Endothelial Barrier Protection Research Articles

Ameliorating lipopolysaccharide induced acute lung injury with Lianhua Qingke: focus on pulmonary endothelial barrier protection.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) has long posed challenges in clinical practice, lacking established preventive and therapeutic approaches. Lianhua Qingke (LHQK), a patented traditional Chinese medicine (TCM), has been found to have anti-inflammatory effects for ameliorating ALI/ARDS induced by lipopolysaccharide (LPS). This study aimed to investigate the effects and potential mechanisms of LHQK on endothelial protection in LPS-induced ALI/ARDS in vivo and in LPS-induced human pulmonary microvascular endothelial cells (HPMECs) injury in vitro. In the animal experiment, we induced an ALI/ARDS model by intratracheal injection of LPS (5 mg/mL). LHQK (3.7 g/kg/d for low dose and 7.4 g/kg/d for high dose) or dexamethasone (DEX) (5 mg/kg/d) was administered to mice 3 days prior to LPS treatment. In the in vitro experiments, HPMECs were pretreated with LHQK at concentrations of 125 and 250 µg/mL for 2 hours before being stimulated with LPS (10 µg/mL). We employed lung function test, measurement of lung index, hematoxylin and eosin (H&E) staining, bronchoalveolar lavage fluid (BALF) cell counts, and inflammatory cytokine levels to assess the therapeutic effect of LHQK. Additionally, the extravasation assay of fluorescein isothiocyanate-dextran (FITC-dextran) dye and the transmembrane electrical resistance (TEER) assay were used to evaluate endothelial barrier. Barrier integrity and relevant protein validation were assessed using immunofluorescence (IF) and Western blot analyses. Furthermore, network pharmacology analysis and cellular level screening were employed to predict and screen the active ingredients of LHQK. Compared to the LPS group, LHQK significantly improved lung function, mitigated lung pathological injuries, reduced inflammatory cells and inflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6] levels in BALF, and inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1), attenuated LPS-induced pulmonary oedema and FITC-dextran permeability, and enhanced the expression of vascular endothelial-cadherin (VE-cadherin) and occludin. In vitro, LHQK attenuated LPS-induced HPMECs injury by elevating TEER values and enhancing VE-cadherin and occludin protein levels. Finally, network pharmacology analysis and cellular level validation identified potential active ingredients of LHQK. In summary, LHQK can mitigate LPS-induced inflammatory infiltration, pulmonary edema, and pulmonary vascular endothelial barrier dysfunction in the context of ALI/ARDS. This is achieved by decreasing the levels of VCAM-1, and increasing the expression levels of barrier-associated junctions, such as VE-cadherin and occludin. Consequently, LHQK exhibits promising therapeutic potential in preventing the progression of ALI/ARDS.

Abstract Tu029: Effects of the phosphodiesterase inhibitors, cilostazol and ensifentrine, on alveolar-capillary dysfunction caused by MRSA

Background: Acute lung injury (ALI) is characterized by excessive inflammation and dysfunction of the alveolar-capillary barrier, which consists of lung vascular endothelial and alveolar epithelial cells. Therapeutic interventions that protect this barrier are promising strategies for ALI treatment. Phosphodiesterase (PDE) inhibitors are potent modulators of inflammation. These include cilostazol, a PDE3 inhibitor, and ensifentrine, a dual PED3/4 inhibitor, which are both clinically available compounds that exhibit cytoprotective and anti-inflammatory properties. However, their roles in regulating alveolar-capillary function in ALI are unknown. The aim of this study is to investigate the effects of cilostazol and ensifentrine on lung endothelial and epithelial dysfunction caused by methicillin-resistant Staphylococcus aureus (MRSA), a common bacterial pathogen involved in ALI pathogenesis. Methods: Human primary lung endothelial cells (EC) and A549 (alveolar epithelial cell line) were pre-treated with cilostazol (CZ; 15 μM) or ensifentrine (Ens; 10 μΜ) for 1 hour and then challenged with heat-killed MRSA (2.5 x10 8 /ml). The Electrical Cell Impedance Sensing (ECIS) assay was used to assess EC barrier integrity. Pro-inflammatory signaling markers were measured by western blotting (WB) and qPCR. Cell conditioned media was analyzed for extracellular vesicle release by flow cytometry and WB. Results: MRSA-induced EC barrier disruption was attenuated by both CZ and Ens, although Ens exhibited superior barrier protective properties. CZ and Ens suppressed MRSA-induced upregulation of the inflammatory adhesion molecules, VCAM-1 and ICAM-1, in EC. Ens, but not CZ, reduced MRSA-induced ICAM-1 upregulation in A549. MRSA caused a significant increase in the release of EC-derived extracellular vesicles, which was partially inhibited by CZ or Ens pretreatment. Conclusions: Both cilostazol and ensifentrine exert anti-inflammatory properties in MRSA-treated lung EC. Compared to cilostazol, ensifentrine exhibits enhanced endothelial barrier protection and inhibits inflammation in both lung endothelial and epithelial cells. Future in vivo studies will evaluate the therapeutic efficacy of these inhibitors in bacterial-induced ALI.

S1PR3 inhibition protects against LPS-induced ARDS by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation

BackgroundInflammation and endothelial barrier dysfunction are the major pathophysiological changes in acute respiratory distress syndrome (ARDS). Sphingosine-1-phosphate receptor 3 (S1PR3), a G protein-coupled receptor, has been found to mediate inflammation and endothelial cell (EC) integrity. However, the function of S1PR3 in ARDS has not been fully elucidated.MethodsWe used a murine lipopolysaccharide (LPS)-induced ARDS model and an LPS- stimulated ECs model to investigate the role of S1PR3 in anti-inflammatory effects and endothelial barrier protection during ARDS.ResultsWe found that S1PR3 expression was increased in the lung tissues of mice with LPS-induced ARDS. TY-52156, a selective S1PR3 inhibitor, effectively attenuated LPS-induced inflammation by suppressing the expression of proinflammatory cytokines and restored the endothelial barrier by repairing adherens junctions and reducing vascular leakage. S1PR3 inhibition was achieved by an adeno-associated virus in vivo and a small interfering RNA in vitro. Both the in vivo and in vitro studies demonstrated that pharmacological or genetic inhibition of S1PR3 protected against ARDS by inhibiting the NF-κB pathway and improving mitochondrial oxidative phosphorylation.ConclusionsS1PR3 inhibition protects against LPS-induced ARDS via suppression of pulmonary inflammation and promotion of the endothelial barrier by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation, indicating that S1PR3 is a potential therapeutic target for ARDS.

Platelet releasates mitigate the endotheliopathy of trauma.

Platelets are well known for their roles in hemostasis, but they also play a key role in thromboinflammatory pathways by regulating endothelial health, stimulating angiogenesis, and mediating host defense through both contact dependent and independent signaling. When activated, platelets degranulate releasing multiple active substances. We hypothesized that the soluble environment formed by trauma platelet releasates (TPR) attenuates thromboinflammation via mitigation of trauma induced endothelial permeability and metabolomic reprogramming. Blood was collected from injured and healthy patients to generate platelet releasates and plasma in parallel. Permeability of endothelial cells when exposed to TPR and plasma (TP) was assessed via resistance measurement by electric cell-substrate impedance sensing (ECIS). Endothelial cells treated with TPR and TP were subjected to mass spectrometry-based metabolomics. TP increased endothelial permeability, whereas TPR decreased endothelial permeability when compared with untreated cells. When TP and TPR were mixed ex vivo, TPR mitigated TP-induced permeability, with significant increase in AUC compared with TP alone. Metabolomics of TPR and TP demonstrated disrupted redox reactions and anti-inflammatory mechanisms. Trauma platelet releasates provide endothelial barrier protection against TP-induced endothelial permeability. Our findings highlight a potential beneficial action of activated platelets on the endothelium in injured patients through disrupted redox reactions and increased antioxidants. Our findings support that soluble signaling from platelet degranulation may mitigate the endotheliopathy of trauma. The clinical implications of this are that activated platelets may prove a promising therapeutic target in the complex integration of thrombosis, endotheliopathy, and inflammation in trauma.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure

BackgroundAtrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. ObjectivesIn this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. MethodsThe authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. ResultsAF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. ConclusionsThese results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

LPA2 Alleviates Septic Acute Lung Injury via Protective Endothelial Barrier Function Through Activation of PLC-PKC-FAK.

Increased endothelial permeability of pulmonary vessels is a primary pathological characteristic of septic acute lung injury (ALI). Previously, elevated lysophosphatidic acid (LPA) levels and LPA2 (an LPA receptor) expression have been found in the peripheral blood and lungs of septic mice, respectively. However, the specific role of LPA2 in septic ALI remains unclear. A lipopolysaccharide (LPS)-induced model of sepsis was established in wild-type (WT) and global LPA2 knockout (Lpar2-/-) mice. We examined mortality, lung injury, assessed endothelial permeability through Evans blue dye (EBD) assay in vivo, and transendothelial electrical resistance (TEER) of mouse lung microvascular endothelial cells (MLMECs) in vitro. Enzyme-linked immunosorbent assay (ELISA), histopathological, immunofluorescence, immunohistochemistry, and Western blot were employed to investigate the role of LPA2 in septic ALI. Lpar2 deficiency increased vascular endothelial permeability, impaired lung injury, and increased mortality. Histological examination revealed aggravated inflammation, edema, hemorrhage and alveolar septal thickening in the lungs of septic Lpar2-/- mice. In vitro, loss of Lpar2 resulted in increased permeability of MLMECs. Pharmacological activation of LPA2 by the agonist DBIBB led to significantly reduced inflammation, edema and hemorrhage, as well as increased expression of the vascular endothelial tight junction (TJ) protein zonula occludens-1 (ZO-1) and claudin-5, as well as the adheren junction (AJ) protein VE-cadherin. Moreover, DBIBB treatment was found to alleviate mortality by protecting against vascular endothelial permeability. Mechanistically, we demonstrated that vascular endothelial permeability was alleviated through LPA-LPA2 signaling via the PLC-PKC-FAK pathway. These data provide a novel mechanism of endothelial barrier protection via PLC-PKC-FAK pathway and suggest that LPA2 may contribute to the therapeutic effects of septic ALI.

The APC-EPCR-PAR1 axis in sickle cell disease.

Sickle Cell Disease (SCD) is a group of inherited hemoglobinopathies. Sickle cell anemia (SCA) is caused by a homozygous mutation in the β-globin generating sickle hemoglobin (HbS). Deoxygenation leads to pathologic polymerization of HbS and sickling of erythrocytes. The two predominant pathologies of SCD are hemolytic anemia and vaso-occlusive episodes (VOE), along with sequelae of complications including acute chest syndrome, hepatopathy, nephropathy, pulmonary hypertension, venous thromboembolism, and stroke. SCD is associated with endothelial activation due to the release of danger-associated molecular patterns (DAMPs) such as heme, recurrent ischemia-reperfusion injury, and chronic thrombin generation and inflammation. Endothelial cell activation is mediated, in part, by thrombin-dependent activation of protease-activated receptor 1 (PAR1), a G protein coupled receptor that plays a role in platelet activation, endothelial permeability, inflammation, and cytotoxicity. PAR1 can also be activated by activated protein C (APC), which promotes endothelial barrier protection and cytoprotective signaling. Notably, the APC system is dysregulated in SCD. This mini-review will discuss activation of PAR1 by APC and thrombin, the APC-EPCR-PAR1 axis, and their potential roles in SCD.

The vasculoprotective effects of resveratrol are mediated via Kruppel-like factor 2 dependent protection of endothelial barrier function

Resveratrol is a naturally occurring polyphenolic compound that is thought to have vasculoprotective properties. Its observed effects are proposed, in part, to be mediated through the induction of endothelial Krϋppel-like factor 2 (KLF2) expression. KLF2 is a nuclear transcription factor that is highly expressed within the vascular endothelium. Studies from our laboratory and others have shown that this protein mediates vascular function through its transactivation domain, and its targeted expression promotes vascular health, notably by acting as an important positive regulator of endothelial barrier function. In this study, we demonstrate that resveratrol possesses endothelial barrier protective effects dependent on the presence of KLF2, with several key endothelial tight junction proteins expressed in a KLF2-dependent manner. Collectively, our findings identify KLF2 as essential for resveratrol-mediated endothelial barrier protection, thus further implicating KLF2 as a critical vasculoprotective factor.

Effect of albumin solutions on endothelial oxidant injury: A microfluidic study

BackgroundStudies have suggested a beneficial effect of early plasma-based resuscitation in patients following trauma-hemorrhagic shock. The underlying mechanism(s) are unknown but may be owing to protective effects of plasma components on the endothelium and its glycocalyx layer. Albumin, the major protein in plasma, influences vascular integrity and has antioxidant properties in vivo. Sphingosine 1-phosphate is a bioactive sphingolipid with diverse signaling functions, which include endothelial barrier protection in part owing to preservation of the glycocalyx. Sphingosine 1-phosphate is bound mainly to albumin and high-density lipids in the plasma. Debate continues about the beneficial effect of albumin solutions in shock resuscitation. Pharmacologic preparations may modify constituents of albumin solutions for clinical use. We examined the relative effects of sphingosine 1-phosphate concentrations in albumin solutions on the endothelial-glycocalyx barrier in an in vitro microfluidic platform. MethodsEndothelial cell monolayers were established in microfluidic perfusion devices and exposed to control or biomimetic shock conditions followed by 5% plasma or different albumin solutions ± exogenous sphingosine 1-phosphate perfusion. Biomarkers of endothelial and glycocalyx activation, damage, and oxidant injury were then determined. ResultsEndothelial cell and glycocalyx barriers were damaged after biomimetic shock conditions. Plasma and sphingosine 1-phosphate loaded albumin solutions protected against barrier injury. Modest protective effects were noted with albumin alone; the efficacy varied with sphingosine 1-phosphate content of the albumin solution. ConclusionThe protective effect of albumin on the endothelia-glycocalyx barrier against oxidant injury was dependent on its sphingosine 1-phosphate concentration. Our data may help explain the discrepancies regarding the effectiveness of albumin solutions in shock resuscitation.

Adipose-Derived Circulating Exosomes Promote Protection of the Pulmonary Endothelial Barrier by Inhibiting EndMT and Oxidative Stress through Down-Regulation of the TGF-β Pathway: A Potential Explanation for the Obesity Paradox in ARDS

The “obesity paradox in acute respiratory distress syndrome” (ARDS) refers to the phenomenon in which obesity is associated with higher morbidity but lower mortality in patients with ARDS. Endothelial-to-mesenchymal transition (EndMT) represents a key link in the interaction between endothelial disruption and mesenchymal fibrosis under inflammatory and oxidative conditions, which represent the intersectional pathophysiology of ARDS. Adipose tissue is considered to constitute the major source of circulating exosomal microRNAs (miRNAs), which act as genetic forms of adipokines for cell–cell crosstalk. We aimed to demonstrate the regulation and mechanism of adipose-derived exosomes in the obesity paradox in ARDS. High-fat-induced obese mice and lean control mice were subjected to ARDS insult to investigate the effects of obesity on ARDS and microarray analysis was performed to screen for differences in circulating miRNAs. In addition, mice and pulmonary endothelial cells were administered with adipose-derived exosomal miR-122-5p to investigate the underlying molecular mechanisms. We found high-fat diet-induced obesity protected against ARDS in mice by reinforcing endothelial barrier and attenuating fibroproliferation. Circulating exosomes produced in the obese state mediated these protective effects by inhibiting EndMT and oxidative stress. Mechanistically, adipose-derived exosomal miR-122-5p promoted the integrity and function of pulmonary endothelial barrier and alleviated fibrogenesis by suppressing EndMT and oxidative stress through down-regulation of the transforming growth factor β1 (TGF-β1)/TGF-β receptor 1 (TGF-βR1)/Smad2 pathway in vivo and in vitro. In conclusion, adipose-derived circulating exosomal miR-122-5p protects against ARDS by reinforcing pulmonary endothelial barrier through inhibition of EndMT and oxidative stress via down-regulation of the TGF-β pathway, which propose a potential explanation for the obesity paradox in ARDS and indicate promising prospects for adipose-derived exosomes in cell-free therapies for ARDS.

Regulation of activated Protein C/PAR1 endothelial cytoprotective signaling by G protein‐coupled receptor kinases

Endothelial dysfunction is a hallmark of inflammation and associated with vascular diseases. Our goal is to define pathways by which the endothelium can resist injury and disruption to facilitate the advancement of new targets for therapeutic development. Activated Protein C (APC) has been shown in preclinical studies to increase endothelial barrier protection and reduce inflammatory responses and signals via protease‐activated receptor‐1 (PAR1). APC enhances endothelial cytoprotection through cleavage of PAR1 at an N‐terminal arginine (R)‐46 site, that is distinct from the canonical thrombin cleavage site at R‐41. This results in the generation of a distinct tethered ligand that promotes PAR1 bias signaling. We discovered that APC/PAR1 signals preferentially via b‐arrestin2 (b‐arr2), rather than heterotrimeric G proteins to promote endothelial barrier protection. The objective of this study is to develop a mechanistic understanding of how APC/PAR1 generates b‐arr2 cytoprotective signaling. G protein‐coupled receptor kinases (GRKs) phosphorylate thrombin‐activated PAR1 and facilitate β‐arrestin‐mediated desensitization. However, contribution of GRKs to β‐arrestin driven APC/PAR1‐induced cytoprotection is unclear. We hypothesize that compartmentalization of APC/PAR1 together with GRK5 is essential to initiate b‐arr2 recruitment and biased signaling. Here we will present studies examining the function and mechanism by which GRKs regulate endothelial cytoprotective responses using GRK‐specific siRNA to assess function as well as confocal immunofluorescence imaging and biochemical sucrose fraction to examine GRK localization and compartmentalization in caveolae. The work is expected to provide important new insight into the role of GRKs in regulating APC/PAR1 cytoprotection in human cultured endothelial cells.

Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg−1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.

Vascular endothelial barrier protection prevents nanoscale cardiac remodeling: a novel strategy to prevent atrial fibrillation

Atrial fibrillation (AF) patients experience inflammation and vascular dysfunction and have elevated levels of cytokines that promote vascular leak and edema, such as vascular endothelial growth factor (VEGF). We previously identified edema-induced disruption of sodium channel (NaV1.5) -rich intercalated disk (ID) nanodomains as a novel arrhythmia mechanism. Therefore, we hypothesized that: (i) VEGF-induced vascular leak acutely slows action potential propagation in the atria and increases arrhythmia risk by disrupting ID nanodomains, and (ii) protection of the vascular barrier can prevent vascular leak-induced atrial arrhythmias.

Immunolocalization of protease-activated receptors in endothelial cells of splenic sinuses.

The immunolocalization of protease-activated receptors (PARs) and related proteins in splenic sinus endothelial cells was examined using immunofluorescence and electron microscopy. Immunofluorescence microscopy showed that PAR1 colocalized with PAR2, PAR3, and PAR4. PAR4 colocalized with PAR3 and P2Y12. Myosin heavy chain IIA localized to the outer shape and at the base of cells, but did not colocalize with α-catenin. The localization of di-phosphorylated myosin regulatory light chains (ppMLC) was partially detected on the outer circumference and conspicuously at the base of cells. Macrophage migration inhibitory factor (MIF) also localized in cells. Immunogold electron microscopy revealed the localization of PAR1 on the caveolar membrane, plasma membrane, and junctional membrane of cells. PAR2 and PAR3 localized to the plasma membrane of cells. PAR4 localized to the plasma membrane, depressions in the plasma membrane, and cytoplasmic vesicles. PpMLC was detected in stress fibers, but rarely near the adherens junctions of neighboring cells. MIF localized in vesicles on the apical and basal sides of the Golgi apparatus. Electron microscopy of endothelial cells with saponin extraction showed the depression of many coated pits formed by clathrin from the plasma membrane. Stress fibers developed at the base of cells; however, few actin filaments were observed near adherens junctions. These results indicate that PARs play important roles in splenic sinus endothelial cells, such as in endothelial barrier protection and the maintenance of firm adhesion to ring fibers.

Distinct Factors Secreted by Adipose Stromal Cells Protect the Endothelium From Barrier Dysfunction and Apoptosis

We have shown previously that adipose stromal cell (ASC)-derived conditioned media (CM) limited lung injury, endothelial barrier dysfunction, and apoptosis. Here, we used endothelial hyperpermeability and apoptosis assays to investigate how concentration processes affect endothelium-directed bioactivity of ASC-CM and to gain information on the nature of bioactive factors. Comparison of ASC-CM concentrated with differential molecular weight (MW) cutoff filters showed that endothelial barrier protection depended on the species-specific factors in ASC-CM fractionated with MW > 50 kDa. Known barrier regulators—keratin growth factor (KGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF)—were detected in ASC-CM fraction of > 100 kDa. Pretreatment of endothelial monolayers with concentrations of KGF, VEGF, and HGF detected in ASC-CM showed that only KGF and HGF protect the endothelium from barrier dysfunction. Depletion of KGF and HGF from ASC-CM attenuated ASC-CM’s ability to protect the endothelial barrier. In contrast to barrier-protective factors, apoptosis-protective factors fractionated with MW < 3 kDa and were not species-specific. Application of donors of apoptosis-mitigating gases showed that the CO donor carbon monoxide-releasing molecule 2 (CORM2) protected the endothelium from apoptosis, while the H2S donor NaSH did not. Knockdown of CO-generating heme oxygenase 1 in ASC attenuated ASC-CM’s ability to protect the endothelium from apoptosis. We have shown that tumor necrosis factor alpha (TNFα)-induced apoptosis in endothelium is c-Jun N-terminal kinase (JNK)-dependent, and JNK activation is inhibited by ASC-CM pretreatment of endothelial cells. ASC-CM from heme oxygenase 1-depleted ASC displayed attenuated ability to suppress endothelial JNK activation, suggesting that CO-mediated protection of the endothelium from apoptosis is achieved by the downregulation of the JNK pathway. Altogether, our results demonstrate that the concentration of ASC-CM with low MW cutoff filters significantly reduces its anti-apoptotic activity while preserving its barrier-protective activity.

C‐terminal residues of activated protein C light chain contribute to its anticoagulant and cytoprotective activities

BackgroundActivated protein C (APC) is an important homeostatic blood coagulation protease that conveys anticoagulant and cytoprotective activities. Proteolytic inactivation of factors Va and VIIIa facilitated by cofactor protein S is responsible for APC's anticoagulant effects, whereas cytoprotective effects of APC involve primarily the endothelial protein C receptor (EPCR), protease activated receptor (PAR)1 and PAR3. ObjectiveTo date, several binding exosites in the protease domain of APC have been identified that contribute to APC's interaction with its substrates but potential contributions of the C‐terminus of the light chain have not been studied in detail. MethodsSite‐directed Ala‐scanning mutagenesis of six positively charged residues within G142‐L155 was used to characterize their contributions to APC's anticoagulant and cytoprotective activities. Results and ConclusionsK151 was involved in protein S dependent‐anticoagulant activity of APC with some contribution of K150. 3D structural analysis supported that these two residues were exposed in an extended protein S binding site on one face of APC. Both K150 and K151 were important for PAR1 and PAR3 cleavage by APC, suggesting that this region may also mediate interactions with PARs. Accordingly, APC's cytoprotective activity as determined by endothelial barrier protection was impaired by Ala substitutions of these residues. Thus, both K150 and K151 are involved in APC's anticoagulant and cytoprotective activities. The differential contribution of K150 relative to K151 for protein S‐dependent anticoagulant activity and PAR cleavage highlights that binding exosites for protein S binding and for PAR cleavage in the C‐terminal region of APC's light chain overlap.

Integration of GPCR‐induced endothelial cytoprotection signaling by β‐arrestin‐2

Vascular dysfunction is a hallmark of inflammation and results in endothelial barrier disruption, vascular leakage and apoptosis. G protein‐coupled receptors (GPCRs) are highly druggable and mediate inflammatory responses associated with endothelial dysfunction. Specifically, two GPCRs, protease‐activated receptor‐1 (PAR1) and sphingosine‐1‐phosphate receptor‐1 (S1PR1) promote endothelial cytoprotection including barrier protection, Akt‐mediated cell survival, and anti‐inflammatory responses. Activated protein C (APC) activates PAR1 to enhance endothelial barrier stability, whereas S1PR1 contributes to basal barrier stability, whether the two receptors coordinate signaling to promote endothelial cytoprotection is not known. Our previous work showed that APC/PAR1 signaling via the β‐arrestin‐2 and Dishevelled‐2 (Dvl2) scaffolds promote Rac1 signaling and endothelial barrier protection. However, whether β‐arrestin‐2 and Dvl2 are required for integration of S1PR1 into APC/PAR1‐mediated cytoprotection is not known. Here, we report that APC/PAR1 protection against TNF‐α‐induced apoptosis requires S1PR1, whereas S1PR1 is not necessary for APC/PAR1‐mediated barrier protection. We further show that APC/PAR1 transactivation of S1PR1 is mediated by β‐arrestin‐2 dependent activation of sphingosine kinase 1 (Sphk1) and is pivotal for anti‐apoptotic Akt signaling. S1PR1, is activated by sphingosine‐1‐phosphate generated by Sphk1 and we hypothesized that β‐arrestin‐2 and Dvl2 were required for Sphk1 activity. We found that APC promotes Sphk1 phosphorylation and translocation to the membrane. In addition, loss of β‐arrestin‐2 but not Dvl2 abolished APC‐mediated Sphk1 activation. Pharmacological antagonism of S1PR1, inhibition of Sphk1, and β‐arrestin‐2 depletion abolished APC/PAR1‐mediated Akt signaling. Moreover, inhibition of Sphk1, and β‐arrestin‐2 depletion also resulted in loss of APC/PAR1‐mediated cell survival suggesting that Sphk1 and β‐arrestin‐2 are involved in APC/PAR1‐mediated cytoprotection. Our mechanistic studies were corroborated in vivo using a Sphk1 gene knockout zebrafish model. Loss of Sphk1 ablated APC/PAR1‐mediated cell survival and we are currently in the process of validating a β‐arrestin‐2 knockout zebrafish line to test its role in vivo in cytoprotection. In summary, our work demonstrates that APC/PAR1 signaling via the β‐arrestin‐2 multi‐functional scaffold segregates into two distinct pathways: 1) β‐arrestin‐2‐Sphk1 activation induces transactivation of S1PR1 to promote Akt‐mediated cell survival and 2) β‐arrestin‐2/Dvl2‐Rac1 activation promotes endothelial barrier protection.Support or Funding InformationSupported by NIH/ NIGMS R35 GM127121 and NIGMS IRACDA K12 GM068524.

G‐protein coupled receptor kinases’ (GRKs) role in APC/PAR1‐induced endothelial cells

Endothelial dysfunction is a hallmark of inflammation and prevalent in vascular diseases. Recent efforts are focused on developing new strategies to enhance endothelial resistance to inflammation. One promising candidate is activated Protein C (APC), which has been shown in preclinical studies to increase endothelial barrier protection and reduce inflammatory responses. APC enhances endothelial cytoprotection by activation of PAR1 through cleavage at an N‐terminal arginine (R)‐46 site, that is distinct from the canonical thrombin cleavage site at R‐41. This results in the generation of a distinct tethered ligand that promotes PAR1 bias signaling. G‐protein coupled receptor kinase (GRK) family plays a role in phosphorylating PAR1 and recruiting b‐arrestins, however, the contribution of GRKs to b‐arrestin driven cytoprotection is unknown. Our study focuses on determining the role and mechanisms by which GRKs regulate APC/PAR1‐induced endothelial cytoprotection to delineate their function in this pathway. Here we will present studies examining the function and mechanism by which GRKs regulate endothelial cytoprotective responses using GRK‐specific siRNA to assess function as well as confocal immunofluorescence imaging and biochemical sucrose fraction to examine GRK localization and compartmentalization in caveolae. The work is expected to provide important new insight into the role of GRKs in regulating APC/PAR1 cytoprotection in human cultured endothelial cells.Support or Funding InformationThis work is funded by NIH R01 GM090689, NIH R35GM127121, and IRACDA K12 GM068524.

Nur77 limits endothelial barrier disruption to LPS in the mouse lung.

Nur77 is an orphan nuclear receptor implicated in the regulation of a wide range of biological processes, including the maintenance of systemic blood vessel homeostasis. Although Nur77 is known to be expressed in the lung, its role in regulating pulmonary vascular functions remains entirely unknown. In this study, we found that Nur77 is expressed at high levels in the lung, and its expression is markedly upregulated in response to LPS administration. While the pulmonary vasculature of mice that lacked Nur77 appeared to function normally under homeostatic conditions, we observed a dramatic decrease in its barrier functions after exposure to LPS, as demonstrated by an increase in serum proteins in the bronchoalveolar lavage fluid and a reduction in the expression of endothelial junctional proteins, such as vascular endothelial cadherin (VE-cadherin) and β-catenin. Similarly, we found that siRNA knockdown of Nur77 in lung microvascular endothelial cells also reduced VE-cadherin and β-catenin expression and increased the quantity of fluorescein isothiocyanate-labeled dextran transporting across LPS-injured endothelial monolayers. Consistent with Nur77 playing a vascular protective role, we found that adenoviral-mediated overexpression of Nur77 both enhanced expression of VE-cadherin and β-catenin and augmented endothelial barrier protection to LPS in cultured cells. Mechanistically, Nur77 appeared to mediate its protective effects, at least in part, by binding to β-catenin and preventing its degradation. Our findings demonstrate a key role for Nur77 in the maintenance of lung endothelial barrier protection to LPS and suggest that therapeutic strategies aimed at augmenting Nur77 levels might be effective in treating a wide variety of inflammatory vascular diseases of the lung.

Prospects of HSP70 as a genetic marker for thermo-tolerance and immuno-modulation in animals under climate change scenario.

Heat stress induced by long periods of high ambient temperature decreases animal productivity, leading to heavy economic losses. This devastating situation for livestock production is even becoming worse under the present climate change scenario. Strategies focused to breed animals with better thermo-tolerance and climatic resilience are keenly sought these days to mitigate impacts of heat stress especially in high input livestock production systems. The 70-kDa heat shock proteins (HSP70) are a protein family known for its potential role in thermo-tolerance and widely considered as cellular thermometers. HSP70 function as molecular chaperons and have major roles in cellular thermotolerance, apoptosis, immune-modulation and heat stress. Expression of HSP70 is controlled by various factors such as, intracellular pH, cyclic adenosine monophosphate (cyclic AMP), protein kinase C and intracellular free calcium, etc. Over expression of HSP70 has been observed under oxidative stress leading to scavenging of mitochondrial reactive oxygen species and protection of pulmonary endothelial barrier against bacterial toxins. Polymorphisms in flanking and promoter regions in HSP70 gene have shown association with heat tolerance, weaning weight, milk production, fertility and disease susceptibility in livestock. This review provides insight into pivotal roles of HSP70 which make it an ideal candidate genetic marker for selection of animals with better climate resilience, immune response and superior performance.