Activation Of Microvascular Endothelial Cells Research Articles

Thymic stromal lymphopoietin and digital microvascular damage in systemic sclerosis patients: A pilot study

BackgroundSystemic sclerosis (SSc) is a complex autoimmune connective-tissue disease, characterised by vasculopathy and fibrosis of the skin and internal organs. Activation of microvascular endothelial cells (ECs) causes the intimal hyperplasia that characterises the vascular remodelling in SSc. The most frequent complication of SSc is the development of digital ulcers (DUs). Thymic stromal lymphopoietin (TSLP) may trigger fibrosis and sustain vascular damage. Aim of this study was to evaluate the correlation between serum level of TSLP and DUs. Methods75 consecutive SSc patients were enrolled and serum TSLP levels were measured. The presence of history of DUs (HDU) was evaluated. Recurrent new DUs were defined as the presence of at least 3 episodes of DUs in a 12-months follow up period. The risk of developing new DUs was calculated by applying the capillaroscopic skin ulcer risk index (CSURI). ResultsThe median value of TSLP was higher in patients with HDU than patients without HDU [181.67 pg/ml (IQR 144.67; 265.66) vs 154.67 pg/ml (IQR 110.67; 171.33), p < 0.01]. The median value of TSLP was higher in patients with an increased CSURI index than patients without an increased CSURI [188 pg/ml (IQR 171.33; 246.33) vs 159.33 pg/ml (IQR 128.67; 218), p < 0.01]. Kaplan-Meier curves demonstrated that free survival from new DUs was significantly (p < 0.01) lower in SSc patients with increased TSLP serum levels. ConclusionTSLP might have a key role in digital microvascular damage of SSc patients.

Sars-Cov-2 Spike Protein 1 Activates Microvascular Endothelial Cells: Understanding the Implications

The spike protein of SARS-CoV-2, particularly its subunit Spike Protein 1 (S1), has been identified as a crucial component in the pathogenesis of COVID-19. Recent studies have revealed that SARS-CoV-2 Spike Protein 1 has the ability to activate microvascular endothelial cells, which play a vital role in maintaining vascular integrity and regulating inflammatory responses. This activation of microvascular endothelial cells by the spike protein has significant implications for COVID-19 pathogenesis. It can lead to endothelial dysfunction, increased vascular permeability, recruitment of immune cells, and the release of inflammatory molecules, contributing to the systemic inflammatory response observed in severe cases of COVID-19. Furthermore, it can exacerbate tissue damage and contribute to the cytokine storm. Understanding the mechanisms and consequences of SARS-CoV-2 Spike Protein 1-mediated activation of microvascular endothelial cells is crucial for developing targeted therapeutic interventions to mitigate the inflammatory response and improve patient outcomes. This knowledge may also guide the exploration of existing medications to modulate endothelial dysfunction and attenuate the pathogenic effects of spike protein activation. Continued research in this field is warranted to uncover precise mechanisms and develop effective strategies to combat this aspect of the disease.

Fatty acid metabolism promotes TRPV4 activity in lung microvascular endothelial cells in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was β-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.

Empagliflozin prevents angiotensin II-induced hypertension related micro and macrovascular endothelial cell activation and diastolic dysfunction in rats despite persistent hypertension: Role of endothelial SGLT1 and 2

SGLT2 inhibitors (SGLT2i) showed pronounced beneficial effects in patients with heart failure but the underlying mechanisms remain unclear. We evaluated the effect of empagliflozin, selective SGLT2i, on hypertension-induced cardiac and vascular dysfunction.Male Wistar rats received diet with or without empagliflozin (30 mg/kg/day). After 1 week, a hypertensive dose of Ang II (0.4 mg/kg/day) was administered using osmotic mini-pumps for 4 weeks. Systolic blood pressure was determined by sphygmomanometry, the cardiac function by echocardiography and ex vivo (coronary microvascular endothelial cell activation, LV remodeling and fibrosis responses), and the systemic micro and macrovascular endothelial cell activation ex vivo. Empagliflozin treatment did not affect the Ang II-induced hypertensive response. Ang II treatment increased LV mass and induced LV diastolic dysfunction, fibrosis, collagen I and ANP expression, and infiltration of macrophages. In the vasculature, it caused eNOS upregulation in the aorta and down-regulation in mesenteric microvessels associated with increased oxidative stress, ACE, AT1R, VCAM-1, MCP-1, MMP-2, and MMP-9 and collagen I expression, increased endothelial SGLT1 staining in the aorta, mesenteric and coronary microvessels, increased SGLT1 and 2 protein levels in the aorta. All Ang II-induced cardiac and vascular responses were reduced by the empagliflozin treatment.Thus, the SGLT2i effectively attenuated the deleterious impact of Ang II-induced hypertension on target organs including cardiac diastolic dysfunction and remodeling, and endothelial cell activation and pro-atherosclerotic, pro-fibrotic and pro-remodeling responses in macro and microvessels despite persistent hypertension.

Metabolic regulation of mitochondrial reactive oxygen species (mtROS) production and transient receptor potential vanilloid‐4 (TRPV4) activation in microvascular endothelial cells in PAH

Rationale In pulmonary arterial hypertension (PAH), highly proliferative microvascular endothelial cells (MVECs) are thought to contribute to vaso-occlusive lesion formation and increased pulmonary artery pressures. Using MVECs isolated from the Sugen/Hypoxia (SuHx) model of PAH, we recently showed that increased mtROS production activates the TRPV4 calcium channel and contributes to increased MVEC proliferation in vitro. However, the mechanistic basis for sustained mtROS production in MVECs isolated from SuHx rats (SuHx-MVECs) was unclear. Along with increased mtROS production, we observed evidence of significant mitochondrial dysfunction, including increased fragmentation and glycolytic shift. Since mtROS production can be increased by changes in the type of fuel (e.g. fatty acids)used to generate TCA metabolites, we hypothesized that use of non-glucose sources (specifically, increased fatty acid oxidation - FAO) could be the source of mitochondrial dysfunction and mtROS production in SuHx-MVECs. Methods Intracellular Ca2+ concentration ([Ca2+]i) was measured using Fura-2AM loaded MVEC in a flow chamber perfused with modified Krebs solution. Mitochondrial morphology was measured computationally using validated algorithms applied to confocal images of mitochondria in N- and SuHx-MVEC stained with MitoTracker. Lipid droplets were measured using BODIPY staining. Targeted metabolomics were performed on MVEC cell lysates using a standardized LC/MS approach as described previously (Suresh et al., AJP-Lung 2019). Human PAH metabolomics and genomics data were obtained from NIH Metabolomics Workbench or GEO Omnibus, respectively, and analyzed using R. Results Despite evidence of glycolytic shift (decreased oxygen consumption rate, increased extracellular acidification rate, increased extracellular lactate levels), levels of TCA metabolites, measured using targeted metabolomics, were not significantly lower in SuHx-MVECs compared to normoxic (N-MVEC) controls, suggesting presence of anaplerosis. Serum free fatty acids (FFA) were increased in SuHx rats at 5 weeks, while intracellular lipid droplet number was increased in SuHx-MVECs. Analysis of human PAH metabolomics and genomics data revealed increased serum FFA levels and increased expression of enzymes involved in breaking down long chain fatty acids for use in the TCA cycle (i.e. FAO). In SuHx-MVECs, untargeted metabolomics and metabolite-specific assays revealed increased levels of beta-hydroxybutyrate (BOHB), a ketone body produced by FAO. In N-MVECs, BOHB supplementation was sufficient to increase TRPV4 sensitivity to pharmacologic agonists. In contrast, inhibition of FAO (with Etoximir) in SuHx-MVECs attenuated basal Ca2+ and mitochondrial fragmentation to a similar extent as TRPV4 inhibition or mtROS quenching. Conclusion We conclude that intermediary signaling molecules such as BOHB and mtROS, produced as a consequence of increased FAO, act in concert to sensitize and activate the TRPV4 channel in lung MVECs. These data suggest an exciting role for metabolism as a regulator of TRPV4 channel activity in PAH.

Beclin-1 overexpression regulates NLRP3 activation by promoting TNFAIP3 in microvascular injury following myocardial reperfusion

Innate immune response contributes significantly to ischemia reperfusion (I/R) injury. Targeting innate immunity seems to be a promising method for protecting the microvascular injury in ST-elevation myocardial infarction (STEMI) patients following myocardial I/R injury (MI/R). NLRP3 inflammasome is a central part of the innate immune system involved in the pathophysiological process of MI/R. However, the mechanisms regulating NLRP3 activation are yet to be clarified. Recently, autophagy has been related to the regulation of NLRP3 activation. Thus, how Beclin-1/Becn1 overexpression influences NLRP3 activation in microvascular endothelial cells (CMECs) after MI/R is yet to be investigated. The present study showed that Becn1 overexpression exhibits a significant increase in NLRP3 and IL-1β in CMEC responses to MI/R. Interestingly, Becn1 overexpression promoted TNFAIP3 expression, which restricted NLRP3 activation in vitro and in vivo. The current study also showed that inflammatory cells (CD68) and B (CDB220) lymphocytes were decreased in transgenic mice with overexpression of Beclin-1 (BECN1-Tg) in the spleen and heart. These findings highlighted Becn1 as a prospective target for treating NLRP3 mediated microvascular injury following MI/R.

Exosomes Derived from CXCR4-Overexpressing BMSC Promoted Activation of Microvascular Endothelial Cells in Cerebral Ischemia/Reperfusion Injury

Background Ischemic stroke is a severe acute cerebrovascular disease which can be improved with neuroprotective therapies at an early stage. However, due to the lack of effective neuroprotective drugs, most stroke patients have varying degrees of long-term disability. In the present study, we investigated the role of exosomes derived from CXCR4-overexpressing BMSCs in restoring vascular function and neural repair after ischemic cerebral infarction. Methods BMSCs were transfected with lentivirus encoded by CXCR4 (BMSCCXCR4). Exosomes derived from BMSCCXCR4 (ExoCXCR4) were isolated and characterized by transmission electron microscopy and dynamic light scattering. Western blot and qPCR were used to analyze the expression of CXCR4 in BMSCs and exosomes. The acute middle cerebral artery occlusion (MCAO) model was prepared, ExoCXCR4 were injected into the rats, and behavioral changes were analyzed. The role of ExoCXCR4 in promoting the proliferation and tube formation for angiogenesis and protecting brain endothelial cells was determined in vitro. Results Compared with the control groups, the ExoCXCR4 group showed a significantly lower mNSS score at 7 d, 14 d, and 21 d after ischemia/reperfusion (P < 0.05). The bEnd.3 cells in the ExoCXCR4 group have stronger proliferation ability than other groups (P < 0.05), while the CXCR4 inhibitor can reduce this effect. Exosomes control (ExoCon) can significantly promote the migration of bEnd.3 cells (P < 0.05), while there was no significant difference between the ExoCXCR4 and ExoCon groups (P > 0.05). ExoCXCR4 can further promote the proliferation and tube formation for the angiogenesis of the endothelium compared with ExoCon group (P < 0.05). In addition, cobalt chloride (COCl2) can increase the expression of β-catenin and Wnt-3, while ExoCon can reduce the expression of these proteins (P < 0.05). ExoCXCR4 can further attenuate the activation of Wnt-3a/β-catenin pathway (P < 0.05). Conclusions In ischemia/reperfusion injury, ExoCXCR4 promoted the proliferation and tube formation of microvascular endothelial cells and play an antiapoptotic role via the Wnt-3a/β-catenin pathway.

Abstract 14723: Autoreactive T Lymphocytes Activate Cardiac Endothelium Independently of Tnf-α and Cause Endothelial Dysfunction Through Exosomes in Experimental Autoimmune Myocarditis

Background: Inflammatory heart diseases represent an important clinical problem, nonetheless data regarding activation of cardiac microvascular endothelial cells (MVECs) are limited. Aim: To examine influence of TNF-α and exosomes produced by heart-reactive CD4+ T lymphocytes on activation of cardiac MVECs. Methods: Experimental autoimmune myocarditis (EAM) was induced in wild-type (WT) and TNF-α-deficient (TNF-KO) mice. CD4+ T lymphocytes were isolated from EAM mice at day 21 and activated in vitro to produce conditioned medium and exosomes. Activation of MVECs was assessed by specific assays and leukocyte-to-endothelial adhesion was analysed under shear flow condition using the BioFlux microfluidic system. Results: TNF-KO mice showed lower prevalence of myocarditis when compared to WT mice (50% vs. 90%). Stimulation of MVECs with secretome of antigen-activated autoreactive T cells resulted in upregulation of adhesion molecules (ICAM-1, VCAM-1 and P-selectin), increased ROS and decreased NO production. Addition of anti-TNF-α neutralizing antibodies effectively blocked adhesion of leukocytes to MVECs activated with the conditioned medium. Endothelial activation and dysfunction induced by the conditioned medium were independent of TNF-α produced by T cells. Stimulation of MVECs with T cell-derived exosomes increased ROS and decreased levels of NO and eNOS activation, but exosomes neither increased expression of adhesion molecules in MVECs nor induced their ability to bind leukocytes. Conclusions: TNF-α promotes MVEC activation and EAM development. In this model, autoreactive T cells activate MVECs, and TNF-a produced by MVECs rather than T cells is essential in this process. On the other hand, endothelial dysfunction caused by T cells seems to be mediated mainly by exosomes.

Aloe‐emodin inhibits hypertension‐induced inter‐endothelial junction dysfunction through endothelial ROS‐TXNIP‐Nlrp3 inflammasome axle

Recent studies have revealed that Aloe‐emodin, a natural compound from the root and rhizome of Rheum palmatum L., exhibits significant pharmacological activities. However, the pharmacological relevance of the compound, particularly for cardiovascular disease, remains largely unknown. Here, we hypothesized that Aloe‐emodin could improve endothelial injury by inhibiting the activation of Nlrp3 inflammasome, and ultimately prevent cardiovascular disease. In vitro, our findings demonstrated that Aloe‐emodin inhibits AngII‐induced Nlrp3 inflammasome formation and activation in Microvascular Endothelial Cells (MECs), as shown by fluorescence confocal microscopy and Western blot. At the same time, the data shown that Aloe‐emodin exerts anti‐oxidation and ROS scavenged effects. Interestingly, thioredoxin‐interacting protein (TXNIP) protein and high mobility group box 1 (HMGB1) markedly decreased with Aloe‐emodin treatment. We also demonstrated that the decreased expression of the tight junction proteins ZO‐1/ZO‐2, which are related to endothelial permeability after stimulation by AngII in endothelial cells. Aloe‐emodin could recover the gap junction protein and decrease monolayer cell permeability in endothelial cells. Similarly, we used confocal microscopy to study the formation and activation of NOD‐like receptor family pyrin domain containing‐3 (Nlrp3) inflammasomes and expression of tight junction proteins in coronary arteries of AngII/DOCA‐treated mice. We found that Aloe‐emodin inhibited the formation and activation of Nlrp3 inflammasome, and also could restore the expression of the endothelial connective protein ZO1/2 and the release of HMGB1. Together with these changes, we revealed a new protection mechanism of Aloe‐emodin that inhibits Nlrp3 inflammasome activation and decreases subsequent caspase‐1 activation, triggering the release of HMGB1 by reducing ROS generation. Our findings indicate that Aloe‐emodin exhibits immense potential and specific therapeutic value in hypertension‐related cardiovascular disease and the development of innovative drugs.Support or Funding InformationThe National Key Research and Development Program of China (No. 2017YFC1700400); the project supported by National Natural Science Foundation of China (No. 81603587); Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2018B030306027); Science and Technology Development Plan of Guangdong Province (No. 2017A020211016)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Craniovertebral Junction in Rheumatoid Arthritis: State of the Art.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder, characterized by polyarticular inflammation causing progressive joint damage and disability. The mechanisms underlying its pathogenesis involve activation of innate and adaptive immunity, microvascular endothelial cell activation, and inflammatory infiltration of lymphocytes and monocytes into the synovium. Spinal involvement in RA is not typical; when it occurs, the main radiological features are (1)atlantoaxial subluxation (AAS), which is the most typical form of cervical spine involvement; (2)cranial settling-also known as basilar impression, atlantoaxial impaction or superior migration of the odontoid-which is the most severe form of associated spinal instability; and (3)subaxial subluxation. A combination of these alterations may occur. Synovitis is characterized by infiltration of innate and adaptive immune cells; joint destruction is a consequence of activation of synovial fibroblasts, which acquire aggressive, inflammatory, invasive features, associated with increased chondrocyte catabolism and synovial osteoclastogenesis.Neck pain is the most frequent symptom of spinal involvement in RA; it occurs in 40-80% of patients and is mostly localized at the craniocervical junction. Other symptoms-caused by compression of neural structures such as the greater occipital nerve (at C2), the nucleus of the spinal trigeminal tract and the greater auricular nerve-are occipital neuralgia, facial pain and ear pain, respectively. Irritation of the lesser occipital nerve (at C1) can cause pain in the suboccipital region. Sometimes patients may complain of a sensation of their head falling down with flexion, weakness, reduced endurance, loss of ability, gait alterations, paraesthesias or other symptoms due to cord and medullary compression, and upper or lower motor neuron signs, or both. Surgical management of RA remains a challenging field.

Effect of tryptase on mouse brain microvascular endothelial cells via protease-activated receptor 2

BackgroundMast cells (MCs), the ‘first responders’ in brain injury, are able to disrupt the blood–brain barrier (BBB), but the underlying mechanism is not well understood. Tryptase is the most abundant MC secretory product. Protease-activated receptor 2 (PAR-2) has been identified as a specific receptor for tryptase, which is abundantly expressed in brain microvascular endothelial cells. The BBB comprises brain microvascular endothelial cells that display specialised molecular properties essential for BBB function and integrity. Therefore, the purpose of the present study was to investigate the effects of tryptase on mouse brain microvascular endothelial cell line bEnd3 and its potential mechanisms of action.MethodsInduction of mouse brain microvascular endothelial cell activation by tryptase was examined. Then, mouse brain microvascular endothelial cells were pretreated with a PAR-2 antagonist and stimulated with tryptase. Cellular activation, proinflammatory cytokine production, expression of PAR-2, Toll-like receptors (TLRs) and mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-kappa B) phosphorylation were assessed.ResultsTryptase upregulated the production of VCAM-1, MMPs (MMP9 and MMP2), TLR4 and TNF-α and downregulated the expression of the tight junction proteins occludin and claudin-5 in mouse brain microvascular endothelial cell. Among the MAPK and NF-kappa B pathway, ERK and NF-kappa B were activated by tryptase. All of these effects could be eliminated by the PAR-2 inhibitor.ConclusionBased on our findings, we conclude that tryptase can trigger brain microvascular endothelial cell activation and proinflammatory mediator release. These findings may further clarify the involvement and mechanism of tryptase in BBB disruption.

Quercetin Attenuates Adhesion Molecule Expression in Intestinal Microvascular Endothelial Cells by Modulating Multiple Pathways.

In inflammatory bowel disease, activation of microvascular endothelial cells and adhesion of immune cells are required for the initiation and maintenance of inflammation. We evaluated the effects and mechanisms of quercetin, a flavone identified in a wide variety of dietary sources, in LPS-induced rat intestinal microvascular endothelial cells (RIMVECs). RIMVECs were pretreated with quercetin of various concentrations (20, 40 and 80μM) followed by LPS (10μg/ml) stimulation. ELISA was used to examine protein levels of intercellular adhesion molecules-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the supernatant. Protein levels of Toll-like receptor 4 (TLR4), nuclear transcription factor kappa B (NF-κB) p65, inhibitors of NF-κB (IκB-α), extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK) p38 and signal transducer and activator of transcription (STAT) in cells were measured by Western blot. Quercetin significantly suppressed protein levels of ICAM-1 and VCAM-1 induced by LPS. Quercetin also inhibited TLR4 expression, NF-κB p65, ERK, JNK and STAT phosphorylation and decreased IκB-α degradation. Moreover, the MAPK p38 signal does not contribute to the anti-inflammatory effects on RIMVECs, although LPS significantly increases its phosphorylation. These results indicate that quercetin may have an anti-inflammatory effect by inhibiting expression of ICAM-1 and VCAM-1 in RIMVECs by suppressing TLR4, NF-κB, ERK, JNK and STAT but not the p38 signaling pathway.

Allogenic Endothelial Cells Differentially Modulate the Expansion of Anti-Inflammatory FoxP3 High Regulatory T Lymphocytes According to Their State of Activation

Endothelial cell activation and microvascular inflammation are hallmarks of antibody-mediated rejection. This activation is evidenced by the reduplication of the capillary basement membrane, endothelial swelling and changes in intragraft endothelial-associated transcripts. During antibody-mediated rejection, donor specific antibodies targeting HLA class II antigens are prevalent and deleterious. The healthy microvascular endothelium readily expresses HLA-DR, yet both HLA-DR and HLA-DQ are significantly upregulated during allograft rejection; this may be associated with endothelial activation. The changing expression of HLA class II by the endothelium may contribute to allorecognition, the generation of de novo DSA and DSA-mediated damage. In an in vitro model of pro-inflammatory activation of microvascular endothelial cells, a relatively short exposure to interferon gamma (IFNγ) was sufficient to produce HLA-DR+ HLA-DQ- activated endothelial cells (aEC), whilst a longer and more intense regimen using IFNγ and tumor necrosis factor alpha (TNFα) was required to produce HLA-DR++ HLA-DQ+ chronically activated endothelial cells (caEC). Additionally, the pro-inflammatory conditions induced concomitant changes in the expression of key costimulatory, adhesion and complement-inhibiting molecules: PD-L1, ICAM-1 and CD59. To evaluate the functional impact of endothelial activation, aEC or caEC were co-cultured with peripheral blood mononuclear cells (PBMC). Alloproliferation and the relative expansion of CD4+ T lymphocyte subpopulations were assessed by flow cytometry and intracellular cytokine staining. Previously, we found that aEC are capable of expanding Th17 and FoxP3high T regulatory cell (Treg) populations (Taflin PNAS 2011, Lion Am J Transplant 2016). The presence of intragraft Th17 correlates with shorter graft survival, whilst the proportion of Treg cells has been implicated in tolerance. aEC and caEC demonstrated a similar capacity to amplify proinflammatory subsets, Th1 and Th17. However caEC have a reduced capacity to expand anti-inflammatory FoxP3high Treg. Quantification of IL-2, IL-6 and TGF beta in the supernatants showed no difference between aEC and caEC co-cultures. Studies using PD-L1 blocking antibodies on the endothelium identified a role for high PD-L1 expression in the suppression of Treg expansion in this allogenic model. The level of endothelial activation, induced by a pro-inflammatory environment, changes endothelial immunogenicity and notably impacts the expression of molecules involved in CD4+ T cell interactions, such as HLA antigens, PD-L1 and ICAM-1. Indeed, CD4+ T cell polarisation was differentially modulated by activated and chronically activated endothelial cells. The impaired expansion of the Treg subset in caEC cocultures results in a more pro-inflammatory profile of alloreactive CD4+ T lymphocytes, and could promote rejection and compromise allograft tolerance. AC was supported by the Société Francophone de Transplantation (SFT).

Inhibition of Toll-Like Receptor-4 (TLR-4) Improves Neurobehavioral Outcomes After Acute Ischemic Stroke in Diabetic Rats: Possible Role of Vascular Endothelial TLR-4.

Diabetes increases the risk of occurrence and poor functional recovery after ischemic stroke injury. Previously, we have demonstrated greater hemorrhagic transformation (HT), edema, and more severe functional deficits after stroke in diabetic animals that also presented with cerebral vasoregression and endothelial cell death in the recovery period. Given that Toll-like receptor 4 (TLR-4) activation in microvascular endothelial cells triggers a robust inflammatory response, we hypothesized that inhibition of TLR-4 signaling prevents endothelial cell death and improves outcomes after stroke. Animals were treated with vehicle or TLR-4 inhibitor TAK242 (3mg/kg; i.p.) following middle cerebral artery occlusion (MCAO). Neurobehavioral deficits were measured at baseline and day 3 after ischemic stroke. Primary brain microvascular endothelial cells (BMVECs) from diabetic animals were subjected to oxygen glucose deprivation re-oxygenation (OGDR) and treated with 0.1mM iron(III)sulfate hydrate (iron) (to mimic the post-stroke bleeding) and TLR-4 inhibitors. Ischemic stroke increased the expression of TLR-4 in both hemispheres and in the microvasculature of diabetic animals. Cerebral infarct, edema, HT, and functional deficits were greater in diabetic compared to control animals. Inhibition of TLR-4 significantly reduced the neurovascular injury and improved functional outcomes. OGDR and iron reduced the cell viability and increased the expression of TLR-4 associated proteins (RIP3, MyD88, phospho-NF-kB, and release of IL-6) in BMVECs from diabetic animals. In conclusion, TLR-4 is highly upregulated in the microvasculature and that beneficial effects of TLR-4 inhibition are more profound in diabetes. This suggests that inhibition of vascular TLR-4 may provide therapeutic benefits for stroke recovery in diabetes.

Brain Microvascular Endothelial Cell Activation and Barrier Disruption in a Mouse Model of Psychological Resilience

Psychological resilience, as measured by the ability of an individual to overcome the development of psychological disorders following exposure to stressful or traumatic events, varies significantly from individual to individual. The mechanisms underlying this variability among individuals have not been fully elucidated. A key factor in the development and progression of psychological disorders such as depression, schizophrenia, and post‐traumatic stress disorder is disruption of the blood brain barrier, thus we hypothesized that the function of brain microvascular endothelial cells (BMECs) of resilient individuals are more resistant to disruption by endothelial cell activators (ethanol (EtOH), corticosterone (CORT), and lipopolysaccharide (LPS)), compared to non‐resilient individuals. We used Electric Cell‐substrate Impedance Signaling (ECIS) technology (Applied BioPhysics, Troy, NY) to measure transendothelial electrical resistance (TEER) of cultured BMECs of the resilient C57BL6 mouse (BMECC57BL6), and the non‐resilient BalbC mouse (BMECBalbC). BMECs were plated at a density of 4.8 × 104 cells/cm2, and allowed to establish a monolayer for 48 hours. BMECs were then treated with either EtOH (0, 1, 10, 25, 50, or 100 mM), CORT (0, 10, 100 or 1000 ng/ml), or LPS derived from E. coli (0, 1, 10, or 100 ng/ml). TEER was continuously recorded for an additional 24 hours. Barrier function (Rb), membrane capacitance (Cm), and constraint on current flow beneath the cells (α) were derived via ECIS mathematical modeling. We found that at baseline, BMECC57BL6 had significantly lower Rb (1.31±0.14 versus 2.64±0.43 Ωcm2, p = 0.009, n = 4) and α (6.13±0.33 versus 7.56±0.53 Ω0.5cm, p = 0.009, n = 4), and significantly higher Cm (2.65±1.19 versus 1.28±0.012 μF/cm2, p = 0.007, n = 4) when compared to BMECBalbC. Contrary to our hypothesis, the effect of EtOH, CORT, or LPS on these baseline parameters did not significantly differ between the two types of BMEC. These findings indicate that variability in psychological resilience among individuals may be associated with functional differences in BMECs, an important component of the blood brain barrier.Support or Funding InformationThe views expressed in this presentation are those of the authors and do not reflect the official policy or position of the Department of the Army, the Department of Defense, or the U.S. government.

Relationship of human microvascular endothelial cell activation and adenosine level under the hypoxia

Background Retinal hypoxia is one of primary causes of retinal neovascularization, and its mechanism is research hot topic.Studies showed that hypoxia stimulates the activation of many trancripation factors and vascular endothelial cells, which leads to angiogenesis.Besides to the vascular endothelial growth factor, the effect of adenosine on angiogenesis is increasingly concerned. Objective This study was to invastigate the relationship of biological behaviour of human microvascular endothelial cells (HMEC-1) under the hypoxia and adenosine, and to explore the effect of adenosine on angiogenesis under hypoxia. Methods HMEC-1 cell line was cultured in vitro, and the cells were divided into normoxia group and hypoxia group.The cells in the normoxia group were cultured under the 5%CO2 environment, and those in the hypoxia group were cultured under the 1% O2, 94%N2 and 5%CO2 environment.The proliferation ability and percentage of the cells were assayed by cell counting kit-8 (CCK8) and EDU.The migration number and invasive number of the cells were detected by transwell chamber.The expressions of CD39 and CD73 proteins in the cells were tested by Western blot and immunofluorescence technique, and the level of adenosine was measured by high performance liquid chromatography (HPLC). Results The proliferation values (absobancy) were 0.715±0.067 and 0.821±0.056 in the normoxia group and the hypoxia group in 12 hours after culture, and those in 24 hours were 0.946±0.028 and 0.998±0.028, showing significant increase in the hypoxia group compared with the normoxia group (t12h= 3.805, t24h= 3.222, all at P<0.01). The precentage of the proliferation in the hypoxia group was evidently higher than that in the normoxia group (t=-6.868, P<0.01). The number of the cell migration and invasion in 24 hours after culture was 185.3±10.594 and 74.2±10.741 respectively in the normoxia group, and that in the hypoxia group was 300.7±22.853 and 107.5±7.007, with significant differences between the two groups (t=-12.124, -6.367, both at P<0.01). The expression levels of CD39 and CD73 proteins in the hypoxia group were significantly higher than those in the normoxia group in both12 hours and 24 hours after culture(all at P<0.05), and the adenosine content in the cells is gradually increased in 2, 6, 12, 24 and 36 hours after culture, with the highest content in 36 hours.The adenosine content was significantly higher in the hypoxia group than in the normoxia group at various time points (t2h=2.469, P=0.017; t6h=5.442, P<0.001; t12h=3.841, P<0.001; t24h=4.458, P<0.001; t36h=2.757, P=0.008; t48h=3.319, P=0.002). Conclusions Compared with the normoxia group, the proliferation, migration and invasion abilities of HMEC-1 are stronger, meanwhile, the expression of CD39 and CD73 as well as the adenosine level in the cells are all increased under the hypoxic condition.It is suggested that the activation of human microvascular endothelial cells might be significantly related to the level of adenosine and its key enzymes. Key words: Neovascularization; Retina; Microvascular endothelial cells; Human; Angiogenesis; adenosine

Assesment of Microcirculatory Function with Retrobulbar Blood Flow Velocity Measurement Predicting Cardiovascular Events

Atherosclerosis first begins in the endothelium of the arterial wall, and is described as an inflammatory disease. Although atherosclerotic lesions occur in large arteries, the increased expression of adhesion molecules characteristic of endothelial cell activation, the decreased endothelium-dependent vasodilatation as well as oxidative stress are not limited to lesion-prone arteries where factors other than endothelial cell activation might progress to detect atheroma formation. Microvascular endothelial cell activation might be directly stimulated by cardiovascular risk factors with consequent release of inflammatory mediators and soluble isoforms of adhesion molecules that detect microvascular dysfunction and the atherosclerosis-associated systemic inflammatory state. The quantification of retrobulbar blood flow velocity has been used to analyze the microvascular circulation of the eye. Structural and functional changes in various microvascular beds can predict CV risk factors and diseases.

Ex-Vivo Cytoadherence Phenotypes of Plasmodium falciparum Strains from Malian Children with Hemoglobins A, S, and C

Sickle hemoglobin (Hb) S and HbC may protect against malaria by reducing the expression of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of parasitized red blood cells (RBCs), thereby weakening their cytoadherence to microvascular endothelial cells (MVECs) and impairing their activation of MVECs to produce pathological responses. Therefore, we hypothesized that parasites causing malaria in HbAS or HbAC heterozygotes have overcome this protective mechanism by expressing PfEMP1 variants which mediate relatively strong binding to MVECs. To test this hypothesis, we performed 31 cytoadherence comparisons between parasites from HbAA and HbAS (or HbAC) Malian children with malaria. Ring-stage parasites from HbAA and HbAS (or HbAC) children were cultivated to trophozoites, purified, and then inoculated in parallel into the same wildtype uninfected RBCs. After one cycle of invasion and maturation to the trophozoite stage expressing PfEMP1, parasite strains were compared for binding to MVECs. In this assay, there were no significant differences in the binding of parasites from HbAS and HbAC children to MVECs compared to those from HbAA children (HbAS, fold-change = 1.46, 95% CI 0.97–2.19, p = 0.07; HbAC, fold-change = 1.19, 95% CI 0.77–1.84, p = 0.43). These data suggest that in-vitro reductions in cytoadherence by HbS and HbC may not be selecting for expression of high-avidity PfEMP1 variants in vivo. Future studies that identify PfEMP1 domains or amino-acid motifs which are selectively expressed in parasites from HbAS children may provide further insights into the mechanism of malaria protection by the sickle-cell trait.

Quantitative &lt;em&gt;In vitro&lt;/em&gt; Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

α1-Antitrypsin Modulates Lung Endothelial Cell Inflammatory Responses to TNF-α

α₁-Antitrypsin (A1AT) is an acute-phase reactant, but also a major protective factor against the development of chronic obstructive pulmonary disease, a complex disease with sustained chronic inflammation. The lung-protective effects of A1AT have been attributed to the inhibition of proteases involved in lung matrix fragmentation, macrophage activation, and endothelial-cell apoptosis. More recently, A1AT has been shown to directly interact with or modulate the actions of cytokines such as TNF-α or IL-1 in inflammatory cells, but its effect on the lung endothelium, an active participant in the amplification and resolution of inflammation, has received little attention. An important role of A1AT in modulating lung endothelial inflammatory responses is expected, given the high concentrations of circulating A1AT during inflammation and its active uptake by endothelial cells. We investigated the role of A1AT in primary lung microvascular endothelial cell activation by relevant cytokines such as TNF-α or IL-1β. Despite an initial marked augmentation of TNF-α self-induced transcription, A1AT inhibited TNF-α receptor 1 up-regulation and significantly reduced TNF-α secretion, effects that were associated with inhibition of TNF-α-converting enzyme activity. Furthermore, A1AT inhibited calpain activity, whose activation by TNF-α contributed to decreased intracellular A1AT concentrations. These data indicate that A1AT initially facilitates acute responses of the endothelium to TNF-α, followed by selective inhibition of TNF-α-induced-self amplification, which may assist the vasculature in the resolution of chronic inflammation.