Endothelial Cell Inflammatory Responses Research Articles

Revealing Leptin and Adiponectin Involvement in Endothelial Cell Response to Disturbed Flow

Background and AimsEndothelial cells (ECs) line on the inner surface of the blood vessels and responsible from sensing and responding to mechanical forces of blood flow, to maintain vascular homeostasis. On the bifurcations and inner curves of arteria, ECs are exposed to multidirectional disturbed flow. Whereas, on the other parts of arteria, blood flow is unidirectional laminar flow. In response to disturbed flow, EC permeability, nitric oxide (NO) bioavailability, and tissue repair mechanisms are affected. As a result, increased inflammatory response occurs. Disturbed flow induced endothelial dysfunction and inflammation can cause atherosclerosis, which is a life threatening, chronic inflammatory disease of arteria characterized by disruption of blood flow via plaque formation. Discovering the molecules that mediate endothelial response leading to inflammatory diseases is important to develop early detection, prevention, and treatment methods. In the present study, two adipokines were investigated: A proinflammatory protein leptin that is related to cardiovascular (CV) risk factors and an anti‐inflammatory protein adiponectin (APN) that has cardioprotective functions. Considering their roles in CV diseases, the present study aimed to investigate whether leptin and APN are involved in EC response to proatherogenic disturbed flow.MethodsOrbital shaker (Grant Instruments, UK) was used to generate laminar and disturbed flow conditions on cultured human endothelial cells (EA.hy926) by applying 10mm diameter circulatory flow with speed of 210rpm. ECs were exposed to fluid flow for different time points (6h, 15h, 24h, and 48h). Real time polymerase chain reaction (RT‐PCR) was performed to study gene expressions.ResultsGeneration of laminar flow condition was confirmed via increased endothelial NO synthase (eNOS) by 1.964 ± 1.015 folds (n=8, p,0.05); disturbed condition via increased expression of inflammatory molecules, such as, e‐Selectin, by 2.075 ± 1.154 folds (n=8, p,0.05) and VCAM1 by 2.329 ± 1.540 folds (n=8, p,0.05). This study showed that mRNA expressions of intracellular leptin as well as APN increased by 3.559 ± 2.207 folds (n=8, p,0.05), and by 2.697 ± 1.713 folds (n=8, p<0.05) respectively, in response to disturbed flow, compared to static and laminar flow conditions. Further, APN receptor‐2 (APNR‐2) expression was downregulated by 0.5761 ± 0.2516 folds (n=8, p,0.05) in ECs following disturbed flow exposure, while leptin receptor expression alterations remained non‐significant.ConclusionThe obtained results of this study indicate that disturbed flow upregulates leptin and APN expressions along with inflammatory molecules (VCAM1 and e‐Selectin). Knowing that leptin plays role in inflammation activation and CV disease development, confirming its relationship with EC inflammatory response to disturbed flow is logic. However, disturbed flow induced expression of cardioprotective, and anti‐inflammatory APN, is contradicting. On the other hand, APNR‐2 reduction shows reduced APN effect.

Fibroblast growth factor 21 improves lipopolysaccharide-induced pulmonary microvascular endothelial cell dysfunction and inflammatory response through SIRT1-mediated NF-κB deacetylation.

Pneumonia is a common infectious disease of the respiratory system in children. It often leads to death in children by causing acute lung injury. Fibroblast growth factor 21 (FGF21) is a peptide hormone that plays an important role in the regulation of energy homeostasis. This study aimed to investigate the role of FGF21 in alleviating the lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cell (HPMEC) injury, as well as the underlying mechanism. The expression of sirtuin 1 (SIRT1), NF-κB p65, Ac-NF-κB p65, apoptosis-related proteins, tight junction proteins and adhesion molecules in HPMECs were analyzed by Western blotting. The viability and apoptosis of HPMECs were detected by CCK-8 and TUNEL assays. Lactate dehydrogenase level and levels of inflammatory factors were respectively determined by assay kits. The mRNA expression of adhesion molecules in HPMECs was analyzed by RT-qPCR. As a result, SIRT1 expression was decreased and the expression of NF-κB p65 and Ac-NF-κB p65 were increased in LPS-induced HPMECs, which were reversed by recombinant FGF21 (rFGF21). rFGF21 increased the viability and inhibited the apoptosis, inflammatory response, permeability, and release of cell adhesion molecules of LPS-induced HPMECs. In addition, EX527 as SIRT1 inhibitor could reverse the effect of rFGF21 on LPS-induced HPMECs. In conclusion, FGF21 improved LPS-induced HPMEC dysfunction and inflammatory response through SIRT1-mediated NF-κB deacetylation.

324 The role of CCN3 in lung endothelial identity and function

OBJECTIVES/GOALS: Idiopathic Pulmonary Fibrosis (IPF) is a fatal disease of lung scarring. Aberrant vascular remodeling is a contributor to IPF progression. We have identified CCN3 as an endothelial gene that is upregulated in resolving but not in persistent lung fibrosis in mice. Here we tested the role of CCN3 in lung microvascular endothelial function. METHODS/STUDY POPULATION: RNAi loss of function experiments were used to evaluate the role of CCN3 in lung endothelial biology. Human lung microvascular endothelial cells (HLMECs) were transfected with human CCN3 siRNA and analyzed via qPCR to assess expression of endothelial transcripts, via wound healing assay for assessment of migratory function, and via 2D tube formation assay for assessment of angiogenic function. To ascertain the effect of HLMECs on Normal Human Lung Fibroblasts (NHLFs), conditioned media (CM) from endothelial cells with control and CCN3 siRNA was applied to TGFβ primed fibroblasts and qPCR was used to measure expression levels of pro-fibrotic transcripts. Recombinant human CCN3 protein was subsequently used to confirm the gain of function role of CCN3 in lung endothelial biology using a subset of these assays. RESULTS/ANTICIPATED RESULTS: CCN3 is a secreted matricellular protein thought to be involved in angiogenesis, cell adhesion, cell migration, and inflammatory responses in endothelial cells. In other organs, CCN3 suppresses expression of fellow matricellular protein, CCN2 (CTGF); importantly, CCN2 is a known pro-fibrotic mediator of aberrant tissue remodeling in the fibrotic lung. Upon CCN3 knockdown in HLMECs, we observed reduced transcripts for inflammatory and pro-fibrotic genes, along with impaired endothelial function in wound healing and angiogenesis assays. CM from CCN3 knockdown endothelial cells enhanced the pro-fibrotic effects of TGFβ in NHLFs. Addition of recombinant CCN3 to HLMECs generated, conditioned media that reduced fibroblast pro-fibrotic activation. DISCUSSION/SIGNIFICANCE: We have shown that matricellular protein- CCN3 plays a fundamental role in endothelial identity and function and could be a promising therapeutic target in IPF. A future goal is to restore levels of genes such as CCN3 in the aged vasculature in the setting of lung fibrosis to test their capacity to promote vascular repair and fibrosis resolution.

Formononetin attenuates Aβ25-35-induced adhesion molecules in HBMECs via Nrf2 activation

Brain vascular inflammation plays a crucial role in the pathogenesis of Alzheimer’s disease (AD). As a central pathogenic factor in AD, the extracellular buildup of amyloid-β (Aβ) induces brain microvascular endothelial cells activation, impairs endothelial structure and function. Formononetin (FMN) has been reported to protect against Alzheimer’s disease (AD) and attenuates vascular inflammation in atherosclerosis. However, its involvement in regulating vascular inflammation of AD has not been investigated. In the study, we found that FMN significantly attenuates Aβ25–35-induced expression of adhesion molecules, including intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the human brain microvascular endothelial cells (HBMECs), suggesting that FMN inhibits Aβ25–35-induced brain endothelial cells inflammatory response. Moreover, we observed that FMN attenuates Aβ25–35-induced translocation of NFκB (p65) into the nucleus of HBMECs, and found that FMN treatment induces Nrf2 expression and attenuates Nrf2-Keap1 association in a dose-dependent manner in HBMECs. Furthermore, we demonstrated that Nrf2 silencing significantly attenuates FMN-reduced NFκB (p65) activation and nuclear translocation. Lastly, our results showed that FMN treatment attenuates Aβ25–35-induced adhesion of THP-1 cell to endothelial cell monolayer. Collectively, these findings suggest that FMN attenuates Aβ25–35-induced activation in human brain microvascular endothelial cells, which at least in part was mediated through Nrf2 pathways.

Multiphysics and multiscale modeling of microthrombosis in COVID-19.

Emerging clinical evidence suggests that thrombosis in the microvasculature of patients with Coronavirus disease 2019 (COVID-19) plays an essential role in dictating the disease progression. Because of the infectious nature of SARS-CoV-2, patients’ fresh blood samples are limited to access for in vitro experimental investigations. Herein, we employ a novel multiscale and multiphysics computational framework to perform predictive modeling of the pathological thrombus formation in the microvasculature using data from patients with COVID-19. This framework seamlessly integrates the key components in the process of blood clotting, including hemodynamics, transport of coagulation factors and coagulation kinetics, blood cell mechanics and adhesive dynamics, and thus allows us to quantify the contributions of many prothrombotic factors reported in the literature, such as stasis, the derangement in blood coagulation factor levels and activities, inflammatory responses of endothelial cells and leukocytes to the microthrombus formation in COVID-19. Our simulation results show that among the coagulation factors considered, antithrombin and factor V play more prominent roles in promoting thrombosis. Our simulations also suggest that recruitment of WBCs to the endothelial cells exacerbates thrombogenesis and contributes to the blockage of the blood flow. Additionally, we show that the recent identification of flowing blood cell clusters could be a result of detachment of WBCs from thrombogenic sites, which may serve as a nidus for new clot formation. These findings point to potential targets that should be further evaluated, and prioritized in the anti-thrombotic treatment of patients with COVID-19. Altogether, our computational framework provides a powerful tool for quantitative understanding of the mechanism of pathological thrombus formation and offers insights into new therapeutic approaches for treating COVID-19 associated thrombosis.

Integrin Beta 4E Promotes Endothelial Phenotypic Changes and Attenuates Lung Endothelial Cell Inflammatory Responses.

We previously reported integrin beta 4 (ITGB4) is an important mediator of lung vascular protection by simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitor. In this study, we report increased endothelial cell (EC) expression specifically of ITGB4E, an ITGB4 mRNA splice variant, by simvastatin with effects on EC protein expression and inflammatory responses. In initial experiments, human pulmonary artery ECs were treated using simvastatin (5 μM, 24 h) prior to immunoprecipitation of integrin alpha 6 (ITGA6), which associates with ITGB4, and Western blotting for full-length ITGB4 and ITGB4E, uniquely characterized by a truncated 114 amino acid cytoplasmic domain. These experiments confirmed a significant increase in both full-length ITGB4 and ITGB4E. To investigate the effects of increased ITGB4E expression alone, ECs were transfected with ITGB4E or control vector, and cells were seeded in wells containing Matrigel to assess effects on angiogenesis or used for scratch assay to assess migration. Decreased angiogenesis and migration were observed in ITGB4E transfected ECs compared with controls. In separate experiments, PCR and Western blots from transfected cells demonstrated significant changes in EC protein expression associated with increased ITGB4E, including marked decreases in platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin) as well as increased expression of E-cadherin and N-cadherin along with increased expression of the Slug and Snail transcription factors that promote endothelial-to-mesenchymal transition (EndMT). We, then, investigated the functional effects of ITGB4E overexpression on EC inflammatory responses and observed a significant attenuation of lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, including decreased phosphorylation of both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as reduced inflammatory cytokines (IL-6 and IL-8), expressed in the media of EC after either LPS or excessive cyclic stretch (CS). Finally, EC expression-increased ITGB4E demonstrated decreased barrier disruption induced by thrombin as measured by transendothelial electrical resistance. Our data support distinct EC phenotypic changes induced by ITGB4E that are also associated with an attenuation of cellular inflammatory responses. These findings implicate ITGB4E upregulation as an important mediator of lung EC protection by statins and may lead to novel therapeutic strategies for patients with or at risk for acute lung injury (ALI).

Effect of dehydroepiandrosterone on atherosclerosis in postmenopausal women.

In China, cardiovascular disease (CVD) has surpassed malignant tumours to become the disease with the highest mortality rate, and atherosclerosis (AS) is an important pathological cause of CVD. Dehydroepiandrosterone (DHEA) is the most abundant steroid hormone in circulating human blood and is a precursor of estrogen and androgen. DHEA is converted into a series of sex hormones in local peripheral tissues where its acts physiologically. DHEA also acts therapeutically, thereby avoiding the adverse systemic reactions to sex hormones. DHEA inhibits AS, thus inhibiting the development of CVD, and it improves the prognosis for CVD. The incidence of CVD in postmenopausal women is substantially higher than that in premenopausal women, and that incidence is believed to be related to a decrease in ovarian function. The current review analyzes the mechanisms of postmenopausal women's susceptibility to AS. They tend to have dyslipidemia, and their vascular smooth muscle cells (VSMCs) proliferate and migrate more. In addition, oxidative stress and the inflammatory response of endothelial cells (ECs) are more serious in postmenopausal women. This review also discusses how DHEA combats AS by countering these mechanisms, which include regulating the blood lipid status, protecting ECs (including coping with oxidative stress and inflammatory reactions of the vascular endothelium, inhibiting apoptosis of ECs, and inducing NO production) and inhibiting the proliferation and migration of VSMCs. As a result, DHEA has great value in preventing AS and inhibiting its progression in postmenopausal women.

Sphingosine Kinase 1 Plays an Important Role in Atorvastatin-Mediated Anti-Inflammatory Effect against Acute Lung Injury.

Atorvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor and inhibits cholesterol synthesis. Recently, atorvastatin also showed anti-inflammatory effect in acute lung injury, ameliorating pulmonary gas-blood exchanging function. Sphingosine kinase 1 plays a central role in endothelial (EC) cytoskeleton rearrangement and EC barrier integrity regulation. In this study, the role of sphingosine kinase 1 in atorvastatin anti-inflammatory effect against acute lung injury was investigated. Both wild-type (WT) and SphK1−/− mice were challenged with high tidal volume ventilation (40 ml/kg body weight, 65 breathing/min, 4 hours). The acute lung injury was evaluated and the mechanisms were explored. In WT mice, atorvastatin treatment significantly decreased acute lung injury responding to high tidal volume ventilation (HT), including protein, cellular infiltration, and cytokine releasing; comparing to WT mice, SphK1−/− mice showed significantly worsen pulmonary injuries on HT model. Moreover, the atorvastatin-mediated anti-inflammatory effect was diminished in SphK1−/− mice. To further confirm the role of SphK1 in VILI, we then compared the inflammatory response of endothelial cells that were isolated from WT and SphK1−/− mice to cyclic stretching. Similarly, atorvastatin significantly decreased cytokine generation from WT EC responding to cyclic stretching. Atorvastatin also significantly preserved endothelial junction integrity in WT EC against thrombin challenge. However, the inhibitory effect of atorvastatin on cytokine generation induced by cyclic stretching was abolished on SphK1−/− mice EC. The endothelial junction integrity effects of atorvastatin also diminished on SphK1−/− mouse EC. Signal analysis indicated that atorvastatin inhibited JNK activation induced by cyclic stretch. SphK1 knockout also blocked atorvastatin-mediated VE-cadherin junction enhancement. In summary, by inhibition of MAPK activity and maintenance of EC junction homeostasis, SphK1 plays a critical role in atorvastatin-mediated anti-inflammatory effects in both cellular and in vivo model. This study also offers an insight into mechanical stress-mediated acute lung injury and potential therapy in the future.

Carbon dioxide inhibits COVID-19-type proinflammatory responses through extracellular signal-regulated kinases 1 and 2, novel carbon dioxide sensors

Mitogen-activated protein kinase (MAPK) signalling pathways are crucial for developmental processes, oncogenesis, and inflammation, including the production of proinflammatory cytokines caused by reactive oxygen species and upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There are no drugs that can effectively prevent excessive inflammatory responses in endothelial cells in the lungs, heart, brain, and kidneys, which are considered the main causes of severe coronavirus disease 2019 (COVID-19). In this work, we demonstrate that human MAPKs, i.e. extracellular signal-regulated kinases 1 and 2 (ERK1/2), are CO2 sensors and CO2 is an efficient anti-inflammatory compound that exerts its effects through inactivating ERK1/2 in cultured endothelial cells when the CO2 concentration is elevated. CO2 is a potent inhibitor of cellular proinflammatory responses caused by H2O2 or the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. ERK1/2 activated by the combined action of RBD and cytokines crucial for the development of severe COVID-19, i.e. interferon-gamma (IFNγ) and tumour necrosis factor-α (TNFα), are more effectively inactivated by CO2 than by dexamethasone or acetylsalicylic acid in human bronchial epithelial cells. Previously, many preclinical and clinical studies showed that the transient application of 5–8% CO2 is safe and effective in the treatment of many diseases. Therefore, our research indicates that CO2 may be used for the treatment of COVID-19 as well as the modification of hundreds of cellular pathways.

RNA Externalized by Neutrophil Extracellular Traps Promotes Inflammatory Pathways in Endothelial Cells.

Neutrophil extracellular traps (NETs) are extracellular lattices composed of nucleic material bound to neutrophil granule proteins. NETs may play pathogenic roles in the development and severity of autoimmune diseases such as systemic lupus erythematosus (SLE), at least in part, through induction of type I interferon (IFN) responses via externalization of oxidized immunostimulatory DNA. A distinct subset of SLE proinflammatory neutrophils (low-density granulocytes [LDGs]) displays enhanced ability to form proinflammatory NETs that damage the vasculature. We undertook this study to assess whether NET-bound RNA can contribute to inflammatory responses in endothelial cells (ECs) and the pathways that mediate this effect. Expression of newly synthesized and total RNA was quantified in NETs from healthy controls and lupus patients. The ability of ECs to take up NET-bound RNA and downstream induction of type I IFN responses were quantified. RNAs present in NETs were sequenced and specific small RNAs were tested for induction of endothelial type I IFN pathways. NETs extruded RNA that was internalized by ECs, and this was enhanced when NET-bound nucleic acids were oxidized, particularly in lupus LDG-derived NETs. Internalization of NET-bound RNA by ECs was dependent on endosomal Toll-like receptors (TLRs) and the actin cytoskeleton and induced type I IFN-stimulated genes (ISGs). This ISG induction was dependent on NET-associated microRNA let-7b, a small RNA expressed at higher levels in LDG-derived NETs, which acted as a TLR-7 agonist. These findings highlight underappreciated roles for small RNAs externalized in NETs in the induction of proinflammatory responses in vascular cells, with implications for lupus vasculopathy.

Comparison of Surface Functionalization of PLGA Composite to Immobilize Extracellular Vesicles

Endothelialization by materials provides a promising approach for the rapid re-endothelialization of a cardiovascular implantation. Although previous studies have focused on improving endothelialization through the immobilization of bioactive molecules onto the surface of biodegradable implants, comparative studies of effective surface modification have not yet been reported. Here, we conducted a comparative study on the surface modification of poly(lactide-co-glycolide) (PLGA)-based composites to graft mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) using three different materials, fibronectin (FN), polyethylenimine (PEI), and polydopamine (PDA), which have different bond strengths of ligand–receptor interaction, ionic bond, and covalent bond, respectively. Further in vitro analysis exhibited that MSC-EVs released from all modified films sustainably, but the MSC-EVs grafted onto the surface coated with PEI are more effective than other groups in increasing angiogenesis and reducing the inflammatory responses in endothelial cells. Therefore, the overall results demonstrated that PEI is a desirable coating reagent for the immobilization of MSC-EVs on the surface of biodegradable implants.

Differential Effects of Ruminant and Industrial 18-Carbon trans-Monounsaturated Fatty Acids (trans Vaccenic and Elaidic) on the Inflammatory Responses of an Endothelial Cell Line.

Endothelial dysfunction and inflammation are recognised factors in the development of atherosclerosis. Evidence suggests that intake of industrial trans fatty acids (TFAs) promotes endothelial dysfunction, while ruminant TFAs may have the opposite effect. The aim of this study was to compare the effects of elaidic acid (EA (18:1n-9t); an industrially produced TFA) and trans vaccenic acid (TVA (18:1n-7t); a natural TFA found in ruminant milk and meat) on inflammatory responses of endothelial cells (ECs). ECs (EA.hy926 cells) were cultured under standard conditions and exposed to TFAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments. TFAs were incorporated into ECs in a dose-dependent manner. Preincubation with EA (50 µM) increased production of MCP-1, RANTES, and IL-8 in response to TNF-α, while preincubation with TVA (1 µM) decreased production of ICAM-1 and RANTES in response to TNF-α. Preincubation with EA (50 µM) upregulated toll-like receptor 4 and cyclooxygenase 2 gene expression in response to TNF-α. In contrast, preincubation with TVA (1 µM) downregulated TNF-α induced nuclear factor kappa B subunit 1 gene expression. Preincubation of ECs with EA (50 µM) increased THP-1 monocyte adhesion. In contrast, preincubation of ECs with TVA (1 µM) reduced THP-1 monocyte adhesion, while preincubation of ECs with TVA (50 µM) decreased the level of surface expression of ICAM-1 seen following TNF-α stimulation. The results suggest that TVA has some anti-inflammatory properties, while EA enhances the response to an inflammatory stimulus. These findings suggest differential effects induced by the TFAs tested, fitting with the idea that industrial TFAs and ruminant TFAs can have different and perhaps opposing biological actions in an inflammatory context.

Vitamin D, Thyroid Hormones and Cardiovascular Risk: Exploring the Components of This Novel Disease Triangle.

The role of thyroid hormones (THs) in the cardiovascular (CV) system, through several direct and indirect effects is recognized. Even very small modification in TH levels (as those observed in subclinical hypothyroidism or hyperthyroidism, and low triiodothyronine syndrome) may adversely affect the CV system, whereas thyroid hormones benefit the CV system and improve the prognosis. There is also evidence of vitamin D effects on cardiometabolic disease (e.g., through modulation of endothelial and smooth muscle cell activity, renin-angiotensin-aldosterone system, nitric oxide, oxidative stress, and inflammatory response), as well as an association between vitamin D [25(OH)D] deficiency and autoimmune thyroid diseases or cancer, and a relationship between vitamin D concentration and titers of antibodies and thyroid autoimmunity replacement. Interestingly, experimental data indicate a direct effect of vitamin D on Type 2 deiodinase expression causing subsequential peripheral conversion of T4 into T3. However, the functional links among THs, vitamin D and the cardiovascular system, and clinical effects of coexisting abnormalities in this new troublesome triad, have not yet been reviewed. The main aim of this review is to discuss pathophysiology of this relationship, proposing new mechanistic insights involving vitamin D in the modulation of cardiometabolic disease and thyroid profile.

Pulmonary delivery of the broad-spectrum matrix metalloproteinase inhibitor marimastat diminishes multiwalled carbon nanotube-induced circulating bioactivity without reducing pulmonary inflammation

BackgroundMultiwalled carbon nanotubes (MWCNT) are an increasingly utilized engineered nanomaterial that pose the potential for significant risk of exposure-related health outcomes. The mechanism(s) underlying MWCNT-induced toxicity to extrapulmonary sites are still being defined. MWCNT-induced serum-borne bioactivity appears to dysregulate systemic endothelial cell function. The serum compositional changes after MWCNT exposure have been identified as a surge of fragmented endogenous peptides, likely derived from matrix metalloproteinase (MMP) activity. In the present study, we utilize a broad-spectrum MMP inhibitor, Marimastat, along with a previously described oropharyngeal aspiration model of MWCNT administration to investigate the role of MMPs in MWCNT-derived serum peptide generation and endothelial bioactivity.ResultsC57BL/6 mice were treated with Marimastat or vehicle by oropharyngeal aspiration 1 h prior to MWCNT treatment. Pulmonary neutrophil infiltration and total bronchoalveolar lavage fluid protein increased independent of MMP blockade. The lung cytokine profile similarly increased following MWCNT exposure for major inflammatory markers (IL-1β, IL-6, and TNF-α), with minimal impact from MMP inhibition. However, serum peptidomic analysis revealed differential peptide compositional profiles, with MMP blockade abrogating MWCNT-derived serum peptide fragments. The serum, in turn, exhibited differential potency in terms of inflammatory bioactivity when incubated with primary murine cerebrovascular endothelial cells. Serum from MWCNT-treated mice led to inflammatory responses in endothelial cells that were significantly blunted with serum from Marimastat-treated mice.ConclusionsThus, MWCNT exposure induced pulmonary inflammation that was largely independent of MMP activity but generated circulating bioactive peptides through predominantly MMP-dependent pathways. This MWCNT-induced lung-derived bioactivity caused pathological consequences of endothelial inflammation and barrier disruption.

Morin hydrate inhibits atherosclerosis and LPS-induced endothelial cells inflammatory responses by modulating the NFκB signaling-mediated autophagy

Atherosclerosis (AS) is a chronic inflammatory disease involving blood vessels. Inflammation affects different cells and increases the expression of adhesion molecules. Morin hydrate (MO) is a naturally occurring bioflavonoid with anti-inflammatory and anti-oxidant effects. Although the exact mechanism has not been fully elucidated, MO possibly influences autophagy pathways in immunity and inflammation. In this study, MO showed the potential to inhibit atherosclerotic and promote vascular endothelial autophagy in apolipoprotein E (ApoE)−/− mice with a high-fat diet. Then, we aimed to explore the anti-inflammatory effects of MO in human umbilical vein endothelial cells (HUVECs) and its relationship with autophagy. We found that MO inhibited lipopolysaccharide (LPS)-induced monocyte adhesion and the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), and matrix metallopeptidase 9 (MMP-9) in HUVECs. Moreover, MO reduced the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) by inhibiting the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor kappa B (NFκB) signaling pathway. MO induced autophagy by inhibiting the NFκB signaling pathway in normal HUVECs and LPS-stimulated HUVECs. When autophagy was inhibited by 3-methyladenine (3-MA) or small interfering RNA (siRNA), the anti-inflammatory effect of MO was reduced. In conclusion, MO inhibits atherosclerosis in ApoE−/− mice and LPS-induced inflammatory responses by inhibiting the activation of the PI3K/Akt1/NFκB signaling pathway in a NFκB signaling-mediated autophagy way.

MiR-302a Limits Vascular Inflammation by Suppressing Nuclear Factor-κ B Pathway in Endothelial Cells.

The inflammatory response of endothelial cells accelerates various vascular diseases. MicroRNAs (miRNAs) participate in diverse cellular processes during inflammation. In the present study, we found that miR-302a is an effective suppressor of vascular inflammation in endothelial cells. It was revealed that miR-302a exhibited a lower level in a lipopolysaccharide (LPS)-induced mouse model and in patients with vascular inflammatory disease. Genetic haploinsufficiency of miR-302 aggravated the LPS-induced vascular inflammatory response in mice, and overexpression of miR-302a attenuated vascular inflammation in mice. Furthermore, overexpression of miR-302a inhibited the synthesis and secretion of adhesion factors in endothelial cells, and suppressed the adhesion of monocytes to endothelium. In the study of molecular mechanism, we found that miR-302a relieved vascular inflammation mainly by regulating the nuclear factor kappa-B (NF-κB) pathway in endothelial cells. The results showed that interleukin-1 receptor-associated kinase4 (IRAK4) and zinc finger protein 91 (ZFP91) were the binding targets of miR-302a. MiR-302a prevented the nuclear translocation of NF-κB by inhibiting phosphorylation of IκB kinase complex β (IKKβ) and inhibitors of κBα (IκBα) via targeting IRAK4. In addition, miR-302a downregulated the expression of NF-κB by directly binding with ZFP91. These findings indicate that miR-302a negatively regulates inflammatory responses in the endothelium via the NF-κB pathway and it may be a novel target for relieving vascular inflammation.

The effect of absent blood flow on the zebrafish cerebral and trunk vasculature.

The role of blood flow in vascular development is complex and context-dependent. In this study, we quantify the effect of the lack of blood flow on embryonic vascular development on two vascular beds, namely the cerebral and trunk vasculature in zebrafish. We perform this by analysing vascular topology, endothelial cell (EC) number, EC distribution, apoptosis, and inflammatory response in animals with normal blood flow or absent blood flow. We find that absent blood flow reduced vascular area and EC number significantly in both examined vascular beds, but the effect is more severe in the cerebral vasculature, and severity increases over time. Absent blood flow leads to an increase in non-EC-specific apoptosis without increasing tissue inflammation, as quantified by cerebral immune cell numbers and nitric oxide. Similarly, while stereotypic vascular patterning in the trunk is maintained, intra-cerebral vessels show altered patterning, which is likely to be due to vessels failing to initiate effective fusion and anastomosis rather than sprouting or path-seeking. In conclusion, blood flow is essential for cellular survival in both the trunk and cerebral vasculature, but particularly intra-cerebral vessels are affected by the lack of blood flow, suggesting that responses to blood flow differ between these two vascular beds.

Activation of κ-opioid receptor inhibits inflammatory response induced by sodium palmitate in human umbilical vein endothelial cells

ObjectivesThe current study aims to investigate the effect of κ-opioid receptor (κ-OR) activation on sodium palmitate (SP)-induced human umbilical vein endothelial cells (HUVECs) inflammatory response and elucidate the underlying mechanisms. MethodsA hyperlipidemic cell model was established and treated with κ-OR agonist (U50,488H), and antagonist (norbinaltorphimine, nor-BNI), or inhibitors targeting PI3K, Akt or eNOS (LY294002, MK2206-2HCl or L-NAME, respectively). Furthermore, the expression levels of NLRP3, caspase-1, p-Akt, Akt, p-eNOS, and total eNOS were evaluated. Additionally, the production of reactive oxygen species, and levels of inflammatory factors, such as TNF-α, IL-1β, IL-6, IL-1 and adhesion molecules, such as ICAM-1, VCAM-1, P-selectin, and E-selectin were determined. The adherence rates of the neutrophils and monocytes were assessed as well. ResultsThe SP-induced hyperlipidemic cell model demonstrated increased expression of NLRP3 and caspase-1 proteins (P < 0.05) and elevated ROS levels (P < 0.01), and decreased phosphorylated-Akt and phosphorylated-eNOS expression (P < 0.05). In addition, SP significantly increased TNF-α, IL-1β, IL-6, ICAM-1, VCAM-1, P-selectin, and E-selectin levels (P < 0.01), decreased IL-10 levels (P < 0.01), and increased the adhesion rates of monocytes and neutrophils (P < 0.01). The SP-induced inflammatory response in HUVECs was ameliorated by κ-OR agonist, U50,488H. However, the protective effect of U50,488H was abolished by κ-OR antagonist, nor-BNI, and inhibitors of PI3K, Akt and eNOS. ConclusionOur findings suggest that κ-OR activation inhibits SP-induced inflammation by activating the PI3K/Akt/eNOS signaling pathway.

S-nitrosylation-mediated coupling of G-protein alpha-2 with CXCR5 induces Hippo/YAP-dependent diabetes-accelerated atherosclerosis

Atherosclerosis-associated cardiovascular disease is one of the main causes of death and disability among patients with diabetes mellitus. However, little is known about the impact of S-nitrosylation in diabetes-accelerated atherosclerosis. Here, we show increased levels of S-nitrosylation of guanine nucleotide-binding protein G(i) subunit alpha-2 (SNO-GNAI2) at Cysteine 66 in coronary artery samples from diabetic patients with atherosclerosis, consistently with results from mice. Mechanistically, SNO-GNAI2 acted by coupling with CXCR5 to dephosphorylate the Hippo pathway kinase LATS1, thereby leading to nuclear translocation of YAP and promoting an inflammatory response in endothelial cells. Furthermore, Cys-mutant GNAI2 refractory to S-nitrosylation abrogated GNAI2-CXCR5 coupling, alleviated atherosclerosis in diabetic mice, restored Hippo activity, and reduced endothelial inflammation. In addition, we showed that melatonin treatment restored endothelial function and protected against diabetes-accelerated atherosclerosis by preventing GNAI2 S-nitrosylation. In conclusion, SNO-GNAI2 drives diabetes-accelerated atherosclerosis by coupling with CXCR5 and activating YAP-dependent endothelial inflammation, and reducing SNO-GNAI2 is an efficient strategy for alleviating diabetes-accelerated atherosclerosis.

MKP-3 suppresses LPS-induced inflammatory responses in HUVECs via inhibition of p38 MAPK/NF-κB pathway

ABSTRACT Endothelial cell dysfunction and inflammatory responses play critical roles in the development of atherosclerosis. Recent data on the processes underlying atherogenesis indicate the substantial role of endotoxins (lipopolysaccharides; LPS) of the intestinal microflora in the initiation and progression of atherosclerosis. Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a cytoplasmic dual-specificity protein phosphatase that specifically binds to and inactivates MAP kinases in mammalian cells, but its biological function in endothelial cell dysfunction and inflammatory responses remains largely unknown. The aim of the present study was to investigate the role of MKP-3 in endotoxin-induced endothelial inflammation by western blotting, quantitative polymerase chain reaction, and immunofluorescence. The results of our study demonstrated that MKP-3 overexpression markedly inhibited the adhesion of human monocytic THP-1 cells to human umbilical vein endothelial cells (HUVECs) by downregulating the expression of vascular cell adhesion protein 1 (VCAM-1) and pro-inflammatory cytokines. In contrast, MKP-3-encoding gene knockdown by small interfering RNA (siRNA) exacerbated LPS-induced endothelial dysfunction. Additionally, we found that MKP-3 overexpression inhibited LPS-induced p38 MAPK phosphorylation and decreased the nuclear translocation of nuclear factor kappa B (NF-κB) after LPS treatment, suggesting its implication in the LPS/Toll-like receptor 4 (TLR4)/p38/NF-κB pathway. Overall, these observations suggest that MKP-3 plays a protective role in endothelial dysfunction and may be a therapeutic target.