Stimulation Of Endothelial Cells Research Articles

Changes in ROS/RNS Levels in Endothelial Cells in Experimental Bacteremia.

A high-precision system was developed for the quantification of biological analytes in single cells (reactive oxygen species (ROS) and reactive nitrogen species (RNS)) based on the electrochemical amperometric method. The efficacy of this system was evaluated using an experimental bacteremia model. Endothelial cells exhibited increased ROS/RNS production when stimulated by Staphylococcus aureus. However, they remained inactive when exposed to either unprimed or primed neutrophils that had been pre-incubated with bacteria. It is noteworthy that the sequential stimulation of endothelial cells with bacteria followed by neutrophils resulted in a significant increase in the ROS/RNS level, which demonstrated a correlation with the number of neutrophils in contact with the endothelial cells. These results highlight the potential of our system to quantitatively assess ROS/RNS dynamics in complex biological systems. They also offer insights into the interplay between various cellular components in experimental bacteremia.

OGG1 prevents atherosclerosis-induced vascular endothelial cell injury through mediating DNA damage repair.

This study was designed to investigate the role of 8-oxoguanine DNA glycosylase 1 (OGG1) in preventing atherosclerosis-induced vascular EC injury, thereby providing a theoretical basis for the exploration of drug targets and treatment methods for atherosclerosis. Human umbilical vein cell line (EA.hy926) was treated with ox-LDL to construct an in vitro atherosclerotic cell model. pcDNA3.1-OGG1 was transfected into EA.hy926 cells to overexpress OGG1. qRT-PCR, CCK-8 assay, flow cytometry, oil red O staining, ELISA, comet assay and western blot were used to evaluate the OGG1 expression, viability, apoptosis level, lipid droplet content, 8-OHdG level and DNA damage of cells in each group. Compared with the Control group, ox-LDL stimulation of endothelial cells significantly decreased cell viability, promoted apoptosis and DNA damage, and increased intracellular levels of 8-OHdG and γH2AX, while decreasing protein levels of PPARγ, FASN, FABP4, RAD51 and POLB. However, overexpression of OGG1 can significantly inhibit ox-LDL damage to endothelial cells, promote lipid metabolism, decrease lipid droplet content, and improve DNA repair function. Over-expression of OGG1 improves DNA repair. Briefly, OGG1 over-expression enhances the DNA damage repair of ECs by regulating the expression levels of γH2AX, RAD51 and POLB, thereby enhancing cell viability and reducing apoptosis.

Identification of an osteopontin-derived peptide that binds neuropilin-1 and activates vascular repair responses and angiogenesis

The osteopontin-derived peptide FOL-005 stimulates hair growth. Using ligand-receptor glyco-capture technology we identified neuropilin-1 (NRP-1), a known co-receptor for vascular endothelial growth factor (VEGF) receptors, as the most probable receptor for FOL-005 and the more stable analogue FOL-026. X-ray diffraction and microscale thermophoresis analysis revealed that FOL-026 shares binding site with VEGF in the NRP-1 b1-subdomain. Stimulation of human umbilical vein endothelial cells with FOL-026 resulted in phosphorylation of VEGFR-2, ERK1/2 and AKT, increased cell growth and migration, stimulation of endothelial tube formation and inhibition of apoptosis in vitro. FOL-026 also promoted angiogenesis in vivo as assessed by subcutaneous Matrigel plug and hind limb ischemia models. NRP-1 knock-down or treatment of NRP-1 antagonist EG00229 blocked the stimulatory effects of FOL-026 on endothelial cells. Exposure of human coronary artery smooth muscle cells to FOL-026 stimulated cell growth, migration, inhibited apoptosis, and induced VEGF gene expression and VEGFR-2/AKT phosphorylation by an NRP-1-dependent mechanism. RNA sequencing showed that FOL-026 activated pathways involved in tissue repair. These findings identify NRP-1 as the receptor for FOL-026 and show that its biological effects mimic that of growth factors binding to the VEGF receptor family. They also suggest that FOL-026 may have therapeutical potential in conditions that require vascular repair and/or enhanced angiogenesis.

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses.

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

Suppression of retinal neovascularization by intravitreal injection of cryptotanshinone

Neovascular eye diseases, including proliferative diabetic retinopathy and retinopathy of prematurity, is a major cause of blindness. Laser ablation and intravitreal anti-VEGF injection have shown their limitations in treatment of retinal neovascularization. Identification of a new therapeutic strategies is in urgent need. Our study aims to assess the effects of Cryptotanshinone (CPT), a natural compound derived from Salvia miltiorrhiza Bunge, in retina neovascularization and explore its potential mechanism. Our study demonstrated that CPT did not cause retina tissue toxicity at the tested concentrations. Intravitreal injections of CPT reduced pathological angiogenesis and promoted physical angiogenesis in oxygen-induced retinopathy (OIR) model. CPT improve visual function in OIR mice and reduced cell apoptosis. Moreover, we also revealed that CPT diminishes the expression of inflammatory cytokines in the OIR retina. In vitro, the administration of CPT effectively inhibited endothelial cells proliferation, migration, sprouting, and tube formation induced by the stimulation of human retinal vascular endothelial cells (HRVECs) with VEGF165. Mechanistically, CPT blocking the phosphorylation of VEGFR2 and downstream targeting pathway. After all, the findings demonstrated that CPT exhibits potent anti-angiogenic and anti-inflammatory effects in OIR mice, and it has therapeutic potential for the treatment of neovascular retinal diseases.

CAMP enriched EVs mediates hypoxic pulmonary hypertension

Our lab reported the novel finding that stimulation of pulmonary endothelial cells with a beta-agonist and a phosphodiesterase inhibitor induced enrichment of cyclic adenosine monophosphate (cAMP) in EVs. cAMP protects the pulmonary vasculature by regulation of permeability, vasoreactivity and proliferation. Extracellular vesicles provide a protected environment for circulating cAMP. Thus, we hypothesized that administration of cAMP, in the protected environment of an EV, could deliver cAMP to improve outcomes in pulmonary hypertension. Briefly, male Sprague Dawley rats, maintained in normoxic or hypoxic conditions for 3 weeks, received 50 ug of cAMP-enriched EVs (cAMP-EVs) per day over 3 days via tail vein injection. cAMP-EV injections improved the Fulton index (non-injected hypoxia rat 0.37 ± 0.039 versus injected hypoxia rat 0.32 ± 0.019, p value 0.032 n = 5) and echocardiography revealed PH rats that received cAMP-EVs had a significant increase in PAAT/PAET ratio compared to those that did not (non-injected hypoxia rat 0.27 ± 0.056 versus injected hypoxia rat 0.32 ± 0.04, p value 0.04, n = 6). Using IVIS analysis of isolated tissues to detect PKH-labeled EVs we found that cAMP-enriched EVs accumulated in the lung but were not detectable in the heart. To confirm this was not strictly a vasoreactivity effect, we also examined PCNA, a marker of proliferation, in histological sections and found it was decreased in the presence of cAMP-EVs. Pulmonary arterial thickness was also significantly improved in the presence of cAMP-EVs. To understand the underlying mechanism for cAMP-EV based signaling, we studied the phosphorylation status of endothelial nitric oxide synthase (eNOS) in isolated paraffn fixed lung slices. We found increased phosphorylation of eNOS in PH rats which received cAMP EVs compared to non-injected PH or control rats. Combined these data suggest that cAMP-EVs may decrease right ventricular hypertrophy, improve pulmonary arterial function, and repair hypoxic vascular injury. Future investigations will determine whether the observed response is cAMP dependent and further evaluate the specificity of pulmonary vascular uptake of cAMP-EVs. 1R01HL133066-01A1 (Bauer). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Chronic Inflammatory Activation of Murine and Human Arterial Endothelial Cells at Normal Lipoprotein and Cholesterol Levels In Vivo and In Vitro.

The activation of endothelial cells is crucial for immune defense mechanisms but also plays a role in the development of atherosclerosis. We have previously shown that inflammatory stimulation of endothelial cells on top of elevated lipoprotein/cholesterol levels accelerates atherogenesis. The aim of the current study was to investigate how chronic endothelial inflammation changes the aortic transcriptome of mice at normal lipoprotein levels and to compare this to the inflammatory response of isolated endothelial cells in vitro. We applied a mouse model expressing constitutive active IκB kinase 2 (caIKK2)-the key activator of the inflammatory NF-κB pathway-specifically in arterial endothelial cells and analyzed transcriptomic changes in whole aortas, followed by pathway and network analyses. We found an upregulation of cell death and mitochondrial beta-oxidation pathways with a predicted increase in endothelial apoptosis and necrosis and a simultaneous reduction in protein synthesis genes. The highest upregulated gene was ACE2, the SARS-CoV-2 receptor, which is also an important regulator of blood pressure. Analysis of isolated human arterial and venous endothelial cells supported these findings and also revealed a reduction in DNA replication, as well as repair mechanisms, in line with the notion that chronic inflammation contributes to endothelial dysfunction.

Pathogenic variants in HGF give rise to childhood-to-late onset primary lymphoedema by loss of function.

Developmental and functional defects in the lymphatic system are responsible for primary lymphoedema (PL). PL is a chronic debilitating disease caused by increased accumulation of interstitial fluid, predisposing to inflammation, infections and fibrosis. There is no cure, only symptomatic treatment is available. Thirty-two genes or loci have been linked to PL, and another 22 are suggested, including Hepatocyte Growth Factor (HGF). We searched for HGF variants in 770 index patients from the Brussels PL cohort. We identified ten variants predicted to cause HGF loss-of-function (six nonsense, two frameshifts, and two splice-site changes; 1.3% of our cohort), and 14 missense variants predicted to be pathogenic in 17 families (2.21%). We studied co-segregation within families, mRNA stability for non-sense variants, and in vitro functional effects of the missense variants. Analyses of the mRNA of patient cells revealed degradation of the nonsense mutant allele. Reduced protein secretion was detected for nine of the 14 missense variants expressed in COS-7 cells. Stimulation of lymphatic endothelial cells with these 14 HGF variant proteins resulted in decreased activation of the downstream targets AKT and ERK1/2 for three of them. Clinically, HGF-associated PL was diverse, but predominantly bilateral in the lower limbs with onset varying from early childhood to adulthood. Finally, aggregation study in a second independent cohort underscored that rare likely pathogenic variants in HGF explain about 2% of PL. Therefore, HGF signalling seems crucial for lymphatic development and/or maintenance in human beings and HGF should be included in diagnostic genetic screens for PL.

Sided Stimulation of Endothelial Cells Modulates Neutrophil Trafficking in an In Vitro Sepsis Model.

While the role of dysregulated polymorphonuclear leukocyte (PMN) transmigration in septic mediated tissue damage is well documented, strategies to mitigate aberrant transmigration across endothelium have yet to yield viable therapeutics. Recently, microphysiological systems (MPS) have emerged as novel in vitro mimetics that facilitate the development of human models of disease. With this advancement, aspects of endothelial physiology that are difficult to assess with other models can be directly probed. In this study, the role of endothelial cell (EC) apicobasal polarity on leukocyte trafficking response is evaluated with the µSiM-MVM (microphysiological system enabled by a silicon membrane - microvascular mimetic). Here, ECs are stimulated either apically or basally with a cytokine cocktail to model a septic-like challenge before introducing healthy donor PMNs into the device. Basally oriented stimulation generated a stronger PMN transmigratory response versus apical stimulation. Importantly, healthy PMNs are unable to migrate towards a bacterial peptide chemoattractant when ECs are apically stimulated, which mimics the attenuated PMN chemotaxis seen in sepsis. Escalating the apical inflammatory stimulus by a factor of five is necessary to elicit high PMN transmigration levels across endothelium. These results demonstrate that EC apicobasal polarity modulates PMN transmigratory behavior and provides insight into the mechanisms underlying sepsis.

PEGylated graphene oxide-mediated stimulation of vascular endothelial cells and responsive release of PD-1/PD-L1 inhibitor for efficient chemo-immunotherapy against cancer

Immune checkpoint blockade (ICB) has emerged as a promising immunotherapeutic modality against cancer in the clinic. However, only 10–30% of patients respond to ICB, primarily due to poor immunogenicity and insufficient T cell infiltration in solid tumors. Herein, we presented an approach for high-performance cancer treatment using the programmed cell death protein-1 and programmed cell death ligand-1 (PD-1/PD-L1) inhibitor (BMS-202)-loaded PEGylated graphene oxide (GPi). On the one hand, GPi dissociated tight junctions of vascular endothelial cells (VECs) in tumor, thus promoting the extravasation and intratumoral accumulation of liposomal doxorubicin (LipDox), which then effectively induced immunogenic cell death of tumor cells. On the other hand, GPi also stimulated VECs to upregulate the expression of cell-cell interaction molecules, such as intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1, which facilitated the infiltration of T cells in tumor. Beyond acting as a stimulator of VECs, GPi could exert responsive release of BMS-202 under the acidic tumor microenvironment and blockade PD-1/PD-L1 axis in tumors. Finally, the alternating administration of GPi and LipDox effectively inhibited tumor growth in a 4T1 tumor model, providing a novel treatment mode for chemo-immunotherapy.

Apelin stimulation of the vascular skeletal muscle stem cell niche enhances endogenous repair in dystrophic mice.

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here, we showed that defects in the endothelial cell (EC) compartment of the vascular stem cell niche in mouse models of Duchenne MD, laminin α2-related MD, and collagen VI-related myopathy were associated with inefficient mobilization of MuSCs after tissue damage. Using chemoinformatic analysis, we identified the 13-amino acid form of the peptide hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. Systemic administration of AP-13 using osmotic pumps generated a pro-proliferative EC-rich niche that supported MuSC function through angiocrine factors and markedly improved tissue regeneration and muscle strength in all three dystrophic mouse models. Moreover, EC-specific knockout of the apelin receptor led to regenerative defects that phenocopied key pathological features of MD, including vascular defects, fibrosis, muscle fiber necrosis, impaired MuSC function, and reduced force generation. Together, these studies provide in vivo proof of concept that enhancing endogenous skeletal muscle repair by targeting the vascular niche is a viable therapeutic avenue for MD and characterized AP-13 as a candidate for further study for the systemic treatment of MuSC dysfunction.

Large-scale phosphoproteomics reveals activation of the MAPK/GADD45β/P38 axis and cell cycle inhibition in response to BMP9 and BMP10 stimulation in endothelial cells

BackgroundBMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.MethodsTo get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry.ResultsLarge-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45β. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1.ConclusionsOverall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.

ECCO Grant Dissecting the role of S1PR1 modulator in the mucosal regeneration of Ulcerative Colitis

Abstract Background and Aims Ozanimod, a potent sphingosine 1-phosphate (S1P) 1/5 receptor agonist, first approved for the treatment of adults with moderately to severely active UC in the USA and in the EU, has showed promising results in terms of both tolerability and efficacy reducing inflammatory processes and inducing disease remission. The selectivity of Ozanimod for S1PR1 is 27 times that for S1PR5. Therefore, is plausible that the beneficial effects of Ozanimod are mediated by S1PR1, which is constitutively expressed not only by immune cells, but also by epithelial, endothelial, and stromal cells. However, its function on non-immune cells has been neglected. Based on preliminary data demonstrating a faster recovery of mucosal damage in mice with a conditional deletion of S1PR1 in the epithelia and endothelia compartments, our hypothesis is that the inactivation of S1PR1 not only inhibits immune cell trafficking, but also promotes tissue repair. Based on this background, the main objectives will be to exploit the Ozanimod effects during the process of mucosa healing, and to investigate the molecular pathways activated by Ozanimod on epithelial and endothelial cells. Methods Pre-clinical validation of Ozanimod, in vivo model of UC in association with conditional knockout mice for S1PR1 on epithelial and endothelial cells, stimulation of primary endothelial cells from UC patients and human induced pluripotent stem cell-derived intestinal organoids (iHOs) will be used to address our aims. Transcriptomics, functional assay, immune characterization, WB validation will be performed. Anticipated Impact In short and medium term, we will improve the knowledge on the role of S1PR1 in the processes of mucosal healing and identify the molecular pathways involved in the response to the mucosal damage mediated by the ozanimod in non-immune cells. The results of this study may improve the treatment options for UC patients.

Knockdown of ChREBP ameliorates retinal microvascular endothelial cell injury and angiogenic responses in diabetic retinopathy

PurposeTo examine whether and how carbohydrate response element-binding protein (ChREBP) plays a role in diabetic retinopathy. MethodsWestern blotting was used to detect ChREBP expression and location following high glucose stimulation of Human Retinal Microvascular Endothelial Cells (HRMECs). Flow cytometry, TUNEL staining, and western blotting were used to evaluate apoptosis following ChREBP siRNA silencing. Cell scratch, transwell migration, and tube formation assays were used to determine cell migration and angiogenesis. Diabetic models for wild-type (WT) and ChREBP knockout (ChKO) mice were developed. Retinas of WT and ChKO animals were cultivated in vitro with vascular endothelial growth factor + high glucose to assess neovascular development. ResultsChREBP gene knockdown inhibited thioredoxin-interacting protein and NOD-like receptor family pyrin domain containing protein 3 expression in HRMECs, which was caused by high glucose stimulation, reduced apoptosis, hindered migration, and tube formation, and repressed AKT/mTOR signaling pathway activation. Compared with WT mice, ChKO mice showed suppressed high glucose-induced alterations in retinal structure, alleviated retinal vascular leakage, and reduced retinal neovascularization. ConclusionsChREBP deficiency decreased high glucose-induced apoptosis, migration, and tube formation in HRMECs as well as structural and angiogenic responses in the mouse retina; thus, it is a potential therapeutic target for diabetic retinopathy.

TLR3-Dependent Induction of TF Is Required to Maintain Lung Hemostasis during Poly IC-Induced ARDS-like Lung Dysfunction in Mice

Background: Respiratory virus infection causes damage to the epithelial-endothelial barrier of the lung. Mice challenged with influenza A virus (IAV) exhibit bleeding in the airspace. IAV increases tissue factor (TF) expression in epithelial cells (EpC) of the lung and deletion of lung EpC TF is associated with increased bleeding. aThe ssRNA viruses IAV undergo a dsRNA genomic phase during viral replication. Poly IC is a chemical mimetic of dsRNA. dsRNA acts like PAMPs and can activate the innate immune receptor, toll-like receptor 3 (TLR3). TLR3 stimulation of endothelial cells increased TF expression. However, the role of TLR3 in virus-induced TF expression in the lung TF has not been studied. Aim: To investigate the role of TLR3 in induction of TF during poly IC induced acute respiratory distress syndrome (ARDS)-like lung dysfunction in mice Methods: The murine lung EpC line, E10, was treated with the dsRNA mimetic (5 µg/mL), poly IC, for 0-6h. TF expression was measured by western blotting and a one-stage plasma clotting assay. Wild-type (WT) mice, mice with lung EpC TF deletion (TF fl/fl;SPCCre +, TFΔEpC) and their controls (TFfl/fl) as wells as TLR3 +/+ and TLR3 -/- mice were used in the study. To induce ARDS, poly IC (100 µg in 50 µL PBS) was administered intranasally daily for 3 days to adult mice (both sexes). Lung function was recorded noninvasively using the Buxco whole-body plethysmography system and plasma and bronchoalveolar lavage fluid (BALF) were collected before and 24 hours after the last administration of poly IC. Cellular composition of the BALF and blood was analyzed by an automated cell counter. Lung TF activity was quantified by one-stage plasma clotting and TF activity of extracellular vesicles (EV-TF) was analyzed by two-stage FXa generation assay. Results: Poly IC stimulation induced NFκB-dependent TF expression in E10 cells in dose and time-dependent manner. In addition, poly IC administration induced ARDS-like lung dysfunction, local lung inflammation and coagulation activation in WT mice compared to PBS-treated WT mice. Lung TF activity was increased 2-fold after poly IC administration compared to PBS-treated mice. Mice with lung EpC TF deficiency (TFΔEpC) did not exhibit an increase in lung TF or EV-TF in BALF after poly IC administration compared to controls. Importantly, TFΔEpC mice exhibited reduced activation of coagulation, impaired pulmonary hemostasis and increased hemorrhage into the airspace after poly IC compared to their littermate controls. In addition, TFΔEpC mice showed increased signs of poly IC-induced ARDS and had reduced lung function, increased lung injury and local inflammation compared to their control mice after poly IC. TLR3 -/- mice had significantly less EV-TF activity and less thrombin-antithrombin complexes (TAT) in BALF compared to TLR3 +/+ mice after poly IC. Conclusion: We show that the dsRNA mimetic poly IC induces lung EpC TF expression in vitro and vivo in part via TLR3-dependent NFκB pathway activation. TLR3 stimulation by intranasal poly IC results in sterile inflammation, and ARDS-like phenotype in mice similarly to that observed after IAV infection. Importantly, a deficiency in lung EpC TF leads to impaired lung hemostasis that is associated with an increased poly IC-induced ARDS phenotype as seen previously in our studies using IAV infection of mice. dsRNA induces induction of lung TF via TLR3. We proposed that induction of TF expression in the lung during respiratory viral infections protects against alveolar hemorrhage but can also contribute to thrombosis.

Lamellipodia dynamics and microrheology in endothelial cell paracellular gap closure

Vascular endothelial cells (ECs) form a semipermeable barrier separating vascular contents from the interstitium, thereby regulating the movement of water and molecular solutes across small intercellular gaps, which are continuously forming and closing. Under inflammatory conditions, however, larger EC gaps form resulting in increased vascular leakiness to circulating fluid, proteins, and cells, which results in organ edema and dysfunction responsible for key pathophysiologic findings in numerous inflammatory disorders. In this study, we extend our earlier work examining the biophysical properties of EC gap formation and now address the role of lamellipodia, thin sheet-like membrane projections from the leading edge, in modulating EC spatial-specific contractile properties and gap closure. Micropillars, fabricated by soft lithography, were utilized to form reproducible paracellular gaps in human lung ECs. Using time-lapse imaging via optical microscopy, rates of EC gap closure and motility were measured with and without EC stimulation with the barrier-enhancing sphingolipid, sphingosine-1-phosphate. Peripheral ruffle formation was ubiquitous during gap closure. Kymographs were generated to quantitatively compare the lamellipodia dynamics of sphingosine-1-phosphate-stimulated and -unstimulated ECs. Utilizing atomic force microscopy, we characterized the viscoelastic behavior of EC lamellipodia. Our results indicate decreased stiffness and increased liquid-like behavior of expanding lamellipodia compared with regions away from the cellular edge (lamella and cell body) during EC gap closure, results in sync with the rapid kinetics of protrusion/retraction motion. We hypothesize this dissipative EC behavior during gap closure is linked to actomyosin cytoskeletal rearrangement and decreased cross-linking during lamellipodia expansion. In summary, these studies of the kinetic and mechanical properties of EC lamellipodia and ruffles at gap boundaries yield insights into the mechanisms of vascular barrier restoration and potentially a model system for examining the druggability of lamellipodial protein targets to enhance vascular barrier integrity.

Action of 3-Hydroxymethylglutaryl CoA Reductase Inhibitors on ABCA-1 Protein (ATP-Binding Cassette Transporter-1) in Endothelial Cells Stimulated with Uremic Serum

Action of 3-Hydroxymethylglutaryl CoA Reductase Inhibitors on ABCA-1 Protein (ATP-Binding Cassette Transporter-1) in Endothelial Cells Stimulated with Uremic Serum

CAPE Triggers a Fast Cytoplasmic Antioxidant Response by TRPV1 Stimulation of Endothelial Cells and Impairs Cellular Migration by Lowering Extracellular H2O2

CAPE Triggers a Fast Cytoplasmic Antioxidant Response by TRPV1 Stimulation of Endothelial Cells and Impairs Cellular Migration by Lowering Extracellular H2O2

S-Nitrosylation in endothelial cells contributes to tumor cell adhesion and extravasation during breast cancer metastasis

BackgroundNitric oxide is produced by different nitric oxide synthases isoforms. NO activates two signaling pathways, one dependent on soluble guanylate cyclase and protein kinase G, and other where NO post-translationally modifies proteins through S-nitrosylation, which is the modification induced by NO in free-thiol cysteines in proteins to form S-nitrosothiols. High levels of NO have been detected in blood of breast cancer patients and increased NOS activity has been detected in invasive breast tumors compared to benign or normal breast tissue, suggesting a positive correlation between NO biosynthesis, degree of malignancy and metastasis. During metastasis, the endothelium plays a key role allowing the adhesion of tumor cells, which is the first step in the extravasation process leading to metastasis. This step shares similarities with leukocyte adhesion to the endothelium, and it is plausible that it may also share some regulatory elements. The vascular cell adhesion molecule-1 (VCAM-1) expressed on the endothelial cell surface promotes interactions between the endothelium and tumor cells, as well as leukocytes. Data show that breast tumor cells adhere to areas in the vasculature where NO production is increased, however, the mechanisms involved are unknown.ResultsWe report that the stimulation of endothelial cells with interleukin-8, and conditioned medium from breast tumor cells activates the S-nitrosylation pathway in the endothelium to induce leukocyte adhesion and tumor cell extravasation by a mechanism that involves an increased VCAM-1 cell surface expression in endothelial cells. We identified VCAM-1 as an S-nitrosylation target during this process. The inhibition of NO signaling and S-nitrosylation blocked the transmigration of tumor cells through endothelial monolayers. Using an in vivo model, the number of lung metastases was inhibited in the presence of the S-nitrosylation inhibitor N-acetylcysteine (NAC), which was correlated with lower levels of S-nitrosylated VCAM-1 in the metastases.ConclusionsS-Nitrosylation in the endothelium activates pathways that enhance VCAM-1 surface localization to promote binding of leukocytes and extravasation of tumor cells leading to metastasis. NAC is positioned as an important tool that might be tested as a co-therapy against breast cancer metastasis.

Vagus nerve stimulation-induced stromal cell-derived factor-l alpha participates in angiogenesis and repair of infarcted hearts.

We aim to explore the role and mechanism of vagus nerve stimulation (VNS) in coronary endothelial cells and angiogenesis in infarcted hearts. Seven days after rat myocardial infarction (MI) was prepared by ligation of the left anterior descending coronary artery, the left cervical vagus nerve was treated with electrical stimulation 1h after intraperitoneal administration of the α7-nicotinic acetylcholine inhibitor mecamylamine or the mAChR inhibitor atropine or 3days after local injection of Ad-shSDF-1α into the infarcted heart. Cardiac tissue acetylcholine (ACh) and serum ACh, tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) levels were detected by ELISA to determine whether VNS was successful. An inflammatory injury model in human coronary artery endothelial cells (HCAECs) was established by lipopolysaccharide and identified by evaluating TNF-α, IL-1β and IL-6 levels and tube formation. Immunohistochemistry staining was performed to evaluate CD31-positive vessel density and stromal cell-derived factor-l alpha (SDF-1α) expression in the MI heart in vivo and the expression and distribution of SDF-1α, C-X-C motif chemokine receptor 4 and CXCR7 in HCAECs in vitro. Western blotting was used to detect the levels of SDF-1α, V-akt murine thymoma viral oncogene homolog (AKT), phosphorylated AKT (pAKT), specificity protein 1 (Sp1) and phosphorylation of Sp1 in HCAECs. Left ventricular performance, including left ventricular systolic pressure, left ventricular end-diastolic pressure and rate of the rise and fall of ventricular pressure, should be evaluated 28days after VNS treatment. VNS was successfully established for MI therapy with decreases in serum TNF-α, IL-1β and IL-6 levels and increases in cardiac tissue and serum ACh levels, leading to increased SDF-1α expression in coronary endothelial cells of MI hearts, triggering angiogenesis of MI hearts with increased CD31-positive vessel density, which was abolished by the m/nAChR inhibitors mecamylamine and atropine or knockdown of SDF-1α by shRNA. ACh promoted SDF-1α expression and its distribution along with the branch of the formed tube in HCAECs, resulting in an increase in the number of tubes formed in HCAECs. ACh increased the levels of pAKT and phosphorylation of Sp1 in HCAECs, resulting in inducing SDF-1α expression, and the specific effects could be abolished by mecamylamine, atropine, the PI3K/AKT blocker wortmannin or the Sp1 blocker mithramycin. Functionally, VNS improved left ventricular performance, which could be abolished by Ad-shSDF-1α. VNS promoted angiogenesis to repair the infarcted heart by inducing SDF-1α expression and redistribution along new branches during angiogenesis, which was associated with the m/nAChR-AKT-Sp1 signalling pathway.