functionality and coregulation are impaired, affecting Ca2+ signaling and endothelial barrier function. Combined channel activation significantly reduces endothelial barrier integrity and increases NO production in EPE. Changes in arachidonic acid metabolism are suggested as a key underlying factor mediating impaired channel functionality in EPE fpECs.

Inhibition of Angiopoietin-2 rescues sporadic brain arteriovenous malformations by reducing pericyte loss.

Brain arteriovenous malformations (bAVMs) are a major cause of hemorrhagic stroke in children and young adults. These lesions are thought to result from somatic KRAS/BRAF mutations in brain endothelial cells (bECs). In this study, we introduce a new bAVM model by inducing a brain endothelial-specific BrafV600E mutation using the Slc1o1c1(BAC)-CreER driver line. The pathological characteristics of this model resemble human bAVMs, including dilated and hyperpermeable vessels, as well as parenchymal hemorrhage. We observed that these lesions showed a typical reduction in pericyte coverage and disruption of the pericyte-endothelial cell connection. Additionally, we found that ANGPT2 levels were significantly increased in the endothelium of bAVM lesions, which may be a critical factor in the pericyte deficits of the malformed vessels. Treatment with an ANGPT2 neutralizing antibody confirmed that blocking ANGPT2 can restore pericyte density in bAVM lesions, improve pericyte coverage around microvessels, enhance tight junction protein coverage related to endothelial cells, and normalize endothelial barrier function. In summary, our findings suggest that increased ANGPT2 expression in endothelial cells with the BrafV600E mutation is a key factor in pericyte deficiencies in bAVMs, highlighting the potential effectiveness of anti-ANGPT2 therapy in treating bAVMs.

CEACAM1 in vascular homeostasis and inflammation.

The glycoprotein Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1), also known as CD66a, is a member of the immunoglobulin superfamily. It is expressed in a variety of tissues including epithelial, immune, as well as endothelial cells, and is crucial to diverse physiological and pathological mechanisms. This review aims to provide a comprehensive understanding of CEACAM1's multifaceted roles in vascular biology and inflammatory processes. Directed literature research was conducted using databases, such as PubMed, and relevant studies were categorized based on the physiological effects of CEACAM1. CEACAM1 plays a pivotal role in vascular homeostasis, particularly influencing the formation, maturation, and aging of blood vessels, as well as the endothelial barrier function. It supports endothelium-dependent vasodilation and nitric oxide formation, thus promoting vascular integrity and regulating blood pressure. Additionally, CEACAM1 is of emerging importance to vascular inflammation and its potential clinical consequences. CEACAM1 is a crucial regulator of vascular homeostasis and inflammation with significant implications for cardiovascular health. Despite the lack of understanding of tissue-specific modulation and isoform-dependent mechanisms, CEACAM1 could be a promising therapeutic target for the prevention of cardiovascular disease in the future.

Pericytes require physiological oxygen tension to maintain phenotypic fidelity

Pericytes function to maintain tissue homeostasis by regulating capillary blood flow and maintaining endothelial barrier function. Pericyte dysfunction is associated with various pathologies and has recently been found to aid cancer progression. Despite having critical functions in health and disease, pericytes remain an understudied population due to a lack of model systems which accurately reflect in vivo biology. In this study we developed a protocol to isolate and culture murine lung, brain, bone, and liver pericytes, that maintains their known phenotypes and functions. We demonstrate that pericytes, being inherently plastic, benefit from controlled oxygen tension culture conditions, aiding their expansion ex vivo. Primary pericytes grown in physiologically relevant oxygen tensions (10% O2 for lung; 5% O2 for brain, bone, and liver) also better retain pericyte phenotypes indicated by stable expression of characteristic transcriptional and protein markers. In functional tube formation assays, pericytes were observed to significantly associate with endothelial junctions. Importantly, we identified growth conditions that limit expression of the plasticity factor Klf4 to prevent spontaneous phenotypic switching in vitro. Additionally, we were able to induce pathological pericyte phenotypic switching in response to metastatic stimuli to accurately recapitulate in vivo biology. Here, we present a robust method for studying pericyte biology in both physiology and disease.

Endothelial RIPK3 minimizes organotypic inflammation and vascular permeability in ischemia-reperfusion injury.

Recent studies have revealed a link between endothelial receptor-interacting protein kinase 3 (RIPK3) and vascular integrity. During mouse embryonic development, hypoxia can trigger elevated endothelial RIPK3 that contributes to lethal vascular rupture. However, it is unknown whether RIPK3 regulate endothelial barrier function in adult vasculature under hypoxic injury conditions such as ischemia-reperfusion (I/R) injury. Here we performed inducible genetic deletion of endothelial Ripk3 (RipkiECKO) in mice, which led to elevated vascular permeability in the small intestine and multiple distal organs after intestinal I/R injury. Mechanistically, this vascular permeability correlated with increased endothelial secretion of IL-6 and organ-specific expression of VCAM-1 and ICAM-1 adhesion molecules. Circulating monocyte depletion with clodronate liposomes reduced permeability in organs with elevated adhesion molecules, highlighting the contribution of monocyte adhesion and extravasation to RipkiECKO barrier dysfunction. These results elucidate mechanisms by which RIPK3 regulates endothelial inflammation to minimize vascular permeability in I/R injury.

9-PAHSA ameliorates microvascular damage during cardiac ischaemia/reperfusion injury by promoting LKB1/AMPK/ULK1-mediated autophagy-dependent STING degradation

BackgroundConsidering that cardiac microvascular injury may play a more critical role than cardiomyocyte injury in the pathology of early ischaemia/reperfusion (I/R) injury, therapeutic strategies targeting the microvasculature are highly desirable. Palmitic acid-9-hydroxystearic acid (9-PAHSA) is a new class of bioactive anti-inflammatory lipids widely distributed in vegetables, fruits and medicinal plants, especially broccoli and apple. However, the pharmacological effects and underlying mechanisms of 9-PAHSA in protecting- against cardiac microvascular I/R injury have rarely been studied. PurposeThis study aimed to explore the potential effects and molecular mechanisms of 9-PAHSA on the coronary microvasculature after cardiac I/R injury. MethodsImmunofluorescence staining, western blotting, and other experimental methods were used to evaluate the role and mechanism of 9-PAHSA in cardiac microvascular I/R injury in vivo and in vitro. Results9-PAHSA administration significantly attenuated myocardial I/R-induced microvascular damage, as indicated by an impaired microvascular structure, reduced regional blood perfusion and decreased endothelial barrier function. In addition, 9-PAHSA administration protected the structure and function of coronary artery endothelial cells (CMECs) to resist I/R damage, an effect that was at least partially mediated by increased autophagy. Mechanistically, 9-PAHSA activated autophagy through the LKB1/AMPK/ULK1 pathway and promoted STING degradation via the autophagic‒lysosomal pathway. ConclusionsTo our best knowledge, this study is the first to report that 9-PAHSA attenuates cardiac microvascular I/R injury, potentially by activating LKB1/AMPK/ULK1-mediated autophagy-dependent STING degradation to suppress apoptosis. Thus, 9-PAHSA may be a promising therapeutic option for alleviating cardiac microvascular I/R injury.

Abstract 4133170: Insulin resistance triggers histamine release from endothelial cells, which disrupts endothelial barrier function and causes vasoconstriction.

Diabetic vascular dysfunction is a serious health concern, affecting ~38 million people in the USA, according to the 2020 CDC-National Diabetes Statistics Reports. Type 2 diabetes (T2D) causes several vascular changes leading to major clinical consequences such as stroke and ~3-fold elevated risk of cardiovascular diseases (CVD) and the underlying causes of vascular dysfunction in T2D are complex. The anatomical position of endothelial cells (ECs) between the circulating blood and vessel walls makes them the first to encounter and respond to cellular stressors in the bloodstream. Here, we investigated the role of long-term increases in plasma histamine levels as observed in T2D and its effects on ECs and vascular contractility. The high-fat diet-low-dose streptozotocin protocol induced T2D in C57BL/6J mice. Endothelial cell function was analyzed using Western blotting, in vitro blood-brain barrier assays, histamine measurements, immunofluorescence, and pressurized artery myography. In our T2D model, EC-mediated histamine synthesis occurred after the onset of IR. A spike in plasma histamine levels was observed immediately following the onset of insulin resistance, followed by a brief plateau. A second spike in plasma histamine levels was noted four weeks later. In isolated control ECs, the addition of external histamine showed a concentration-dependent increase in histidine decarboxylase expression, and cellular histamine levels were observed, which was inhibited by the H2R blocker, famotidine. The tight junction marker, JAM-A (CD321), expression in diabetic cerebral arteries was ~42% less compared to the non-diabetic controls. Transendothelial Electrical Resistance testing showed a ~2-fold higher permeability with diabetic ECs than controls. Pressure myography on isolated cerebral arteries indicated that external histamine passed through the lumen of arteries from T2D mice 4 weeks after onset of IR constricted whereas control arteries dilated. Additionally, T2D mice cerebral artery myogenic tone at 60 mmHg was 70% greater compared to control arteries. These data indicate that the onset of IR triggers an increase in circulating histamine levels, which in turn stimulates histamine synthesis and release from endothelial cells leading to the loss of endothelial barrier function, blood-brain barrier leakage, and vasoconstriction. We are currently investigating the molecular pathways that precede the increase in circulating histamine levels during T2D.

The Adhesion GPCR ADGRL2/LPHN2 Can Protect Against Cellular and Organismal Dysfunction.

The most common trigger of sepsis and septic shock is bacterial lipopolysaccharide (LPS). Endothelial cells are among the first to encounter LPS directly. Generally, their function is closely linked to active endothelial NO Synthase (eNOS), which is significantly reduced under septic conditions. LPS treatment of endothelial cells leads to their activation and apoptosis, resulting in loss of integrity and vascular leakage, a hallmark of septic shock. Hence, therapies that prevent endothelial leakage or restore the endothelial barrier would be invaluable for patients. Adhesion GPCRs (aGPCRs) have been largely overlooked in this context, although particularly one of them, ADGRL2/LPHN2, has been implicated in endothelial barrier function. Our study shows that overexpression of ADGRL2 protects endothelial cells from LPS-induced activation, apoptosis, and impaired migration. Mechanistically, ADGRL2 preserves eNOS activity by shifting its binding from Caveolin-1 to Heat Shock Protein 90. Furthermore, ADGRL2 enhances antioxidative responses by increasing NRF2 activity. Notably, we found that this function may be evolutionarily conserved. In the absence of lat-2, a homolog of ADGRL2 in Caenorhabditis elegans, worms show higher ROS levels and altered stress response gene expression. Additionally, lat-2 mutants have a significantly reduced lifespan, altogether indicating a protective role of ADGRL2 against oxidative stress across species.

Kindlin-2 Phase Separation in Response to Flow Controls Vascular Stability.

Atheroprotective shear stress preserves endothelial barrier function, while atheroprone shear stress enhances endothelial permeability. Yet, the underlying mechanisms through which distinct flow patterns regulate EC integrity remain to be clarified. This study aimed to investigate the involvement of Kindlin-2, a key component of focal adhesion and endothelial adherens junctions crucial for regulating endothelial cell (EC) integrity and vascular stability. Mouse models of atherosclerosis in EC-specific Kindlin-2 knockout mice (Kindlin-2iΔEC) were used to study the role of Kindlin-2 in atherogenesis. Pulsatile shear (12±4 dynes/cm2) or oscillatory shear (0.5±4 dynes/cm2) were applied to culture ECs. Live-cell imaging, fluorescence recovery after photobleaching assay, and OptoDroplet assay were used to study the liquid-liquid phase separation (LLPS) of Kindlin-2. Co-immunoprecipitation, mutagenesis, proximity ligation assay, and transendothelial electrical resistance assay were used to explore the underlying mechanism of flow-regulated Kindlin-2 function. We found that Kindlin-2 localization is altered under different flow patterns. Kindlin-2iΔEC mice showed heightened vascular permeability. Kindlin-2iΔEC were bred onto ApoE-/- mice to generate Kindlin-2iΔEC; ApoE-/- mice, which displayed a significant increase in atherosclerosis lesions. In vitro data showed that in ECs, Kindlin-2 underwent LLPS, a critical process for proper focal adhesion assembly, maturation, and junction formation. Mass spectrometry analysis revealed that oscillatory shear increased arginine methylation of Kindlin-2, catalyzed by PRMT5 (protein arginine methyltransferase 5). Functionally, arginine hypermethylation inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation. Notably, we identified R290 of Kindlin-2 as a crucial residue for LLPS and a key site for arginine methylation. Finally, pharmacologically inhibiting arginine methylation reduces EC activation and plaque formation. Collectively, our study elucidates that mechanical force induces arginine methylation of Kindlin-2, thereby regulating vascular stability through its impact on Kindlin-2 LLPS. Targeting Kindlin-2 arginine methylation emerges as a promising hemodynamic-based strategy for treating vascular disorders and atherosclerosis.

Rap1A Modulates Store-Operated Calcium Entry in the Lung Endothelium: A Novel Mechanism Controlling NFAT-Mediated Vascular Inflammation and Permeability.

Store-operated calcium entry mediated by STIM (stromal interaction molecule)-1-Orai1 (calcium release-activated calcium modulator 1) is essential in endothelial cell (EC) functions, affecting signaling, NFAT (nuclear factor for activated T cells)-induced transcription, and metabolic programs. While the small GTPase Rap1 (Ras-proximate-1) isoforms, including the predominant Rap1B, are known for their role in cadherin-mediated adhesion, EC deletion of Rap1A after birth uniquely disrupts lung endothelial barrier function. Here, we elucidate the specific mechanisms by which Rap1A modulates lung vascular integrity and inflammation. The role of EC Rap1A in lung inflammation and permeability was examined using in vitro and in vivo approaches. We explored Ca2+ signaling in human ECs following siRNA-mediated knockdown of Rap1A or Rap1B. Rap1A knockdown, unlike Rap1B, significantly increased store-operated calcium entry in response to a GPCR (G-protein-coupled receptor) agonist, ATP (500 µmol/L), or thapsigargin (250 nmol/L). This enhancement was attenuated by Orai1 channel blockers 10 μmol/L BTP2 (N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide), 10 μmol/L GSK-7975A, and 5 μmol/L Gd3+. Whole-cell patch clamp measurements revealed enhanced Ca2+ release-activated Ca2+ current density in siRap1A ECs. Rap1A depletion in ECs led to increased NFAT1 nuclear translocation and activity and elevated levels of proinflammatory cytokines (CXCL1 [C-X-C motif chemokine ligand 1], CXCL11 [C-X-C motif chemokine 11], CCL5 [chemokine (C-C motif) ligand 5], and IL-6 [interleukin-6]). Notably, reducing Orai1 expression in siRap1A ECs normalized store-operated calcium entry, NFAT activity, and endothelial hyperpermeability in vitro. EC-specific Rap1A knockout (Rap1AiΔEC) mice displayed an inflammatory lung phenotype with increased lung permeability and inflammation markers, along with higher Orai1 expression. Delivery of siRNA against Orai1 to lung endothelium using lipid nanoparticles effectively normalized Orai1 levels in lung ECs, consequently reducing hyperpermeability and inflammation in Rap1AiΔEC mice. Our findings uncover a novel role of Rap1A in regulating Orai1-mediated Ca2+ entry and expression, crucial for NFAT-mediated transcription and endothelial inflammation. This study distinguishes the unique function of Rap1A from that of the predominant Rap1B isoform and highlights the importance of normalizing Orai1 expression in maintaining lung vascular integrity and modulating endothelial functions.

Human epicardial adipose tissue secretome alters metabolomic profile and induces endothelial-to-mesenchymal transition in human lymphatic endothelial cells

Abstract Background Cardiac lymphatic systems play an important role in maintaining myocardial fluid homeostasis during inflammation. We have recently reported the epicardial adipose tissue (EAT)-derived factor induces inflammation and fibrosis in atrial myocardium, which lead to atrial fibrillation (AF). However, the direct effect of EAT on the cardiac lymphatic endothelial cells (LECs) remains unknown. Objective To elucidate the effect of EAT on cardiac LECs, as one of the potential causes of AF. Methods Human EAT samples were collected from 18 consecutive patients with or without AF patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=9, 72.7±8.6 years) and the AF group (n=9, 73.3±7.4 years). Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation to mature adipocytes, cell culture supernatant was collected, and cultured with human LECs. Cell permeability was measured by using FITC-dextran assay. Tube forming ability was analyzed by seeding LECs on the extracellular matrix gels. Metabolomic profiling of LECs was performed by GC-MS system. Results RT-PCR analysis revealed that cell culture supernatant from mature adipocytes of AF patients significantly increased mRNA expression of mesenchymal marker, TAGLN/SM22a (p<0.01) in human LECs, whereas mRNA expression of lymphatic endothelial markers, LYVE1 (p=0.0552) and PROX1 (p=0.0231) were decreased in human LECs, compared to those from mature adipocytes of SR patients. These data suggest cell culture supernatant from mature adipocytes of AF patients highly induce endothelial-to-mesenchymal transition (EndMT) of LECs. When assessing the endothelial barrier function by using FITC-dextran, cell culture supernatant from mature adipocytes of AF patients significantly decreased cell barrier function (p<0.01) compared to those from mature adipocytes of SR patients. Furthermore, cell culture supernatant from mature adipocytes of AF patients significantly decreased the tube forming ability of LECs (p<0.01) compared to those from mature adipocytes of SR patients. To assess the metabolomic changes in human LECs by EAT-secretome, we conducted the GC/MS analysis. Cell culture supernatant from mature adipocytes of AF patients significantly decreased several amino acids including branched-chain amino acid (BCAA; valine, leucine and isoleucine), compared to those from mature adipocytes of SR patients. Conclusion Our study with human EAT and LECs demonstrated that EAT-secretome from AF patients altered metabolomic profile in LECs, and highly caused EndMT in LECs, leading to impaired barrier function and the tube forming ability. Defects of lymphangiogenesis could be a therapeutic target for atrial cardiomyopathy.

Alleviation of LPS-induced Endothelial Injury due to GHRH Antagonist Treatment.

GHRH is produced in the hypothalamus and affects various tissues beyond the pituitary, including the lungs. GHRH antagonists exert anti-inflammatory properties in several experimental models of disease, but their role inprotecting the endothelial barrier during inflammation is less understood. This study investigates the effects ofGHRHAnt on LPS-induced endothelial dysfunction. BPAEC and HMVEC-L cells were exposed to LPS to induce endothelial injury. GHRHAnt was administered eitherpre- or post-LPS treatment. Western blot analysis was used to evaluate protein expression levels. Paracellularpermeability was assessed utilizing FITC-dextran assay to evaluate endothelial barrier function. GHRHAnt post-treatment significantly reduced LPS-induced MLC2 phosphorylation and cofilin activation inBPAECs. Furthermore, pretreatment with GHRHAnt enhanced barrier function and ameliorated LPS-inducedhyperpermeability in both human and bovine endothelial cells. GHRHAnt treatment mitigates LPS-induced endothelial barrier dysfunction. These findings suggest that GHRHAntcould serve as potential therapeutic agents towards endothelial dysfunction-related illness (e.g. sepsis).

AAMP and MTSS1 Are Novel Negative Regulators of Endothelial Barrier Function Identified in a Proteomics Screen.

Cell-cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity. Here, we sought to identify other barrier regulators, which are characterized by a short half-life, using a proteomics approach. Following short-term inhibition of ubiquitination with E1 ligase inhibitor MLN7243 or Cullin E3 ligase inhibitor MLN4924 in primary human endothelial cells, we identified sixty significantly differentially expressed proteins. Intriguingly, our data showed that AAMP and MTSS1 are novel negative regulators of endothelial barrier function and that their turnover is tightly controlled by ubiquitination. Mechanistically, AAMP regulates the stability and activity of RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, possibly regulating F-actin dynamics. Taken together, these findings demonstrate the critical role of protein turnover of specific proteins in the regulation of endothelial barrier function, contributing to our options to target dysregulation of vascular permeability.