Response Of Endothelial Cells Research Articles

Aging Is Associated With Organ-Specific Alterations in the Level and Expression Pattern of von Willebrand Factor.

VWF (von Willebrand factor) is an endothelial-specific procoagulant protein with a major role in thrombosis. Aging is associated with increased circulating levels of VWF, which presents a risk factor for thrombus formation. Circulating plasma, cellular protein, and mRNA levels of VWF were determined and compared in young and aged mice. Major organs were subjected to immunofluorescence analyses to determine the vascular pattern of VWF expression and the presence of platelet aggregates. An in vitro model of aging, using extended culture time of endothelial cells, was used to explore the mechanism of age-associated increased VWF levels. Increased circulating plasma levels of VWF with elevated levels of larger multimers, indicative of VWF functional activity, were observed in aged mice. VWF mRNA and cellular protein levels were significantly increased in the brains, lungs, and livers but not in the kidneys and hearts of aged mice. Higher proportion of small vessels in brains, lungs, and livers of aged mice exhibited VWF expression compared with young, and this was concomitant with increased platelet aggregate formation. Prolonged culture of endothelial cells resulted in increased cell senescence that correlated with increased VWF expression; VWF expression was specifically detected in senescent cultured endothelial cells and abolished in response to p53 knockdown. A significantly higher proportion of VWF expressing endothelial cells in vivo exhibited senescence markers SA-β-Gal (senescence-associated β-galactosidase) and p53 in aged mouse brains compared with that of the young. Aging elicits a heterogenic response in endothelial cells with regard to VWF expression, leading to organ-specific increase in VWF levels and alterations in vascular tree pattern of expression. This is concomitant with increased platelet aggregate formation. The age-associated increase in VWF expression may be modulated through the process of cell senescence, and p53 transcription factor contributes to its regulation.

Simultaneous Targeting of IL-1-Signaling and IL-6-Trans-Signaling Preserves Human Pulmonary Endothelial Barrier Function During a Cytokine Storm.

Systemic inflammatory diseases, such as sepsis and severe COVID-19, provoke acute respiratory distress syndrome in which the pathological hyperpermeability of the microvasculature, induced by uncontrolled inflammatory stimulation, causes pulmonary edema. Identifying the inflammatory mediators that induce human lung microvascular endothelial cell barrier dysfunction is essential to find the best anti-inflammatory treatments for critically ill acute respiratory distress syndrome patients. We have compared the responses of primary human lung microvascular endothelial cells to the main inflammatory mediators involved in cytokine storms induced by sepsis and SARS-CoV2 pulmonary infection and to sera from healthy donors and severely ill patients with sepsis. Endothelial barrier function was measured by electric cell-substrate impedance sensing, quantitative confocal microscopy, and Western blot. The human lung microvascular endothelial cell barrier was completely disrupted by IL (interleukin)-6 conjugated with soluble IL-6R (IL-6 receptor) and by IL-1β, moderately affected by TNF (tumor necrosis factor)-α and IFN (interferon)-γ and unaffected by other cytokines and chemokines, such as IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and MCP-3. The inhibition of IL-1 and IL-6R simultaneously, but not separately, significantly reduced endothelial hyperpermeability on exposing human lung microvascular endothelial cells to a cytokine storm consisting of 8 inflammatory mediators or to sera from patients with sepsis. Simultaneous inhibition of IL-1 and JAK (Janus kinase)-STAT (signal transducer and activator of transcription protein), a signaling node downstream IL-6 and IFN-γ, also prevented septic serum-induced endothelial barrier disruption. These findings strongly suggest a major role for both IL-6 trans-signaling and IL-1β signaling in the pathological increase in permeability of the human lung microvasculature and reveal combinatorial strategies that enable the gradual control of pulmonary endothelial barrier function in response to a cytokine storm.

Syphilis and the host: multi-omic analysis of host cellular responses to Treponema pallidum provides novel insight into syphilis pathogenesis.

Syphilis is a chronic, multi-stage infection caused by the extracellular bacterium Treponema pallidum ssp. pallidum. Treponema pallidum widely disseminates through the vasculature, crosses endothelial, blood-brain and placental barriers, and establishes systemic infection. Although the capacity of T. pallidum to traverse the endothelium is well-described, the response of endothelial cells to T. pallidum exposure, and the contribution of this response to treponemal traversal, is poorly understood. To address this knowledge gap, we used quantitative proteomics and cytokine profiling to characterize endothelial responses to T. pallidum. Proteomic analyses detected altered host pathways controlling extracellular matrix organization, necroptosis and cell death, and innate immune signaling. Cytokine analyses of endothelial cells exposed to T. pallidum revealed increased secretion of interleukin (IL)-6, IL-8, and vascular endothelial growth factor (VEGF), and decreased secretion of monocyte chemoattractant protein-1 (MCP-1). This study provides insight into the molecular basis of syphilis disease symptoms and the enhanced susceptibility of individuals infected with syphilis to HIV co-infection. These investigations also enhance understanding of the host response to T. pallidum exposure and the pathogenic strategies used by T. pallidum to disseminate and persist within the host. Furthermore, our findings highlight the critical need for inclusion of appropriate controls when conducting T. pallidum-host cell interactions using in vitro- and in vivo-grown T. pallidum.

Butyrate Protects against SARS-CoV-2-Induced Tissue Damage in Golden Hamsters.

Butyrate, produced by gut microbe during dietary fiber fermentation, has anti-inflammatory and antioxidant effects on chronic inflammation diseases, yet it remains to be explored whether butyrate has protective effects against viral infections. Here, we demonstrated that butyrate alleviated tissue injury in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected golden hamsters supplemented with butyrate before and during the infection. Butyrate-treated hamsters showed augmentation of type I interferon (IFN) response and activation of endothelial cells without exaggerated inflammation. In addition, butyrate regulated redox homeostasis by enhancing the activity of superoxide dismutase (SOD) to inhibit excessive apoptotic cell death. Therefore, butyrate exhibited effective prevention against SARS-CoV-2 by upregulating antiviral immune responses and promoting cell survival.

A 96-wells fluidic system for high-throughput screenings under laminar high wall shear stress conditions

The ability of endothelial cells to respond to blood flow is fundamental for the correct formation and maintenance of a functional and hierarchically organized vascular network. Defective flow responses, in particular related to high flow conditions, have been associated with atherosclerosis, stroke, arteriovenous malformations, and neurodegenerative diseases. Yet, the molecular mechanisms involved in high flow response are still poorly understood. Here, we described the development and validation of a 96-wells fluidic system, with interchangeable cell culture and fluidics, to perform high-throughput screenings under laminar high-flow conditions. We demonstrated that endothelial cells in our newly developed 96-wells fluidic system respond to fluid flow-induced shear stress by aligning along the flow direction and increasing the levels of KLF2 and KLF4. We further demonstrate that our 96-wells fluidic system allows for efficient gene knock-down compatible with automated liquid handling for high-throughput screening platforms. Overall, we propose that this modular 96-well fluidic system is an excellent platform to perform genome-wide and/or drug screenings to identify the molecular mechanisms involved in the responses of endothelial cells to high wall shear stress.

TNFα increases the degradation of pyruvate dehydrogenase kinase 4 by the Lon protease to support proinflammatory genes

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.

Quantum Sensing for Detection of Zinc‐Triggered Free Radicals in Endothelial Cells

AbstractOxidative stress originating from the overproduction of free radicals poses a major threat to cell fate, therefore it is of great importance to address the formation of free radicals in cells subjected to various pathological stimuli. Here we investigate the free radical response of endothelial cells to biodegradable zinc. In addition to the standard free radical assays, relaxometry was used for determining the production of free radicals in cells exposed to non‐physiological concentrations of zinc ions. The cellular morphology, intracellular zinc accumulation, as well as the levels of reactive oxygen/nitrogen species, are determined using standard fluorescent methods. For endothelial cells subjected to 50% zinc extracts, deviations from the normal cell shape and cell agglomeration tendency are observed. The culture medium containing the highest amount of zinc ions caused nuclei fragmentation, blebbing, and cell shrinkage, indicating cell death. A potential explanation for the observed phenomena is an overproduction of free radicals. In the case of 1% and 10% zinc extracts, the formation of free radicals is clearly confirmed by relaxometry, while the results obtained by using fluorescent techniques are unambiguous. It is revealed that high concentrations of zinc ions released from biodegradable samples induce a deleterious effect on endothelial cells.

Exploring the Effects of Human Bone Marrow-Derived Mononuclear Cells on Angiogenesis In Vitro.

Cell therapies involving the administration of bone marrow-derived mononuclear cells (BM-MNCs) for patients with chronic limb-threatening ischemia (CLTI) have shown promise; however, their overall effectiveness lacks evidence, and the exact mechanism of action remains unclear. In this study, we examined the angiogenic effects of well-controlled human bone marrow cell isolates on endothelial cells. The responses of endothelial cell proliferation, migration, tube formation, and aortic ring sprouting were analyzed in vitro, considering both the direct and paracrine effects of BM cell isolates. Furthermore, we conducted these investigations under both normoxic and hypoxic conditions to simulate the ischemic environment. Interestingly, no significant effect on the angiogenic response of human umbilical vein endothelial cells (HUVECs) following treatment with BM-MNCs was observed. This study fails to provide significant evidence for angiogenic effects from human bone marrow cell isolates on human endothelial cells. These in vitro experiments suggest that the potential benefits of BM-MNC therapy for CLTI patients may not involve endothelial cell angiogenesis.

Serum salusin-α and -β levels in patients with parkinson's disease.

The etiology of Parkinson's disease (PD) is not well known and there is increasing evidence that oxidative stress also plays an important role in its pathogenesis. Salusins alpha (salusin-α) and beta (salusin-β) affect the central nervous system, vasculature, and kidneys to increase the inflammatory response in endothelial cells, stimulate oxidative stress, and increase monocyte-endothelial adhesion. Neuroinflammation and oxidative stress play roles in the etiopathogenesis of PD. To investigate whether salusin-α and -β are related to PD and whether they are correlated with the development of atherosclerosis, body mass index, disease duration, and the Parkinson's Hoehn and Yahr stage. The low-density lipoprotein cholesterol (LDL-C), total cholesterol, and salusin-β levels were significantly lower and age was significantly higher in Parkinson patients compared to healthy controls (ρ < 0.005). We found a negative linear correlation between salusin-β and the Hoehn and Yahr stage (ρ < 0.001, r = -0.515) in the patients. There was a relationship between salusin-β and PD and a correlation between the salusin-β levels and Parkinson's stage. A possible underlying disease mechanism is an increase in oxidative stress and decrease in neuroprotective effects due to low salusin-β levels. Therefore, the effects of salusin-β in treating Parkinson disease should be evaluated. Further studies are needed to understand the effects of salusin-β treatment on preventing or slowing the course of PD.

Microporous Hydroxyapatite-Based Ceramics Alter the Physiology of Endothelial Cells through Physical and Chemical Cues.

Incorporation of silicate ions in calcium phosphate ceramics (CPC) and modification of their multiscale architecture are two strategies for improving the vascularization of scaffolds for bone regenerative medicine. The response of endothelial cells, actors for vascularization, to the chemical and physical cues of biomaterial surfaces is little documented, although essential. We aimed to characterize in vitro the response of an endothelial cell line, C166, cultivated on the surface CPCs varying either in terms of their chemistry (pure versus silicon-doped HA) or their microstructure (dense versus microporous). Adhesion, metabolic activity, and proliferation were significantly altered on microporous ceramics, but the secretion of the pro-angiogenic VEGF-A increased from 262 to 386 pg/mL on porous compared to dense silicon-doped HA ceramics after 168 h. A tubulogenesis assay was set up directly on the ceramics. Two configurations were designed for discriminating the influence of the chemistry from that of the surface physical properties. The formation of tubule-like structures was qualitatively more frequent on dense ceramics. Microporous ceramics induced calcium depletion in the culture medium (from 2 down to 0.5 mmol/L), which is deleterious for C166. Importantly, this effect might be associated with the in vitro static cell culture. No influence of silicon doping of HA on C166 behavior was detected.

Statement of Retraction: Two mixed-ligand coordination polymers: treatment activity on coronary artery heart disease by reducing the inflammatory response in the vascular endothelial cells

Statement of Retraction: Two mixed-ligand coordination polymers: treatment activity on coronary artery heart disease by reducing the inflammatory response in the vascular endothelial cells

Abstract P431: SARS-CoV-2-induced Inflammation In Human Endothelial Cells Involves ACE2

COVID-19 is associated with hypertension, where endotheliitis and endothelial dysfunction are risk factors. Considering the relationship between the viral spike protein and ACE2, it is unclear whether this interaction is merely a mechanism of infection or whether contributes to vascular injury induced by SARS-CoV-2. Here we tested whether ACE2 acts as a receptor for SARS-CoV-2 pro-inflammatory responses, independently of ACE2 enzymatic function, in human endothelial cells (ECs). ECs and ACE2-expressing HEK cells were exposed to recombinant SARS-CoV-2 spike protein [(rS1p) 0.66 μg/mL] for 24h. Gene expression was assessed by RT-PCR and cytokine release was assessed by ELISA. Protein expression was assessed by immunoblotting. ACE2 was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Label-free quantitation of full proteome of ECs after rS1p exposure or ACE2 co-immunoprecipitation was performed. Co-localization studies were performed by confocal microscopy. Proteomics identified 1150 proteins associated with NFκB signalling and inflammation. 21 proteins were found to be differentially expressed vs vehicle-treated cells (fold change &gt;2, p&lt;0.05), including upregulation of ICAM-1. rS1p increased levels of IL6 (1221.2±18.3 vs. C:22.77±3.2 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU); PAI1 (5.6±0.7 vs. C: 2.9±0.2) and NFκB activation (0.13±0.004 vs. C:0.06±0.01 AU); p&lt;0.05. MLN4760 and ACE2 siRNA blocked rS1P effects. rS1p did not alter ACE2 enzymatic activity. HEK-ACE2 cells had increased IL-8 (78.7±8 vs. C:56.2±4 pg/mL), while rS1p induced IL-8 production only in HEK expressing ACE2 (ACE2:112.8±5.3 vs. C:60.3±3.3 pg/mL); p&lt;0.05. ACE2 CO-IP proteomics identified 216 proteins (filtered with ≥1 unique peptide, 1% FDR) along with ACE-2 (11 peptides). Potential candidate interacting proteins include caveolin 1 (Cav 1). Co-localization studies confirmed interactions between ACE2/Cav-1 in ECs, which was lost after rS1p stimulation. SARS-CoV2 spike protein induces pro-inflammatory responses in ECs via receptor-like function of ACE2. These molecular processes may contribute to vascular damage after COVID-19.

Abstract 124: Renin Cell Identity And Blood Pressure Homeostasis: Exploring The Role Of Endothelial Cell Cross-talk

Our previous studies using an Aortic Coarctation (AoCo) mouse model identified that renin-expressing juxta-glomerular (JG) cells respond to changes in perfusion pressure and Lamin A/C as a component of the renin cell baroreceptor. However, as the mechanisms for transmitting the extracellular pressure to the JG cells are unclear, we hypothesized that the endothelial cells (ECs) act as transducers of signals to the JG cells. Therefore in the current study, we aim 1) to identify the concurrent transcriptomic and epigenomic changes in JG cells and ECs in response to the changes in the perfusion pressure by single-nuclei multiomics approach 2) to define the contribution of EC-specific Lamin A/C (Lmna) in the EC-JG-cells cross-talk. We performed the AoCo surgeries on 3 months (M) old VE-Cad-Cre;Foxd1-Cre;mTmG mice where GFP is expressed simultaneously in Foxd1 and EC-lineage cells. We compared the single-cell gene expression and chromatin accessibility between the left (LK) and the right kidneys (RK) at 72h post-surgeries. Our study revealed: 1) JG cells increased significantly in the LK along with renin gene expression 2) ECs significantly increased by 10.73% in LK with a higher expression for genes promoting EC proliferation, morphogenesis, and immune response 3) JGs of the LK showed enrichment for the motifs Mbd1, Smad1, Smad5 indicating epigenetic changes promoting transcriptional regulation and TgfB1 signaling. Enrichment of Mybl1, Myb, and E2f2 motifs in the ECs of the LK suggests positive regulation of EC proliferation in response to low blood pressure (BP). Furthermore, deletion of the Lmna gene in the EC-lineage affected the BP responses in 4M old Lmna fl/fl ;VE-Cad.Cre mice. The difference between the carotid and femoral mean BP (MBP) measurements was more prominent in the wild type (WT) compared to the mutants at 72h post-AoCo [WT (n=7), Carotid MBP: 69.16±2.24 Femoral MBP: 63.28±2.33, P&lt;0.00001 Mutants (n=4); Carotid MBP: 75.98±3.06 Femoral MBP: 69.75±3.10, P&lt;0.05)]. Our current study shows that ECs undergo transcriptional and epigenetic alterations in response to changes in perfusion pressure. Deletion of Lmna in ECs reduces the differences in the BP between LK and RK, suggesting that it is important for sensing the changes in extracellular pressure.

Cathelicidin (LL-37) causes expression of inflammatory factors in coronary artery endothelial cells of Kawasaki disease by activating TLR4-NF-κB-NLRP3 signaling.

Kawasaki disease (KD) is a type of vasculitis with an unidentified etiology. Cathelicidin (LL-37) may be involved in the development of the KD process; therefore, further research to investigate the molecular mechanism of LL-37 involvement in KD is warranted. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, NLRP3, and LL-37 in the sera of healthy subjects, children with KD, and children with pneumonia. Subsequently, human recombinant LL-37 or/and toll-like receptors 4 (TLR4)-specific inhibitor TAK-242 stimulated human coronary artery endothelial cells (HCAECs), CCK-8 was used to detect cell proliferation, flow cytometry to detect apoptosis, transmission electron microscopy to observe cytoskeletal changes, Transwell to measure cell migration ability, ELISA to detect inflammatory factor levels, Western blot analysis to analyze protein levels of toll-like receptors 4 (TLR4) and NF-κB p-65, and quantitative real-time polymerase chain reaction (qRT-PCR) to determine LL-37, NLRP3 mRNA levels. In this study, we found that the level of LL-37 was highly expressed in the serum of children with KD, and after LL-37 stimulation, apoptosis was significantly increased in HCAECs, and the expression levels of TLR4, NLRP3 and inflammatory factors in cells were significantly enhanced. Intervention with the TLR4-specific inhibitor TAK-242 significantly alleviated the LL-37 effects on cellular inflammation, TLR4, NLRP3 promotion effect. Our data suggest that LL-37 induces an inflammatory response in KD coronary endothelial cells via TLR4-NF-κB-NLRP3, providing a potential target for the treatment of KD.

Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

Uncovering the heterogeneity and cell fate decisions of endothelial cells after myocardial infarction by single‐cell sequencing

AbstractBackgroundThe plasticity of endothelial cells (ECs) is crucial for tissue response to injury. Myocardial infarction can profoundly affect EC function, leading to a shift toward mesenchymal differentiation.MethodsWe utilized human single‐nucleus RNA sequencing data to investigate the dynamic changes and cellular interactions between normal and post‐infarction ECs.ResultsWe identified two distinct subpopulations of ECs: A transient subpopulation characterized by short‐term mesenchymal gene expression and a long‐term subpopulation characterized by myocardial gene expression. Trajectory analysis revealed the differentiation pathways and potential roles over time and space. Furthermore, we uncovered the proliferation, differentiation, hypoxic, and inflammatory responses of ECs to injury.ConclusionsOur study provides a comprehensive and detailed characterization of endothelial cell states, highlighting the role of activated endothelial cell subpopulations in promoting inflammation and tissue repair after infarction.

Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression.

Introduction: Dysfunction of the endothelium impairs its' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.

Cytocompatibility Evaluation of PEG-Methylsulfone Hydrogels.

Methylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide-thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide-thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide-thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future.

The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4.

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the anti-inflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomain-containing protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent anti-inflammatory effects of depolymerizing microtubule-targeting agents.

Role of human dural fibroblasts in the angiogenic responses of human endothelial cells: An in vitro dural model and the application of lab-on-a-chip for EDAS.

Encephaloduroarteriosynangiosis (EDAS), an indirect anastomosis procedure, is widely accepted as a primary treatment for moyamoya disease (MMD) to improve collateral blood flow. During surgical intervention, dural fibroblasts (DuF) are thought to produce various proteins that create an angiogenic microenvironment. However, the biophysiological evidence supporting the angiogenic properties of this surgical technique has not been thoroughly elucidated. The purpose of these studies was to determine whether DuF releases pro-angiogenic factors and chemokines and promotes angiogenic properties in human endothelial cells (ECs) under IL-1β-mediated wound conditions, which are expected to occur during the process of neo-vascularization within the dura mater. Furthermore, a microfluidic chemotaxis platform was implemented to investigate the angiogenic activity of ECs in response to a reconstituted dura model. Transcriptome sequencing revealed that IL-1β stimulation on DuF induced a significant upregulation of various pro-angiogenic genes, including IL-6, IL-8, CCL-2, CCL-5, SMOC-1, and SCG-2 (p < 0.05). Moreover, compared to ECs cultured in naïve media or naïve DuF media, those exposed to IL-1β-DuF conditioned media expressed higher mRNA and protein levels of these pro-angiogenic factors (p < 0.001). ECs co-cultured with IL-1β-DuF also exhibited considerable migration on the microfluidic chemotaxis platform. Furthermore, the chemotactic effects on the ECs were reduced upon neutralization of IL-8 or inhibition of NF-κB signaling. Our findings demonstrate that IL-1β-DuFs release factors that activate and enhance the angiogenic properties of ECs. These results suggest a potential interaction between DuF and ECs following EDAS for MMD, and these components could be targeted for the development of therapeutic biomarkers.