Activation and Contraction of Human "Vascular" Smooth Muscle Cells Grown From Circulating Blood Progenitors.

Blood outgrowth smooth muscle cells (BO-SMCs) offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering, and personalized medicine. However, little is known about these cells which meant that their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of BO-SMCs and vessel-derived smooth muscle cells to sense the thromboxane mimetic U46619 by measuring intracellular calcium elevation and contraction. U46619 (10-6 M) increased cytosolic calcium in BO-SMCs and vascular smooth muscle cells (VSMCs) but not in fibroblasts. Increased calcium signal peaked between 10 and 20 s after U46619 in both smooth muscle cell types. Importantly, U46619 (10-9 to 10-6 M) induced concentration-dependent contractions of both BO-SMCs and VSMCs but not in fibroblasts. In summary, we show that functional responses of BO-SMCs are in line with VSMCs providing critical evidence of their application in biomedical research.

Incidence of haematological malignancies in Kosovo-A post "uranium war" concern.

BACKGROUND: During the Kosovo War (1998-99) approximately 31,000 rounds with Depleted Uranium (DU) were fired on 85 targets in Kosovo. The number of haematological malignancies (HM) increased after the war and the concern was the use of DU during the war. The aim of this study was to analyse the incidence rates of HM in Kosovo throughout a 20-year that includes pre- and post- war period (1995-2015); and to examine if there is any association between the use of DU rounds and incidence rates of HM in different regions of Kosovo. METHODS: In this retrospective register-based study, 1,798 new patients diagnosed with leukaemia, Hodgkin lymphoma, non-Hodgkin lymphoma and Multiple myeloma were analysed over a 20 year period. Incidence rates were calculated focusing on specific time periods, regions and age-groups. In addition, the correlation between the use of DU in different regions and their incidence of HM was analysed. RESULTS: The average annual crude rate of all HM in Kosovo was 5.02 cases per 100,000 persons. Incidence rates of HM in first post-war period (2000-2003) increased by 0.37 cases/100,000 persons (9.51%) compared to the pre-war period (1995-1998) whereas in the last post-war period (2012-2015), incidence of HM increased by 3.19/100,000 persons (82%). Gjakova and Peja, the first and third most exposed regions to DU ordnance ranked first and second in difference in HM. Prishtina, Gjilan and Ferizaj, regions with the least number of rounds/km2, were characterized by a decline of incidence rates. CONCLUSIONS: After the war, the increase in incidence rate of HM was higher in two regions with most DU rounds/km2 expended Despite these findings, this study warrants further investigation and does not lead us to a conclusive finding on the existence of a causal relationship between the use of DU during the war and the rise in incidence of HM in Kosovo.

A bioassay system of autologous human endothelial, smooth muscle cells, and leukocytes for use in drug discovery, phenotyping, and tissue engineering.

Blood vessels are comprised of endothelial and smooth muscle cells. Obtaining both types of cells from vessels of living donors is not possible without invasive surgery. To address this, we have devised a strategy whereby human endothelial and smooth muscle cells derived from blood progenitors from the same donor could be cultured with autologous leukocytes to generate a same donor "vessel in a dish" bioassay. Autologous sets of blood outgrowth endothelial cells (BOECs), smooth muscle cells (BO-SMCs), and leukocytes were obtained from four donors. Cells were treated in monoculture and cumulative coculture conditions. The endothelial specific mediator endothelin-1 along with interleukin (IL)-6, IL-8, tumor necrosis factor α, and interferon gamma-induced protein 10 were measured under control culture conditions and after stimulation with cytokines. Cocultures remained viable throughout. The profile of individual mediators released from cells was consistent with what we know of endothelial and smooth muscle cells cultured from blood vessels. For the first time, we report a proof of concept study where autologous blood outgrowth "vascular" cells and leukocytes were studied alone and in coculture. This novel bioassay has usefulness in vascular biology research, patient phenotyping, drug testing, and tissue engineering.

Mechanistic definition of the cardiovascular mPGES-1/COX-2/ADMA axis.

AIMS: Cardiovascular side effects caused by non-steroidal anti-inflammatory drugs (NSAIDs), which all inhibit cyclooxygenase (COX)-2, have prevented development of new drugs that target prostaglandins to treat inflammation and cancer. Microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors have efficacy in the NSAID arena but their cardiovascular safety is not known. Our previous work identified asymmetric dimethylarginine (ADMA), an inhibitor of endothelial nitric oxide synthase, as a potential biomarker of cardiovascular toxicity associated with blockade of COX-2. Here, we have used pharmacological tools and genetically modified mice to delineate mPGES-1 and COX-2 in the regulation of ADMA. METHODS AND RESULTS: Inhibition of COX-2 but not mPGES-1 deletion resulted in increased plasma ADMA levels. mPGES-1 deletion but not COX-2 inhibition resulted in increased plasma prostacyclin levels. These differences were explained by distinct compartmentalization of COX-2 and mPGES-1 in the kidney. Data from prostanoid synthase/receptor knockout mice showed that the COX-2/ADMA axis is controlled by prostacyclin receptors (IP and PPARß/δ) and the inhibitory PGE2 receptor EP4, but not other PGE2 receptors. CONCLUSION: These data demonstrate that inhibition of mPGES-1 spares the renal COX-2/ADMA pathway and define mechanistically how COX-2 regulates ADMA.

Development of a novel UHPLC-MS/MS-based platform to quantify amines, amino acids and methylarginines for applications in human disease phenotyping.

Amine quantification is an important strategy in patient stratification and personalised medicine. This is because amines, including amino acids and methylarginines impact on many homeostatic processes. One important pathway regulated by amine levels is nitric oxide synthase (NOS). NOS is regulated by levels of (i) the substrate, arginine, (ii) amino acids which cycle with arginine and (iii) methylarginine inhibitors of NOS. However, biomarker research in this area is hindered by the lack of a unified analytical platform. Thus, the development of a common metabolomics platform, where a wide range of amino acids and methylarginines can be measured constitutes an important unmet need. Here we report a novel high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) platform where ≈40 amine analytes, including arginine and methylarginines can be detected and quantified on a molar basis, in a single sample of human plasma. To validate the platform and to generate biomarkers, human plasma from a well-defined cohort of patients before and after coronary artery bypass surgery, who developed systemic inflammatory response syndrome (SIRS), were analysed. Bypass surgery with SIRS significantly altered 26 amine analytes, including arginine and ADMA. Consequently, pathway analysis revealed significant changes in a range of pathways including those associated with NOS.

Antrolone, a Novel Benzoid Derived from Antrodia cinnamomea, Inhibits the LPS-Induced Inflammatory Response in RAW264.7 Macrophage Cells by Balancing the NF-[Formula: see text]B and Nrf2 Pathways.

Antrodia cinnamomea, a medicinal mushroom, has previously demonstrated anti-inflammatory activity, although the specific compound responsible for the effect remains unclear. The present study was designed to investigate the anti-inflammatory property of antrolone, a novel benzoid derived from A. cinnamomea mycelium, and to clarify the underlying mechanisms of action. To this end, murine macrophage RAW264.7 cells were treated with antrolone (0.1-30[Formula: see text][Formula: see text]M) 30[Formula: see text]min prior to stimulation with lipopolysaccharides (LPS, 0.1[Formula: see text][Formula: see text]g/ml) for 24[Formula: see text]h. Cell viability, nitric oxide (NO) and prostaglandin E2 (PGE2) production, levels of pro-inflammatory cytokines and chemokines, and the signaling pathways involved in the inflammatory cascades were then investigated. Our results show that antrolone significantly decreased LPS-induced NO, PGE2, pro-inflammatory cytokine, and keratinocyte chemoattractant CXCL1 (KC) production and reduced levels of the proteins inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). These effects were independent of the effect of antrolone on macrophage cytotoxicity. Moreover, antrolone significantly inhibited the activation of the NF[Formula: see text]B, MAPK, and AKT pathways, while it increased nuclear factor erythroid-2-related factor (Nrf2) and heme oxygenase-1 (HO-1) levels. Our findings suggest that antrolone exhibits potent anti-inflammatory activity and may, therefore, be a lead compound for the development of an anti-inflammatory drug.

Cyclooxygenase-2 Selectively Controls Renal Blood Flow Through a Novel PPARß/δ-Dependent Vasodilator Pathway.

Cyclooxygenase-2 (COX-2) is an inducible enzyme expressed in inflammation and cancer targeted by nonsteroidal anti-inflammatory drugs. COX-2 is also expressed constitutively in discreet locations where its inhibition drives gastrointestinal and cardiovascular/renal side effects. Constitutive COX-2 expression in the kidney regulates renal function and blood flow; however, the global relevance of the kidney versus other tissues to COX-2-dependent blood flow regulation is not known. Here, we used a microsphere deposition technique and pharmacological COX-2 inhibition to map the contribution of COX-2 to regional blood flow in mice and compared this to COX-2 expression patterns using luciferase reporter mice. Across all tissues studied, COX-2 inhibition altered blood flow predominantly in the kidney, with some effects also seen in the spleen, adipose, and testes. Of these sites, only the kidney displayed appreciable local COX-2 expression. As the main site where COX-2 regulates blood flow, we next analyzed the pathways involved in kidney vascular responses using a novel technique of video imaging small arteries in living tissue slices. We found that the protective effect of COX-2 on renal vascular function was associated with prostacyclin signaling through PPARß/δ (peroxisome proliferator-activated receptor-ß/δ). These data demonstrate the kidney as the principle site in the body where local COX-2 controls blood flow and identifies a previously unreported PPARß/δ-mediated renal vasodilator pathway as the mechanism. These findings have direct relevance to the renal and cardiovascular side effects of drugs that inhibit COX-2, as well as the potential of the COX-2/prostacyclin/PPARß/δ axis as a therapeutic target in renal disease.

Astragaloside II promotes intestinal epithelial repair by enhancing L-arginine uptake and activating the mTOR pathway.

Astragaloside II (AS II) extracted from Astragalus membranaceus has been reported to promote tissue wound repair. However, the effect of AS II on inflammatory bowel disease is unknown. We investigated the effects and mechanism of AS II on intestinal wound healing in both in vitro and in vivo models. Human intestinal Caco-2 cells were treated with multiple concentrations of AS II to assess cell proliferation, scratch wound closure, L-arginine uptake, cationic amino acid transporter activity, and activation of the mTOR signaling pathway. These effects were also measured in a mouse model of colitis. AS II promoted wound closure and increased cell proliferation, L-arginine uptake, CAT1 and CAT2 protein levels, total protein synthesis, and phosphorylation of mTOR, S6K, and 4E-BP1 in Caco-2 cells. These effects were suppressed by lysine or rapamycin treatment, suggesting that the enhanced arginine uptake mediates AS II-induced wound healing. Similar results were also observed in vivo. Our findings indicate that AS II can contribute to epithelial barrier repair following intestinal injury, and may offer a therapeutic avenue in treating irritable bowel disease.

Rhodiola crenulata Attenuates High Glucose Induced Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells.

Rhodiola crenulata root extract (RCE), a traditional Chinese medicine, has been shown to regulate glucose and lipid metabolism via the AMPK pathway in high glucose (HG) conditions. However, the effect of RCE on HG-induced endothelial dysfunction remains unclear. The present study was designed to examine the effects and mechanisms of RCE against hyperglycemic insult in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were pretreated with or without RCE and then exposed to 33[Formula: see text]mM HG medium. The cell viability, nitrite production, oxidative stress markers, and vasoactive factors, as well as the mechanisms underlying RCE action, were then investigated. We found that RCE significantly improved cell death, nitric oxide (NO) defects, and oxidative stress in HG conditions. In addition, RCE significantly decreased the HG-induced vasoactive markers, including endothelin-1 (ET-1), fibronectin, and vascular endothelial growth factor (VEGF). However, the RCE-restored AMPK-Akt-eNOS-NO axis and cell viability were abolished by the presence of an AMPK inhibitor. These findings suggested that the protective effects of RCE were associated with the AMPK-Akt-eNOS-NO signaling pathway. In conclusion, we showed that RCE protected endothelial cells from hyperglycemic insult and demonstrated its potential for use as a treatment for endothelial dysfunction in diabetes mellitus.

Tipifarnib prevents development of hypoxia-induced pulmonary hypertension.

Aims: RhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect. Methods and results: The analysis of lung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia-induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia-induced vascular cell proliferation, increased endothelium-dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerization and increased eNOS expression in vitro and in vivo. Farnesylated-only RhoB (F-RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated-only RhoB (GG-RhoB), and tipifarnib had an inhibitory effect. Label-free proteomics linked F-RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F-RhoB-regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins. Conclusions: Our study demonstrates the importance of protein farnesylation in pulmonary vascular remodelling and provides a rationale for selective targeting of this pathway in pulmonary hypertension.