Exposure Of Smooth Muscle Cells Research Articles

Gene expression of proinflammatory cytokines in human coronary artery smooth muscle cells exposed to alkylating mutagen

Background. It is known that DNA damage in smooth muscle cells can trigger their clonal expansion and transformation into foam cells. Thus, the study of the molecular genetic mechanisms of the vascular smooth muscle cells response to genotoxic exposure is important and relevant in the context of an in-depth understanding of atherogenesis.Aim. To study mRNA level and concentration of proinflammatory cytokines IL6 and IL8 in the human coronary artery smooth muscle cells exposed to alkylating mutagen.Methods. Gene expression signature of studied cytokines in the human coronary artery smooth muscle cells was accessed by quantitative polymerase chain reaction in the two timepoints – immediately after six-hour exposure to mitomycin C (point 1) and after six-hour exposure to mitomycin C followed by 24 hours of cells being cultivated on mitomycin C-free cell growth medium (point 2). Smooth muscle cells cultured according to the above scheme without genotoxin were used as controls. HPRT1, GAPDH and B2M were used as the reference genes. Gene expression level was calculated by ΔCt method. IL6 and IL8 concentration was evaluated in the culture media in points 1 and 2 by enzyme-linked immunosorbent assay. Statistical analysis was performed in GraphPad Prism 9 software.Results. Immediately after mutagenic exposure (point 1) we discovered no significant changes in the expression level of IL6 and IL8 in the mitomycin C exposed smooth muscle cells compared to controls. Removal of mutagen increased expression of IL6 and IL8 in the experimental group (0,36- and 0,67-fold, respectively). At the same time, we discovered no significant differences in the studied cytokines concentration in the culture medium of mutagen-exposed cells compared to the nonexposed controls.Conclusion. Genotoxic stress in human coronary artery smooth muscle cells exposed to alkylating mutagen (mitomycin C) leads to differential expression but not secretion of proinflammatory cytokines IL6 and IL8. Thus, exposure of smooth muscle cells to mitomycin C do not trigger their proinflammatory phenotype.

Heat Shock Protein 70 Is Associated With Cardioversion Outcome and Recurrence of Symptomatic Recent Onset Atrial Fibrillation in Hypertensive Patients.

Accumulating evidence indicates that heat shock proteins (HSPs) may represent a suitable biomarker to predict atrial fibrillation (AF). We investigated the relation of circulating serum HSP70 (sHSP70) with inflammatory cytokines and recurrence of symptomatic recent onset AF (ROAF). We enrolled 90 patients with ROAF (the duration from onset of symptoms ≤24 hours) and 30 controls. Patients received amiodarone for cardioversion and rhythm control. The association of serum HSP70, serum interleukin-2 (sIL-2), and serum interleukin-4 (sIL-4) with the presence of cardioversion and AF recurrence within a year was investigated. Toll-like receptor 4 (TLR4) signaling dependence for IL-2 and IL-4 induction in response to stimulation with HSP70 was tested in rat aortic vascular smooth muscle cell cultures. Patients had higher sHSP70 and sIL-2 and lower sIL-4 compared with controls. Serum HSP70 was independently associated with ROAF (P = 0.005) and correlated with sIL-2 (r = 0.494, P < 0.001) and sIL-4 (r = -0.550, P < 0.001). By 48 hours, 71 of the 90 patients were cardioverted, with noncardioverted patients having higher sHSP70 and sIL-2 and lower sIL-4, which were the only independent factors associated with cardioversion. AF recurred in 38 of the 71 cardioverted patients in 1 year. A cutoff value of sHSP70 ≥0.65 ng/mL and sIL-2 ≥0.21 pg/mL was the only independent factor associated with AF recurrence (hazard ratio: 3.311, 95% confidence interval: 1.503-7.293, P = 0.003 and hazard ratio: 3.144, 95% confidence interval: 1.341-7.374, P = 0.008, respectively). The exposure of smooth muscle cell to HSP70 in vitro increased the expression of IL-2 (5×) and IL-4 (1.5×) through TLR4-dependent and receptor-independent mechanisms. In conclusion, sHSP70 and sIL-2 might constitute a prognostic tool for determining the cardioversion and recurrence likelihood in ROAF.

86 - Are S-Nitrosothiols Predominantly Stable End-Effectors of Protein Function or Transient Intermediates Leading to Disulfide Formation?

S-nitrosation is considered to be a ubiquitous, stable post-translational modification that directly targets and modulates a vast number of proteins. This widespread role appears incompatible with the inherent lability of the nitroso bond and its propensity to rapidly react with thiols to generate disulfide bonds. We present evidence that S-nitrosothiols primarily exist as transient intermediates leading to disulfides. Exposure of primary rat aortic smooth muscle cells (SMCs) to the nitrosating reagent S-nitrosocysteine (CysNO) generated robust and widespread protein S-nitrosation. However, CysNO also induced disulfides in multiple candidate proteins which preceded a significant increase in widespread protein S-nitrosation. Quantitative analysis of SMCs exposed to CysNO demonstrated that disulfides, not S-nitrosothiols, were the dominant modification that accumulated. S-glutathiolation and S-cysteinylation were significantly increased after exposure of SMCs to biotinylated forms of CysNO or S-nitrosoglutathione respectively. 10 μM CysNO depleted non-protein thiols, predominantly reduced glutathione, 7-fold as measured by the Ellman's assay. Furthermore, this intervention also reduced the activity of NAPDH-dependent enzymes (MTS assay) 2-fold, which together indicates CysNO compromises the cells reducing system (p

Angiotensin II-induced arterial thickening, fibrosis and stiffening involves elevated arginase function.

BackgroundArterial stiffness (AS) is an independent risk factor for cardiovascular morbidity/mortality. Smooth muscle cell (SMC) proliferation and increased collagen synthesis are key features in development of AS. Arginase (ARG), an enzyme implicated in many cardiovascular diseases, can compete with nitric oxide (NO) synthase for their common substrate, L-arginine. Increased arginase can also provide ornithine for synthesis of polyamines via ornithine decarboxylase (ODC) and proline/collagen via ornithine aminotransferase (OAT), leading to vascular cell proliferation and collagen formation, respectively. We hypothesized that elevated arginase activity is involved in Ang II-induced arterial thickening, fibrosis, and stiffness and that limiting its activity can prevent these changes.Methods and ResultsWe tested this by studies in mice lacking one copy of the ARG1 gene that were treated with angiotensin II (Ang II, 4 weeks). Studies were also performed in rat aortic Ang II-treated SMC. In WT mice treated with Ang II, we observed aortic stiffening (pulse wave velocity) and aortic and coronary fibrosis and thickening that were associated with increases in ARG1 and ODC expression/activity, proliferating cell nuclear antigen, hydroxyproline levels, and collagen 1 protein expression. ARG1 deletion prevented each of these alterations. Furthermore, exposure of SMC to Ang II (1 μM, 48 hrs) increased ARG1 expression, ARG activity, ODC mRNA and activity, cell proliferation, collagen 1 protein expression and hydroxyproline content. Treatment with ABH prevented these changes.ConclusionArginase 1 is crucially involved in Ang II-induced SMC proliferation and arterial fibrosis and stiffness and represents a promising therapeutic target.

High-glucose-increased expression and activation of NADPH oxidase in human vascular smooth muscle cells is mediated by 4-hydroxynonenal-activated PPARα and PPARβ/δ.

High glucose induces vascular smooth muscle cell (SMC) dysfunction by generating oxidative stress attributable, in part, to the up-regulated NADPH oxidases (Nox). We have attempted to elucidate the high-glucose-generated molecular signals that mediate this effect and hypothesize that products of high-glucose-induced lipid peroxidation regulate Nox by activating peroxisome proliferator-activated receptors (PPARs). Human aortic SMCs were exposed to glucose (5.5-25 mM) or 4-hydroxynonenal (1-25 μM, 4-HNE). Lucigenin assay, real-time polymerase chain reaction, western blot, and promoter analyses were employed to investigate Nox. We found that high glucose generated an increase in Nox activity and expression. It also promoted oxidative stress that consequently induced lipid peroxidation, which resulted in the production of 4-HNE. Pharmacological inhibition of Nox activity significantly reduced the formation of high-glucose-induced 4-HNE. Exposure of SMCs to non-cytotoxic concentrations (1-10 μM) of 4-HNE alone mimicked the effect of high glucose incubation, whereas scavenging of 4-HNE by N-acetyl L-cysteine completely abolished both the effects of high glucose and 4-HNE. The latter exerted its effect by activating PPARα and PPARβ/δ, but not PPARγ, as assessed pharmacologically by the inhibitory effect of selective antagonists and following the silencing of the expression of these receptors. These new data indicate that 4-HNE, generated following Nox activation, functions as an endogenous activator of PPARα and PPARβ/δ. The newly discovered "lipid peroxidation products-PPARs-Nox axis" represents a novel mechanism of Nox regulation and an additional therapeutic target for oxidative stress in diabetes.

TRANSFORMING GROWTH FACTOR BETA DRIVES A DISTINCT DIABETES PHENOTYPE IN HUMAN SAPHENOUS VEIN SMOOTH MUSCLE CELLS VIA UPREGULATION OF MICRORNA-143/145

IntroductionPatients with Type 2 diabetes mellitus (T2DM) suffer premature cardiovascular disease and poor prognosis after revascularisation procedures. Following saphenous vein bypass grafting, smooth muscle cell (SMC) plasticity is vital to...

Monocytes and smooth muscle cells cross-talk activates STAT3 and induces resistin and reactive oxygen species and production

During the early phase of atherosclerosis, monocytes attach to and migrate through the vessel wall where they activate and communicate with smooth muscle cells (SMC) affecting plaque progression by largely unknown mechanisms. Activation of STAT3 transcription factor is suggested to be critically involved in dedifferentiation, migration, and proliferation of SMC in the neointima formation after vascular injury. Monocytes-SMC cross-talk induces an inflammatory phenotype of the resident SMC, but the involvement of STAT3 in phenotype switching is not known. Resistin is a cytokine found in human atheroma associated to monocytes/macrophages with role in inflammation associated with cardiovascular disease. The aim of this study was to follow the effect of activated monocytes-SMC cross-talk on STAT3 activation and subsequent resistin and reactive oxygen species (ROS) production. Our results showed that the interaction of activated monocytes with SMC determines: (i) phosphorylation of STAT3 and reduction of SOCS3 expression in both cell types; (ii) intracellular ROS production dependent on NADPH oxidase (by increased Nox1 expression) and STAT3 activation in SMC; (iii) up-regulation of resistin expression in monocytes dependent on STAT3 activation. Furthermore, exposure of SMC to resistin induces ROS by increasing NADPH oxidase activity and the p22phox and Nox1 expression. In conclusion, the cross-talk between SMC and monocytes activates STAT3 transcription factor and lead to resistin up-regulation in monocytes and ROS production in SMC. Moreover, resistin increases the ROS levels in SMC. These data indicate that monocyte-SMC communication may represent an important factor for progression of the atherosclerotic lesion. J. Cell. Biochem. 114: 2273–2283, 2013. © 2013 Wiley Periodicals, Inc.

Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αVβ3 and RhoA/ROCK-mediated mechanisms

Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVβ3-dependent, but transforming growth factor β-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVβ3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.

Abstract 196: Effects of an Engineered Herpes Simplex Virus (HSV-1) on Vascular Cell Adhesion

Introduction The genetically engineered HSV R7020 is known to block vascular smooth muscle cell (SMC) proliferation while allowing for re-endothelialization after balloon angioplasty. This HSV-1 anti-proliferative effect requires MEK activation. However, it is unknown what effect R7020 has on vascular inflammation and monocyte adhesion. We hypothesized that exposure of SMCs to HSV R7020 would not affect vascular adhesion molecules. Methods Human aortic SMCs were exposed to R7020 for 10 minutes in the absence and presence of TNF-α (10 ng/ml). After washing cells were cultured for 24 hours in the absence and presence of TNF-α. Vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecue-1 (ICAM-1) levels were determined using immunocytochemistry and image J. The adhesion of calcein AM labeled PBMCs to these cells were also analyzed at 24 hours. VCAM-1 and ICAM-1 levels were measured in the carotids of New Zealand White Rabbits that underwent balloon injury and exposed to R7020 (n=4) or saline (n=4) for 10 minutes using microarray analysis. Results There was no significant difference in ICAM-1 levels in the R7020 treated and control SMCs (219±11 vs 223±1; p=0.758); there was an 8 fold increase in ICAM-1 in the TNF-α treated SMCs (219±11 vs 1587±68; p=1.2x10 -13 ). Results were similar for VCAM-1. R7020 caused an increase in the adhesion strength of SMCs to collagen coated glass slides at 24 hours. Microscopic experiments (n=3) showed PBMCs adhered similarly to SMCs treated with TNF-α in the absence and presence of the virus (4 times that of control cells as measured by PBMC adhesion assay). Interestingly, after adhesion, Calcein AM was frequently observed in virus infected SMCs in the presence and absence of TNF-α. In cells not exposed to R7020, this rarely occurred. Microarray analysis demonstrated a 2.3 fold reduction in VCAM-1 expression in the R7020 treated rabbits (p=2.8x10 -14 ) and no significant difference in expression in ICAM gene expression (p=0.3). Conclusions In contrast to TNF-α, R7020 does not increase ICAM-1 or VCAM-1. Both treatments domonstrate PBMC SMC adhesion with no additive or synergistic effect. However, R7020 results in intracellular transfer from PBMCs to SMCs suggesting the virus changes PBMC-SMC interplay.

The Oxysterol 24 (S) ,25‐Epoxycholesterol Attenuates Human Smooth Muscle–Derived Foam Cell Formation Via Reduced Low‐Density Lipoprotein Uptake and Enhanced Cholesterol Efflux

BackgroundFoam cell formation by intimal smooth muscle cells (SMCs) inhibits the elaboration of extracellular matrix, which is detrimental to plaque stabilization. In the present study, we examined the lipoproteins and receptors involved in human SMC foam cell formation and investigated the ability of 24(S),25-epoxycholesterol [24(S),25-EC], an oxysterol agonist of the liver X receptor, to attenuate SMC foam cell formation.Methods and ResultsIncubation of human internal thoracic SMCs with atherogenic lipoproteins demonstrated that low-density lipoprotein (LDL), but not oxidized or acetylated LDL, was the primary lipoprotein taken up, resulting in marked cholesteryl ester deposition (6-fold vs 1.8-fold; P<0.05; n=4). Exposure of SMCs to exogenous or endogenously synthesized 24(S),25-EC attenuated LDL uptake (−90% and −47% respectively; P<0.05; n=3) through decreased sterol regulatory element–binding protein-2 expression (−30% and −17%, respectively; P<0.001; n=3), decreased LDL receptor expression (−75% and −40%, respectively; P<0.05; n=3) and increased liver X receptor–mediated myosin regulatory light chain interacting protein expression (7- and 3-fold, respectively; P<0.05; n=4). Furthermore, exogenous 24(S),25-EC increased adenosine triphosphate–binding cassettes A1– and G1–mediated cholesterol efflux to apolipoprotein AI (1.9-fold; P<0.001; n=5) and high-density lipoprotein3 (1.3-fold; P<0.05; n=5). 24(S),25-EC, unlike a nonsteroidal liver X receptor agonist, T0901317, did not stimulate sterol regulatory element–binding protein-1c–mediated fatty acid synthesis or triglyceride accumulation. 24(S),25-EC preserved the assembly of fibronectin and type I collagen by SMCs.ConclusionsThe oxysterol 24(S),25-EC prevented foam cell formation in human SMCs by attenuation of LDL receptor–mediated LDL uptake and stimulation of cholesterol efflux, restoring the elaboration of extracellular matrix. In contrast to T0901317, 24(S),25-EC prevented the development of a triglyceride-rich foam cell phenotype. (J Am Heart Assoc. 2012;1:e000810 doi: 10.1161/JAHA.112.000810.)

Von Willebrand Factor Permeates Small Vessels in CADASIL and Inhibits Smooth Muscle Gene Expression

CADASIL (cerebral autosomal dominant arteriopathy subcortical infarcts and leukoencephalopathy) is a genetic disorder hallmarked by ischemic stroke and vascular dementia. Characteristic pathological changes in the vasculature include thickening of small arteries and accumulation of heterogeneous material within the vessel wall. We tested whether endothelial von Willebrand factor (vWF) accumulates in CADASIL vessels and whether exposure of smooth muscle cells to vWF alters the expression of smooth muscle gene expression. Brain sections obtained at autopsy from six North American CADASIL patients were examined using immunohistochemistry for vWF and IgG. Rat aortic smooth muscle cells (A7R5 cells) were tested for binding to infrared-tag labeled vWF. Finally, A7R5 cells were exposed to vWF, and expression of mature smooth muscle marker genes was analyzed by quantitative reverse transcriptase PCR. vWF is expressed in the penetrating arterial walls in all CADASIL samples. IgG, a marker of serum extravasation, was present only in a minority of arterial walls. vWF binds to smooth muscle cells in vitro, and low concentrations of vWF rapidly activate c-fos, EGR, TSP1, and c-myc while specifically inhibiting RNA encoding smooth muscle actin, calponin, and SM22. These data demonstrate that vWF, likely produced by the endothelium, permeates the vessel wall of CADASIL brains. Exposure of smooth muscle cells to vWF results in reduction of specific RNAs required for normal vascular homeostasis. This is the first report of accumulation of a protein within CADASIL vessels that inhibits vascular gene expression and implicates a role for vWF beyond hemostasis.

Role of ZFM1 in smooth muscle cell phenotype regulation

Vascular smooth muscle cells (SMC) can rapidly develop a synthetic phenotype in response to growth factors ‐ a phenomenon with major impact on atherosclerosis. Despite its clinical relevance little is known about the mechanisms of this potentially reversible process.Using primary human cultured SMC and platelet‐derived growth factor (PDGF; 60 U/mL) we here show that the transcriptional repressor ZFM1 is a phenotype‐stabilizing factor in these cells. Exposure of SMC to PDGF leads to proliferation and synthetic gene expression. Interestingly, PDGF causes a rapid repression of ZFM1 gene transcription through replacement of Sp1 from the ZFM1 gene promoter by the transcription factor early growth response‐1. The subsequent drop in ZFM1 abundance disinhibits both proliferation and gene expression and is a prerequisite for PDGF‐induced effects in SMC. Consequently, siRNA‐induced ZFM1 knock down alone is sufficient to increase proliferation and expression of PDGF‐sensitive gene products. Conversely, over‐expression of ZFM1 effectively suppresses all effects of PDGF exposure.ZFM1 expression levels pivotally determine the SMC phenotype: Whereas its down‐regulation is necessary for PDGF‐induced changes, the maintenance of ZFM1 expression is sufficient to maintain a contractile SMC phenotype.

Ability of Hypochlorous Acid and N-Chloramines to Chlorinate DNA and Its Constituents

Myeloperoxidase is a heme enzyme released by activated phagocytes that is responsible for the generation of the strong oxidant hypochlorous acid (HOCl). Although HOCl has potent bactericidal properties and plays an important role in the human immune system, this oxidant also causes damage to tissues, particularly under inflammatory conditions. There is a strong link between chronic inflammation and the incidence of many cancers, which may be associated with the ability of HOCl and related oxidants such as N-chloramines to damage DNA. However, in contrast to HOCl, little is known about the reactivity of N-chloramines with DNA and its constituents. In this study, we examine the ability of HOCl and various N-chloramines to form chlorinated base products on nucleosides, nucleotides, DNA, and in cellular systems. Experiments were performed with N-chloramines formed on Nalpha-acetyl-histidine (His-C), Nalpha-acetyl-lysine (Lys-C), glycine (Gly-C), taurine (Tau-C), and ammonia (Mono-C). Treatment of DNA and related materials with HOCl and His-C resulted in the formation of 5-chloro-2'-deoxycytidine (5CldC), 8-chloro-2'-deoxyadenosine (8CldA) and 8-chloro-2'-deoxyguanosine (8CldG). With the nucleosides, 8CldG was the favored product in each case, and HOCl was the most efficient chlorinating agent. 5Cl(d)C was the most abundant product on exposure of the nucleotides and DNA to HOCl and His-C, with only low levels of chlorinated products observed with Lys-C, Gly-C, Tau-C, and Mono-C. 5CldC was also formed on exposure of smooth muscle cells to either HOCl or His-C. Cellular RNA was also a target for HOCl and His-C, with evidence for the formation of 5-chloro-cytidine (5ClC). This study shows that HOCl and the model N-chloramine, His-C, are able to chlorinate cellular genetic material, which may play a role in the development of various inflammatory cancers.

Inducible nitric oxide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 induction by inflammatory stimuli

Cyclooxygenase (COX)-2 and inducible nitric oxide (NO) synthase (iNOS) are responsive to a wide array of inflammatory stimuli, have been localized to vascular smooth muscle cells (SMCs), and are intimately linked to the progression of vascular disease, including atherosclerotic lesion formation. We and others have shown that the production and subsequent impact of COX products appear to be correlative with the status of NO synthesis. This study examined the impact of inflammation-driven NO production on COX-2 expression in SMCs. Concurrent stimulation of quiescent rat aortic SMCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma increased COX-2, iNOS, and nitrite production. Pharmacological inhibition of NO synthase (N(G)-monomethyl-l-arginine) concentration- and time-dependently magnified LPS + IFN-gamma-mediated COX-2 mRNA and protein induction in a cGMP-independent manner. COX-2 induction was associated with activation of the ERK, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. Interestingly, NO synthase inhibition enhanced ERK, p38, and to a lesser extent JNK phosphorylation but suppressed MAPK phosphatase (MKP)-1 induction in response to LPS + IFN-gamma. Similarly, the exposure of SMCs from iNOS(-/-) mice to LPS + IFN-gamma produced an enhancement of COX-2 induction, p38, and JNK phosphorylation and an attenuated upregulation of MKP-1 versus their wild-type counterparts. Taken together, our data indicate that NO, in part derived from iNOS, negatively regulates the immediate early induction of COX-2 in response to inflammatory stimuli.

High-fat diet induces lung remodeling in ApoE-deficient mice: an association with an increase in circulatory and lung inflammatory factors

Hypercholesterolemia is increasingly considered the basis for not only cardiovascular pathologies but also several complications affecting other organs such as lungs. In this study, we examined the effect of hypercholesterolemia on lung integrity using a mouse model (ApoE−/−) of high-fat (HF) diet-induced atherosclerosis. A 12-week HF diet regimen induced systemic production of TNF-α, IFN-γ, GMC-SF, RANTES, IL-1α, IL-2 and IL-12 with TNF-α as the predominant cytokine in ApoE−/− mice. Concomitantly, TNF-α, IFN-γ and MIP-1α were detected in brochoalveolar lavage (BAL) fluids of these mice, coinciding with lung inflammation consisting primarily of monocytes/macrophages. Such lung inflammation correlated with marked collagen deposition and an increase in matrix metalloproteinase-9 activity in ApoE−/−mice without mucus production. Although TGF-β1 was undetectable in the BAL fluid of ApoE−/− mice on HF diet, it showed a much wider tissue distribution compared with that of control animals. Direct exposure of smooth muscle cells to oxidized-LDL, in vitro, induced a time-dependent expression of TNF-α. Direct intratracheal TNF-α-administration induced a lung inflammation pattern in wild-type mice that was strikingly similar to that induced by HF diet in ApoE−/− mice. TNF-α administration induced expression of several factors known to be critically involved in lung remodeling, such as MCP-1, IL-1β, TGF-β1, adhesion molecules, collagen type-I and TNF-α itself in the lungs of treated mice. These results suggest that hypercholesterolemia may promote chronic inflammatory conditions in lungs that are conducive to lung remodeling potentially through TNF-α-mediated processes.

Long-term high glucose concentration influences Akt, ERK1/2, and PTP1B protein expression in human aortic smooth muscle cells

Hyperglycemia stimulates a plethora of intracellular signaling pathways within the cells of the vascular wall resulting in dysfunction-associated pathologies. Most of the studies reported so far explored the effect of rather short-time exposure of smooth muscle cells to high glucose concentrations. To mimic situation in Type 2 diabetes in which vascular wall is constantly exposed to circulating hyperglycemia, we report here the long-term (7 days) effect of high glucose concentration on human media artery smooth muscle cells. This consists in up-regulation of PTP1B protein expression, down-regulation of basal Akt phosphorylation, and elevation of basal ERK1/2 activation. Acute stimulation of cells in high glucose with insulin down-regulated PTP1B expression, slightly decreased ERK1/2 activity, and activated Akt, whereas oxidative stress up-regulated Akt and ERK1/2 phosphorylation. In conclusion, long-term high glucose and acute oxidative stress and insulin stimulation imbalance the expression of activated kinases Akt and ERK1/2 and of dephosphorylating PTP1B in the insulin signaling pathway.

Inducible nitric oxide synthase contributes to MAPK‐phosphatase induction and abundance of cyclooxygenase‐2 expression in an inflammatory setting

Cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) are responsive to inflammatory stimuli and have been localized to vascular smooth muscle cells (SMCs) in pathophysiological settings, e.g. atherosclerosis. Furthermore, the production of, and subsequent impact of COX products appear to be correlative with the status of NO synthesis and bioavailability. This study examined the role of inflammation‐driven NO synthesis on COX‐2 expression in rat aortic SMCs. Concurrent stimulation of quiescent SMCs with lipopolysaccharide (LPS) and interferon (IFN)‐γ induced iNOS and COX‐2 expression maximally at 2‐4hr. Inhibition of NO synthesis (LNMMA) significantly reduced nitrite (140±8 vs. 19±7 nmol/mg protein) and enhanced LPS+IFN‐γ‐mediated COX‐2 mRNA (156‐ vs. 43‐fold increase) and protein expression (5‐fold) at 30 and 60min. COX‐2 induction was associated with activation of the ERK, p38 and JNK pathways. Importantly, LNMMA enhanced ERK and p38 phosphorylation, and suppressed MAPK‐phosphatase (MKP)‐1 induction. Consistent with these data, exposure of SMCs from iNOS‐/‐ mice to LPS+IFN‐γ produced a more robust induction (p&lt;0.05) of COX‐2 and a dramatically attenuated upregulation of MKP‐1 vs. WT. These findings provide pharmacological and genetic evidence implicating an inhibitory influence of iNOS‐derived species on phosphorylation cascades associated with COX‐2‐induction in SMCs.

Glucose-Oxidized Low-Density Lipoproteins Enhance Insulin-Like Growth Factor I-Stimulated Smooth Muscle Cell Proliferation by Inhibiting Integrin-Associated Protein Cleavage

Prior published reports have demonstrated that glucose-oxidized low-density lipoproteins (g-OxLDL) enhance the proliferative response of vascular smooth muscle cells (SMC) to IGF-I. Our previous studies have determined that the regulation of cleavage of integrin-associated protein (IAP) by matrix-metalloprotease-2 (MMP-2) in diabetic mice in response to hyperglycemia is a key regulator of the response of SMC to IGF-I. Because chronic hyperglycemia enhances glucose-induced LDL oxidation, these studies were conducted to determine whether g-OxLDL modulates the response of SMC to IGF-I by regulating MMP-2-mediated cleavage of IAP. We determined that exposure of SMC to g-OxLDL, but not native LDL, was sufficient to facilitate an increase in cell proliferation in response to IGF-I. Exposure to an anti-CD36 antibody, which has been shown to inhibit g-OxLDL-mediated signaling, inhibited the effects of g-OxLDL on IGF-I-stimulated SMC proliferation. The effect of g-OxLDL could be attributed, in part, to an associated decrease in proteolytic cleavage of IAP leading to increase in the basal association between IAP and Src homology 2 domain-containing protein tyrosine phosphatase substrate-1, which is required for IGF-I-stimulated proliferation. The inhibitory effect of g-OxLDL on IAP cleavage appeared to be due to its ability to decrease the amount of activated MMP-2, the protease responsible for IAP cleavage. In conclusion, these data provide a molecular mechanism to explain previous studies that have reported an enhancing effect of g-OxLDL on IGF-I-stimulated SMC proliferation.

Elevated globotriaosylsphingosine as a hallmark of Fabry disease

Fabry disease is an X-linked lysosomal storage disease caused bydeficiency of -galactosidase A that affects males and showsdiseaseexpressioninheterozygotes.Thecharacteristicprogressiverenal insufficiency, cardiac involvement, and neuropathology usu-ally are ascribed to globotriaosylceramide accumulation in theendothelium. However, no direct correlation exists between lipidstorageandclinicalmanifestations,andtreatmentofpatientswithrecombinant enzymes does not reverse several key signs despiteclearanceoflipidfromtheendothelium.Wethereforeinvestigatedthe possibility that globotriaosylceramide metabolites are a miss-ing link in the pathogenesis. We report that deacylated globotri-aosylceramide, globotriaosylsphingosine, and a minor additionalmetabolitearedramaticallyincreasedinplasmaofclassicallyaffectedmale Fabry patients and plasma and tissues of Fabry mice. Plasmaglobotriaosylceramide levels are reduced by therapy. We showthat globotriaosylsphingosine is an inhibitor of -galactosidase Aactivity. Furthermore, exposure of smooth muscle cells, but notfibroblasts, to globotriaosylsphingosine at concentrations ob-served in plasma of patients promotes proliferation. The increasedintima-media thickness in Fabry patients therefore may be relatedto the presence of this metabolite. Our findings suggest thatmeasurement of circulating globotriaosylsphingosine will be use-ful to monitor Fabry disease and may contribute to a betterunderstanding of the disorder.

Phospholipase D signaling in serotonin-induced mitogenesis of pulmonary artery smooth muscle cells

We have previously reported the participation of mitogen-activated protein, Rho, and phosphoinositide-3 (PI3) kinases in separate pathways in serotonin (5-HT)-induced proliferation of pulmonary artery smooth muscle cells (SMCs). In this study, we investigated the possible participation of phospholipase D (PLD) and phosphatidic acid (PA) in this growth process. 5-HT stimulated a time-dependent increase in [(3)H]phosphatidylbutanol and PA generation. Exposure of SMCs to 1-butanol or overexpression of an inactive mutant of human PLD1R898R blocked 5-HT-induced proliferation. Furthermore, 1-butanol inhibited 5-HT activation of S6K1 and S6 protein, downstream effectors of mammalian target of rapamycin (mTOR), by 80 and 72%, respectively, and partially blocked activation of extracellular signal-regulated kinase (ERK) by 30% but had no effect on other associated signaling pathways. Exogenous PA caused cellular proliferation and revitalized cyclin D1 expression by 5-HT of the 1-butanol-treated cells. PA also reproduced activations by 5-HT of mTOR, S6K1, and ERK. Transfection with inactive human PLD1 reduced 5-HT-induced activation of S6K1 by approximately 50%. Inhibition of 5-HT receptor 2A (R 2A) with ketaserin blocked PLD activation by 5-HT. Inhibition with PI3-kinase inhibitor failed to block either activation of PLD by 5-HT or PA-dependent S6K1 phosphorylation. Taken together, these results indicate that ligation of the 5-HTR 2A by 5-HT initiates PLD activation in SMCs, and that its product, PA, is an early signaling molecule in 5-HT-induced pulmonary artery SMC proliferation. Signaling by PA produces its downstream effects primarily through the mTOR/S6K1 pathway and to a lesser extent through the ERK pathway. Hydrolysis of cell membrane lipid may be important in vascular effects of 5-HT.