Bone morphogenetic protein (BMP-2) is involved in mononuclear cell function and their interaction with endothelial cells

In this study, we designed a bifurcated flow channel with a bifurcation angle of 140° and successfully generated a shear stress gradient (SSG) of 280–4800 Pa/m without vortex formation. In areas with high SSG, the cultured endothelial cells (ECs) remained round and randomly oriented even after 24 h of flow exposure. In addition, increased expression of intercellular adhesion molecule 1, a marker of inflammatory response in ECs, was observed. Overall, our findings revealed that disturbed and directional changes caused by flow and wall SSG induce inflammatory responses in ECs.

Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-beta-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway.

The aim of the study was to evaluate the vascular health properties of extracts from biofortified bread, obtained by adding different durum wheat milling by-products rich in phenolic compounds, by analyzing their effects on overwhelming inflammatory response in endothelial cells and monocytes, two main players of atherogenesis. Human umbilical vein endothelial cells or U937 monocytes were incubated with increasing concentrations (1, 5, 10 μg/mL) of biofortified bread polyphenol extracts or corresponding pure phenolic acids before stimulation with lipopolysaccharide (LPS). We analyzed the endothelial-monocyte adhesion and related endothelial adhesion molecules. The expression of chemokines and pro-inflammatory cytokines was also measured in LPS-stimulated endothelial cells and monocytes as well as intracellular oxidative stress. Biofortified bread extracts inhibited monocyte adhesion to LPS-stimulated endothelial cells, in a concentration-dependent manner by reducing mainly endothelial VCAM-1 expression. Phenolic acid extracts contained in 10 mg biofortified bread downregulated the LPS-induced expression of chemokines MCP-1, M-CSF, and CXCL-10 as well as pro-inflammatory cytokines TNF-α and IL-1β, in endothelial cells and monocytes, with CXCL-10 as the most reduced inflammatory mediator. Among phenolic acids of biofortified bread, ferulic, sinapic, and p-coumaric acids significantly inhibited the LPS-stimulated CXCL-10 expression in vascular cells. The reduced pro-inflammatory response was related to a slightly but significant reduction of intracellular oxidative stress. Our findings suggest the bread biofortified with selected durum wheat milling by-products as a source of phenolic acids with multiple anti-inflammatory and anti-atherosclerotic properties, which could help to counteract or prevent inflammatory vascular diseases.

Oxidative stress and vascular inflammatory response are key mediators of endothelial dysfunction that leads to cardiovascular diseases. A novel peptide, IRW, was previously characterized from egg protein with angiotensin converting enzyme inhibitory activity. The purpose of the study was to investigate the effects and molecular mechanisms of IRW on regulating inflammatory response in endothelial cells. The results showed that tumor necrosis factor-α (TNF-α) significantly increased the protein levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemotactic protein-1 (MCP-1), whereas pretreatment with IRW inhibited TNF-α-induced increases of ICAM-1, VCAM-1, and MCP-1 production in a concentration-dependent manner. IRW also reduced the levels of superoxide ions in the presence and absence of TNF-α. These results indicate the potential role of IRW in preventing cardiovascular disease as a functional food ingredient or nutraceutical.

USP11 Exacerbates Radiation-Induced Pneumonitis by Activating Endothelial Cell Inflammatory Response via OTUD5-STING Signaling

In type 2 diabetes (T2D), aggregation of human islet amyloid polypeptide (hIAPP) occurs within the islet extracellular matrix (ECM) and is toxic to β cells. We recently showed that hIAPP aggregation also induces a pro-inflammatory response in islet endothelial cells (ECs). In most tissues, this inflammatory response in ECs is mediated by upregulating production of ECM components, such as hyaluronan (HA). Indeed, we find that HA accumulates in the islet ECM of hIAPP transgenic mice, where it was colocalized with amyloid deposits, while no HA accumulation occurred in non-transgenic littermate controls (which do not develop islet amyloid). Therefore, we hypothesized that hIAPP aggregation results in HA production from islet ECs, and that this HA production exacerbates hIAPP deposition/toxicity. To test this hypothesis, we utilized immortalized murine islet ECs (MS-1 cells), which under normal conditions express low levels of HA synthase (Has isoforms 2 and 3). Treatment of these cells for 48 h with hIAPP (20 µM) increased Has2 and Has3 mRNA levels (23±4 and 8±1 fold over vehicle; n=4, p<0.05 for both), which was abrogated upon co-incubation with Congo red, an inhibitor of hIAPP aggregation (1.4±0.3 and 1.4±0.2 for Has2 and Has3, respectively; n=4). Next, we used a thioflavin T assay, wherein fluorescence is proportional to fibril formation from amyloidogenic peptides, to determine the kinetics of hIAPP aggregation ± HA. We found co-incubation of HA with synthetic hIAPP peptide resulted in a marked acceleration (log time of 1 vs. 40 hours) and increase in maximal (greater than 2-fold) hIAPP accretion. In sum, exposure of islet ECs to aggregated hIAPP increases HA synthesis by these cells, which in turn further exacerbates aggregation of hIAPP to form amyloid. Thus, interventions targeting endothelial cell inflammation and/or HA production may be useful in blocking amyloid formation and its toxic effects in T2D. Disclosure J.J. Castillo: None. M.F. Hogan: None. A. Aplin: None. N. Esser: None. A.T. Templin: None. R.L. Hull: None. Funding National Institutes of Health (T32HL007028)

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are one of the first-line treatments for patients with human immunodeficiency virus (HIV). However, epidemiological studies suggest an increased prevalence of cardiovascular diseases (CVD) in NNRTIs-treated HIV-infected patients. Since inflammation is a risk factor of CVD, the present study aimed to determine whether or not NNRTIs, efavirenz (EFV) and nevirapine (NPV), stimulate vascular inflammation.
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\nHuman umbilical vein endothelial cells (HUVECs) were incubated with EFV (5 or 25 μM), NPV (5 or 25 μM) or their vehicle (dimethyl sulfoxide, 0.1%) for 2 or 24 hours in the absence (the control conditon), or presence of hydrogen peroxide [H2O2 (50 μM), to mimic oxidative stress] or lipopolysaccharides [LPS (10 μg/ml), to mimic bacterial infection]. The releases of inflammatory mediators by HUVECs, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8) and C-reactive proteins (CRP), were determined by measuring their levels in the medium with ELISA. The mRNA levels of TNF-α, IL-6 and IL-8, and the protein presence of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-1 and -2 in HUVECs were measured with real time-polymerase chain reaction assay and Western immunoblotting, respectively.
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\nAfter incubation with EFV (25 μM) for 2 or 24 hours, the number of viable cells, measured by the colorimetric assay with MTT, was significantly reduced. HUVECs treated with this concentration of EFV for 24 hours also showed reduced protein expression of eNOS. Despite these seemingly impairment of endothelial function, the release of CRP and TNF-α, mRNA level of IL-6 and IL-8, and the protein expressions of iNOS and COX-2, in the absence or presence of H2O2 or LPS, were not affected by EFV. As such, it is unlikely that EFV can induce significant inflammatory responses in endothelial cells. In view of the plasma level of EFV after an oral intake (12.89 μM) was lower than the high concentration (25 μM) used in the study, it is unlikely that the clinical use of EFV would cause endothelial cell death.
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\nBy contrast, NPV, at both concentrations tested, did not affect the protein expressions of iNOS, COX-1 and COX-2, and the mRNA level of TNF-α, IL-6 and IL-8 in HUVECs, but after 24 hours reduced CRP release in the presence of H2O2 (at high concentration) and IL-6 release in the presence of LPS (at low concentration). These findings suggest that NPV unlikely causes endothelial inflammation. NPV also did not affect the number of viable cells, thus indicating that it does not have cytotoxic effects on endothelial cells. Nevertheless, NPV down-regulated eNOS in the presence of H2O2 after 24 hours; this effect was observed at both concentrations tested, which are within the plasma concentrations in human, thus suggesting that it may have the potential to impair endothelial function during oxidative stress.
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\nIn summary, the present findings suggest that NNRTIs would not cause significant endothelial inflammation. However, they may impair endothelial function thereby increasing the risk of CVD after chronic treatment.

Pulmonary arterial hypertension (PAH) is a syndrome in which pulmonary arterial obstruction increases pulmonary vascular resistance, which leads to right ventricular (RV) failure and a 15% annual mortality rate. The present review highlights recent advances in the basic science of PAH. New concepts clarify the nature of PAH and provide molecular blueprints that explain how PAH is initiated and maintained. Five basic science concepts provide a framework to understand and treat PAH: (1) Endothelial dysfunction creates an imbalance that favors vasoconstriction, thrombosis, and mitogenesis. Restoration of this balance by inhibition of endothelin and thromboxane or augmentation of nitric oxide (NO) and prostacyclin is the paradigm on which most current therapy is based. (2) PAH has a genetic component. Mutations (bone morphogenetic protein receptor-2 [BMPR2]) and single-nucleotide polymorphisms (SNPs; ion channels and transporter genes) predispose to PAH. (3) Excess proliferation, impaired apoptosis, and glycolytic metabolism in pulmonary artery smooth muscle, fibroblasts, and endothelial cells suggest analogies to cancer. Many experimental therapies reduce PAH by decreasing the proliferation/apoptosis ratio; these include inhibitors of pyruvate dehydrogenase kinase (PDK), serotonin transporters (SERT), survivin, 3-hydroxy-3-methylglutaryl coenzyme A reductase, transcription factors (hypoxia-inducible factor [HIF]-1α and nuclear factor of activated T lymphocytes [NFAT]), and tyrosine kinases. Augmentation of voltage-gated K+ channels (Kv1.5) and BMPR2 signaling also addresses this imbalance. Tyrosine kinase inhibitors used to treat cancer are currently in phase 1 PAH trials. (4) Refractory vasoconstriction may occur due to rho kinase activation. Fewer than 20% of PAH patients respond to conventional vasodilators; however, refractory vasoconstriction may respond to rho kinase inhibitors. (5) The RV can be targeted therapeutically. Although increased afterload initiates RV failure, which is the major cause of death/dysfunction in PAH, the RV may be amenable to cardiac-targeted therapies. The RV in PAH has features of ischemic, hibernating myocardium. Guided by these new …

BACKGROUNDPulmonary hypoplasia accompanied by pulmonary hypertension resistant to treatment is an important feature of congenital diaphragmatic hernia (CDH). The pathogenesis of the pulmonary vascular abnormalities in CDH remains to be...

BackgroundIt is widely accepted the key role of endothelium in the onset of many chronic and acute vascular and cardiovascular diseases.In the last decade, traditional compounds utilized in “folk medicine” were considered with increasing interest to discover new bioactive molecules potentially effective in a wide range of diseases including cardiovascular ones. Since ancient times different parts of the Cucumis sativus L. plant were utilized in Ayurvedic medicine, among these, fruits were traditionally used to alleviate skin problem such as sunburn irritation and inflammation. The main purpose of the present research was, in a well-defined in vitro model of endothelial cells, to investigate whether a water/ethanol extract of Cucumis sativus L. (CSE) fruit can attenuate the damaging effect of pro-inflammatory lipopolysaccharide (LPS).MethodsCell viability, gene expression of endothelial cell markers, cytokines secretion and in vitro angiogenesis assay were performed on porcine Aortic Endothelial Cells exposed to increasing doses (0.02; 02; 2 mg/ml) of CSE in the presence of pro-inflammatory lipopolysaccharide (LPS 10 μg/ml).ResultsCSE reduced LPS-induced cytotoxicity and decreased the cellular detachment, restoring the expression of tight junction ZO-1. The increase of TLR4 expression induced by LPS was counterbalanced by the presence of CSE, while the protective gene Hemeoxygenase (HO)-1 was increased. Cucumis sativus L. inhibited the early robust secretion of inflammatory IL-8 and GM-CSFs, furthermore inhibition of inflammatory IL-6 and IL-1α occurred late at 7 and 24 h respectively. On the contrary, the secretion of anti-inflammatory IL-10, together with IL-18 and IFN-γ was increased. Moreover, the in vitro angiogenesis induced by inflammatory LPS was prevented by the presence of Cucunis sativus L. extract, at any doses tested.ConclusionsOur results have clearly demonstrated that Cucumis sativus L. extract has attenuated lipopolysaccharide-induced inflammatory response in endothelial cells.

Inhibitory effect of Malvidin on TNF-α-induced inflammatory response in endothelial cells

Hemodynamic blood flow‐induced laminar shear flow exerts atheroprotective responses in endothelial cells (ECs) have been intensively studied. Our earlier studies indicated that laminar shear flow attenuated cytokine‐induced inflammatory responses in ECs. However, the effects of laminar shear flow on mitochondrial homeostasis and functions in ECs and during the inflammation remain unclear. We examined the dynamic changes of mitochondrial phenotype and its regulatory proteins associated with mitochondrial functions in ECs under physiological shear flow. Cultured human ECs were subjected to a constant shear stress (12 dynes/cm2) with a parallel‐plate flow chamber system. Mitochondrial biogenesis, dynamics and function were analyzed from ECs after shear treatment. Shear‐treated ECs increased mitochondrial biogenesis as indicated by the elevated levels of mitochondrial transcription factor (TFAM), transcriptional coactivator (PGC1‐α), nuclear respiratory factor (NRF1) and mitochondrial proteins (SDHA and COX4). Shear flow triggered a time‐dependent increment of mitochondrial tubular formation (fusion). This phenomenon was coupled with an increased expression of fusion proteins (MFN2, OPA1) but decreased fission protein (FIS1). Laminar shear flow also increased the long form of OPA‐1 that promotes fusion. Consistently, an increase of phosphorylation at S637 but decrease at S616 on Drp1 and the decrease of mitochondria‐associated Drp1 were observed in ECs after flow treatment. Laminar flow increased the expression of mitochondrial antioxidant enzymes MnSOD2, Thioredoxin (TRX2), Peroxiredoxins (PRX3 and PRX5). As a consequence, ECs exposed to laminar shear flow improve mitochondrial function as revealed by a decreased mitochondrial reactive oxygen species levels (mROS), and an increase of mitochondrial membrane potential and ATP production. Furthermore, ECs exposed to TNFα decreased OPA1 and MFN2 expression and increased mitochondrial fission as revealed by the mitochondrial fragmentation. Consequently, TNFα treatment resulted in a decreased mitochondrial function in ECs. However, ECs under shear flow attenuated this TNFα‐induced decrease of OPA1 and mitochondrial dysfunction. ECs treated with TNFα decreased OPA1 and MFN2 expression, whiles under shear condition, these effects were attenuated. Our results suggest that steady laminar flow promotes endothelial function and helps to prevent the inflammation‐induced mitochondrial dysfunction in ECs. Our study further supports that laminar shear flow exerts atheroprotective role to ECs.

Introduction: We have reported that intermittent hypoxia (IH) relevant to sleep apnea increases oxidative stress causing vascular remodeling and heart failure. The serine protease factor Xa in the coagulation cascade elicits inflammatory responses in endothelial cells. Hypothesis: We hypothesize that rivaroxaban might attenuate cardiac remodeling induced by IH in sleep apnea model mice. Methods: Male C57BL/6J mice at 8 weeks of age were exposed to IH (repeated cycles of 1.5 minute of 5% oxygen followed by 5 minutes of 21% oxygen) for 56 days with/without treatment by rivaroxaban (1.2 g riv./kg chow). After echocardiography, heart was examined by light and electron microscopy, immunohistochemistry, and RT-PCR. HPAECs were cultured under hypoxic conditions (37%, 1% O 2 , 5% CO 2 ) with/without rivaroxaban (1 μM). Results: IH caused cardiac remodeling (Figure), associated with both systolic and diastolic dysfunction compared with those of normoxia (IH vehicle vs. Normoxia; EF: 51.8% vs. 59.4% and E/e’: 29.4 vs. 22.3), which were attenuated by rivaroxaban (EF: 55.8%, E/e’: 24.6). In right atrial myocardium, IH caused endothelial cell degeneration, dissociation of intercalated discs, vacuolar formation and loss of ANP specific granules. Furthermore, 4-hydroxy-2-nonenal protein adducts and the expression of protease-activated receptor-1 (PAR-1) and NF-kB mRNA were increased by IH. Treatment with rivaroxaban significantly suppressed PAR-1 and NF-kB mRNA expression, increased ANP granules and reduced perivascular fibrosis, preventing cardiac remodeling due to IH. Conclusions: Rivaroxaban may attenuate atrial and ventricular remodeling induced by IH at least partly through its anti-inflammatory effect in sleep apnea model mice.

To better understand the molecular basis of vascular cell system behavior in inflammation, we used gene expression microarrays to analyze the expression of 7,075 genes and their response to IL-1beta and TNFalpha in cultures of coronary artery endothelium and smooth muscle derived from a single coronary artery. The most noticeable difference between the cell types was the considerably greater magnitude and complexity of the transcriptional response in the endothelial cells. Two hundred and nine genes were regulated in the endothelium and only 39 in vascular smooth muscle. Among the 209 regulated genes in the endothelium, 99 have not been previously associated with endothelial cell activation and many implicate the endothelium in unconventional roles. For example, the induced genes include several that have only been associated with leukocyte function (e.g., IL-7 receptor, EBI-3 receptor) and others related to antiviral and antibacterial defense (e.g., oligoadenylate synthetase, LMP7, toll-like receptor 4, complement component 3). In addition, 43 genes likely to participate in signal transduction (eg. IL-18 receptor, STK2 kinase, STAF50, ANP receptor, VIP receptor, RAC3, IFP35) were regulated providing evidence that a major effect of TNFalpha and IL-1beta is to alter the potential of the endothelial cell to respond to various other external stimuli.

BackgroundG protein-coupled receptors (GPCRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory.ObjectivesTo determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory.MethodssiRNA-mediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells.ResultsG protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization.ConclusionsG protein-coupled receptor kinase 2 deficiency in endothelial cells results in increased pro-inflammatory signaling and enhanced leukocyte recruitment to activated endothelial cells. The ability of GRK2 to modulate initiation of inflammatory responses in endothelial cells as well as leukocytes now places GRK2 at the apex of control of this finely balanced process.