Human Aortic Vascular Smooth Muscle Research Articles

Calcium Phosphate Crystals Induce Cell Death in Human Vascular Smooth Muscle Cells

Vascular calcification is associated with an increased risk of myocardial infarction; however, the mechanisms linking these 2 processes are unknown. Studies in macrophages have suggested that calcium phosphate crystals induce the release of proinflammatory cytokines; however, no studies have been performed on the effects of calcium phosphate crystals on vascular smooth muscle cell function. In the present study, we found that calcium phosphate crystals induced cell death in human aortic vascular smooth muscle cells with their potency depending on their size and composition. Calcium phosphate crystals of approximately 1 microm or less in diameter caused rapid rises in intracellular calcium concentration, an effect that was inhibited by the lysosomal proton pump inhibitor, bafilomycin A1. Bafilomycin A1 also blocked vascular smooth muscle cell death suggesting that crystal dissolution in lysosomes leads to an increase in intracellular calcium levels and subsequent cell death. These studies give novel insights into the bioactivity of calcified deposits and suggest that small calcium phosphate crystals could destabilize atherosclerotic plaques by initiating inflammation and by causing vascular smooth muscle cell death.

LPS induces inflammatory responses in human aortic vascular smooth muscle cells via Toll-like receptor 4 expression and nitric oxide production

Inflammation is an important event in the development of vascular diseases such as hypertension, atherosclerosis, and restenosis. In addition, the stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) induces the release of critical proinflammatory cytokines that activate potent immune responses. In this study, LPS was found to induce TLR4 expression and increased nitric oxide (NO) production by increasing the expression of inducible nitric oxide synthase (iNOS). Furthermore, LPS was found to induce interleukin (IL)-8 and vascular endothelial growth factor (VEGF) production, as well as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. Taken together, these results indicate that LPS induces inflammatory responses in HASMC. Moreover, NOS inhibitor (L-NAME) and anti-TLR 4 mAb reduced the LPS-induced NO, IL-8 and VEGF production and ICAM-1 expression. Additionally, TLR4 expression was reduced by NOS inhibitor. Taken together, these results indicate that LPS-induced inflammatory responses are regulated by TLR4 expression and NO production.

Cocoa procyanidins inhibit expression and activation of MMP-2 in vascular smooth muscle cells by direct inhibition of MEK and MT1-MMP activities

Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC. Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 microg/mL and 5 microM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1). Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.

Glucose lowers the threshold for human aortic vascular smooth muscle cell migration: inhibition by protein phosphatase-2A

Atherosclerosis, which occurs prematurely in individuals with diabetes, incorporates vascular smooth muscle cell (VSMC) chemotaxis. Glucose, through protein kinase C-beta(II) signalling, increases chemotaxis to low concentrations of platelet-derived growth factor (PDGF)-BB. In VSMC, a biphasic response in PDGF-beta receptor (PDGF-betaR) level occurs as PDGF-BB concentrations increase. The purpose of this study was to determine whether increased concentrations of PDGF-BB and raised glucose level had a modulatory effect on the mitogen-activated protein kinase/extracellular-regulated protein kinase pathway, control of PDGF-betaR level and chemotaxis. Cultured aortic VSMC, exposed to normal glucose (NG) (5 mmol/l) or high glucose (HG) (25 mmol/l) in the presence of PDGF-BB, were assessed for migration (chemotaxis chamber) or else extracted and immunoblotted. At concentrations of PDGF-BB <540 pmol/l, HG caused an increase in the level of PDGF-betaR in VSMC (immunoblotting) versus NG, an effect that was abrogated by inhibition of aldose reductase or protein kinase C-beta(II). At higher concentrations of PDGF-BB (>540 pmol/l) in HG, receptor level was reduced but in the presence of aldose reductase or protein kinase C-beta(II) inhibitors the receptor levels increased. It is known that phosphatases may be activated at high concentrations of growth factors. At high concentrations of PDGF-BB, the protein phosphatase (PP)2A inhibitor, endothall, caused an increase in PDGF-betaR levels and a loss of biphasicity in receptor levels in HG. At higher concentrations of PDGF-BB in HG, the chemoattractant effect of PDGF-BB was lost (chemotaxis chamber). Under these conditions inhibition of PP2A was associated with a restoration of chemotaxis to high concentrations of PDGF-BB. The biphasic response in PDGF-betaR level and in chemotaxis to PDGF-BB in HG is due to PP2A activation.

The C type natriuretic peptide receptor associates with AHNAK in human aortic vascular smooth muscle cells

We recently identified AHNAK as a binding partner for the C type natriuretic peptide receptor (NPR‐C) in normal rat gastric mucosa (RGM1) cells. To investigate the role of AHNAK in NPR‐C function in different cell types and species we examined the expression and interaction of both proteins in multiple cell lines. Antibodies to NPR subtypes and AHNAK domains were used to examine expression by immunoblotting, colocalization by immunohistochemistry, and interaction by immunoprecipitation in multiple mammalian cell lines. Mass spectrometry was used to confirm the identity of the proteins from the immunoprecipitation studies. GST fusion proteins of the N‐terminal, internal repeating, and C‐terminal domains of AHNAK were purified and used to perform GST pulldown assays to verify the association of the two proteins and determine specificity. Coding sequences of human NPR‐C and AHNAK were subcloned into mammalian two hybrid expression vectors to ascertain the interaction in situ. NPR‐C was found to be the most abundantly expressed NPR subtype and AHNAK was found to also be expressed in human aortic vascular smooth muscle cells (AoVSMCs). NPR‐C and AHNAK colocalized to the plasma membrane of AoVSMCs. NPR‐C bound to AHNAK isoform 1 in vitro. The interaction was attributed to a single AHNAK domain. This research was supported by an American Heart Association Predoctoral fellowship and a Veterans Affairs Merit grant.

Potential Role of CYLD (Cylindromatosis) as a Deubiquitinating Enzyme in Vascular Cells

Data from several studies suggest that the ubiquitin-proteasome system may play a role in the progression of atherosclerosis. Here, we examined the potential role of the deubiquitinating enzyme CYLD (cylindromatosis), mutation of which has been reported to cause familial cylindromatosis. Northern blot analysis revealed expression of CYLD mRNA in the aorta, as well as in cultured human aortic endothelial cells (ECs) and vascular smooth muscle cells. Treatment with recombinant tumor necrosis factor (TNF)-alpha significantly increased CYLD expression in ECs and vascular smooth muscle cells. Immunostaining showed CYLD expression in atherosclerotic lesions from human carotid arteries and up-regulation of CYLD expression in the neointima of rat carotid arteries after balloon injury. Overexpression of CYLD in ECs resulted in inhibition of TNF-alpha-induced nuclear factor-kappaB activity through deubiquitination of TNFR-associated factor 2 (TRAF2), whereas overexpression of catalytically inactive CYLD had no effect. CYLD overexpression also inhibited expression of cyclin D1 and activation of the E2F pathway through deubiquitination of the upstream molecule Bcl-3 and inhibition of its translocation into the nucleus. Overexpressed CYLD also significantly inhibited cell viability. Furthermore, overexpression of CYLD in rat balloon-injured carotid artery attenuated neointimal formation through inactivation of nuclear factor-kappaB and E2F. In conclusion, these data demonstrate that the deubiquitinating enzyme CYLD may inhibit inflammation and proliferation in vascular cells and may represent a novel target for the treatment or prevention of atherosclerosis.

QS154. Effect of Estrogen and Progesterone on Matrix Metalloproteinase Gene Regulation in Human Aortic Vascular Smooth Muscle Cells

QS154. Effect of Estrogen and Progesterone on Matrix Metalloproteinase Gene Regulation in Human Aortic Vascular Smooth Muscle Cells

A Novel Histone Deacetylase Inhibitor Reduces Abdominal Aortic Aneurysm Formation in Angiotensin II-Infused Apolipoprotein E-Deficient Mice

Background/Aims: Aberrant expression of components of the matrix metalloproteinase (MMP) enzyme system is implicated in abdominal aortic aneurysm (AAA) formation. We aimed to investigate the influence of a novel histone deacetylase (HDAC) inhibitor (HDACi) metacept-1 (MCT-1), previously documented to reduce MMP expression, on HDAC activity and MMP expression in aortic smooth muscle cells and the in vivo incidence of AAAs. Methods: Western blot and gelatin zymography were used to determine HDAC activity and MMP-2 expression and activity in rat (rVSMCs) and human aortic vascular smooth muscle cells (hVSMCs) in vitro. In vivo AAAs were generated using apolipoprotein E-deficient mice infused with angiotensin (Ang) II. Immunohistochemistry detected MMP-2 and -9 expression in AAA tissue samples. Results: In vitro, MCT-1 inhibited HDAC activity in rVSMCs, and MMP-2 expression and proteolytic activity in hVSMCs.In vivo, Ang II treatment alone exhibited an AAA incidence of 84%. Doxycycline decreased the incidence of AAAs to 50%. Importantly, MCT-1 reduced AAA incidence to approximately 44%. MMP-2 and -9 immunoreactivity was reduced in MCT-1-treated aortic tissue. Conclusion: The novel HDACi MCT-1 inhibits MMP expressionand AAA incidence suggesting this compound may warrant further investigation in the context of AAA biology.

Identification of two GTP‐independent alternatively spliced forms of tissue transglutaminase in human leukocytes, vascular smooth muscle, and endothelial cells

Tissue transglutaminase (tTG) is a multifunctional enzyme with transglutaminase crosslinking (TGase), GTP binding, and hydrolysis activities that play a role in many different disorders. We identified, characterized, and investigated the function and stability of two alternatively spliced forms of tTG using biochemical, cellular, and molecular biological approaches. Using a human aortic vascular smooth muscle cells (VSMC) cDNA library, we identified two cDNAs encoding C-terminal truncated forms, tTG(V1) and tTG(V2). tTG(V1,2) mRNAs were synthesized by a rare splicing event using alternate splice sites within exons 12 and 13 of the tTG gene, respectively. Quantitative PCR and immunoblotting demonstrated that there was unique expression and localization of tTG(V1,2) compared with tTG in human umbilical vein endothelial cells (HUVECs), VSMC, and leukocytes. The loss of C-terminal 52 amino acid residues (AAs) in tTG(V1,2) altered GTP binding, enhanced GTP hydrolysis, rendered the variants insensitive to GTP inhibition, and resulted in <10% residual Ca(+2)-dependent TGase activity. Transfection in HEK293 demonstrated a 28- and 5-fold reduction in the expression of tTG(V1) and tTG(V2), respectively, demonstrating that the C-terminal GTP-binding domain is important in stabilizing and promoting the half-life of tTG. The altered affinity for GTP allowed tTG(V1,2) to exhibit enhanced TGase activity when there is a transient increase in Ca(+2) levels. The abundance of tTG(V1,2) and its distinct intracellular expression patterns in human vascular cells and leukocytes indicate these isoforms likely have unique physiological functions.

Systemic Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Delivery Shows Antiatherosclerotic Activity in Apolipoprotein E–Null Diabetic Mice

Although in vitro studies have suggested that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might be involved in vascular biology, its potential role in the pathogenesis and/or treatment of atherosclerosis has not been investigated. Both recombinant human TRAIL and an adeno-associated virus vector expressing human TRAIL were used to deliver TRAIL in apolipoprotein E (apoE)-null mice in which diabetes mellitus was induced by destruction of islet cells with streptozotocin. Diabetes in apoE-null mice was associated with a significant increase in atherosclerotic plaque area and complexity in the aorta as assessed by a marked increase in interstitial collagen, cellular proliferation, and macrophage infiltration and a focal loss of endothelial coverage. Repeated intraperitoneal injections of recombinant human TRAIL and a single intravenous injection of adeno-associated virus-human TRAIL significantly attenuated the development of atherosclerotic plaques in apoE-null animals. TRAIL also markedly affected the cellular composition of plaque lesions by inducing apoptosis of infiltrating macrophages and increasing the vascular smooth muscle cell content. Moreover, TRAIL promoted the in vitro migration of cultured human aortic vascular smooth muscle cells but not of monocytes or macrophages. Conversely, TRAIL selectively induced apoptosis of human cultured macrophages but not of vascular smooth muscle cells. Overall, data from the present study indicate that atherosclerosis in diabetic apoE-null mice is ameliorated by systemic TRAIL administration and that adeno-associated virus-mediated TRAIL gene delivery might represent an innovative method for the therapy of diabetic vascular diseases.

Human Vascular Smooth Muscle Cells Express a Urate Transporter

An elevated serum uric acid is associated with the development of hypertension and renal disease. Renal regulation of urate excretion is largely controlled by URAT1 (SLC22A12), a member of the organic anion transporter superfamily. This study reports the specific expression of URAT1 on human aortic vascular smooth muscle cells, as assessed by reverse transcription-PCR and Western blot analysis. Expression of URAT1 was localized to the cell membrane. Evidence that the URAT1 transporter was functional was provided by the finding that uptake of 14C-urate was significantly inhibited in the presence of probenecid, an organic anion transporter inhibitor. It is proposed that URAT1 may provide a mechanism by which uric acid enters the human vascular smooth muscle cell, a finding that may be relevant to the role of uric acid in cardiovascular disease.

Sodium Azide in Commercially Available C-Reactive Protein Preparations Does Not Influence Matrix Metalloproteinase-2 Synthesis and Release in Cultured Human Aortic Vascular Smooth Muscle Cells

Detection of circulating concentrations of the acute-phase reactant C-reactive protein (CRP), which is synthesized in response to proinflammatory cytokines, is a relevant tool for identifying the involvement of low-grade inflammation in atherosclerosis and for predicting future atherothrombotic events (1). Whether CRP is only a marker or is also an active player in atherosclerotic injury is a matter of intense debate (2). CRP is present in atherosclerotic lesions (3) and can contribute directly to atherothrombosis (4). In particular, CRP induces expression of proatherogenetic molecules in endothelial cells and promotes LDL uptake by macrophages (4). We recently observed that CRP increases synthesis and secretion of matrix metalloproteinase 2 (MMP-2) from cultured human vascular smooth muscle cells (hVSMCs) (5), a mechanism potentially involved in plaque destabilization. Recently, however, the reliability of results concerning CRP obtained in vitro has been …

Internalization and Recycling of the Human Prostacyclin Receptor Is Modulated through Its Isoprenylation-dependent Interaction with the δ Subunit of cGMP Phosphodiesterase 6

Prostacyclin, the major cyclooxygenase-derived product of arachidonic acid formed in the vasculature, mediates its potent anti-thrombotic and anti-proliferative effects through its G protein-coupled receptor (GPCR) termed the IP. Unlike many GPCRs, agonist-induced internalization of the IP occurs in an arrestin/GPCR kinase-independent manner. However, deletion of the IP COOH-terminal region prevented internalization suggesting that protein interactions at this region are involved in IP regulation. Using the COOH-terminal region of IP as bait we identified the delta subunit of cGMP phosphodiesterase 6 (PDE6delta) as a novel hIP-interacting protein in two independent yeast two-hybrid screens. Interaction of IP and PDE6delta was confirmed by co-immunoprecipitation in HEK293 cells, and in HEPG2 cells, which endogenously express neither IP nor PDE6delta. IP isoprenylation was critical for this interaction, as PDE6delta was unable to associate with an isoprenylation-deficient mutant IP (IPSSLC). PDE6delta overexpression altered the temporal pattern of agonist-induced internalization of IP, but not IPSSLC, in HEPG2 cells, increasing initial internalization but facilitating the return of IP to the cell surface despite the continued presence of agonist. Depletion of PDE6delta using short interfering RNA abolished cicaprost-induced IP internalization in human aortic smooth muscle cells. Recycling of IP, but not IPSSLC, upon agonist removal was facilitated by overexpression of PDE6delta. Thus PDE6delta interacts specifically with IP to modulate receptor trafficking.

Manipulating Glutathione-S-Transferases May Prevent the Development of Tolerance to Nitroglycerin

Tolerance to clinically important organic nitrates such as nitroglycerin (NTG) has been experimentally related to endothelial dysfunction and vascular oxidative stress. Anti-oxidant enzymes such as the glutathione-S-transferases GSTs) could potentially play a protective role in NTG tolerance. Our previous work showed that an alpha-class glutathione-S-transferase (GSTA4-4) defends against oxidative damage in the vascular wall; therefore, we asked whether overexpression of GSTA4-4 in endothelial cells and smooth muscle cells might alter the development of tolerance to NTG. Stable transfections of mouse pancreatic islet endothelial cells (MS1) with cDNA of mGSTA4-4, and human fetal aortic vascular smooth muscle cells (FLTR) with cDNA of hGSTA4-4 were established. MTT cytotoxicity, apoptosis, nitric oxide (NO) synthases, both endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) and cyclic guanosine mono-phosphate (cGMP) were measured. Endothelial cells overexpressing mGSTA4-4, and smooth muscle cells overexpressing hGSTA4-4 were more resistant to cytotoxic injury by NTG, assessed at 24 h (p < 0.05). In both endothelial and smooth muscle cells, NTG-induced apoptosis was inhibited by GST overexpression. Following dosing in a relevant tolerance-inducing NTG protocol, we found that GSTA4-4-overexpressing cells demonstrated significant downregulation of NOS enzymes; NO release, unchanged by the tolerance protocol in both wild-type and vector-transfected cells, was augmented in GST-overexpressing cells (p < 0.01); cGMP levels in control cells fell, whereas it rose in GSTA4-4-overexpressing cells (p < 0.05). Our results demonstrate that overexpression of GST isozymes can protect endothelial cells and smooth muscle cells against oxidative stress associated with NTG, and markedly alter cellular responses to repeated doses, or tolerance. By manipulating GSTs, physiological tolerance to NTG may be diminished or eliminated.

Attenuation of Vascular Smooth Muscle Cell Proliferation by 1-Cyclohexyl-3-dodecyl Urea Is Independent of Soluble Epoxide Hydrolase Inhibition

Epoxyeicosatrienoic acid(s) (EET) have variable hemodynamic, anti-inflammatory, and growth regulatory effects, and inhibitors of their regulatory enzyme, soluble epoxide hydrolase (sEH), can mimic these effects. For this reason, sEH inhibitors are being studied as potential pharmaceuticals for the treatment of hypertension, atherosclerosis, and inflammatory diseases. We now show that a highly selective urea-based sEH inhibitor 1-cyclohexyl-3-dodecyl urea (CDU) attenuates human aortic vascular smooth muscle (HVSM) cell proliferation independently of any effect on sEH. CDU also inhibits endothelial cells when stimulated with basic fibroblast growth factor or serum. In addition, we demonstrate that EET, as well as several newer generation sEH inhibitors and a urea-based weak sEH inhibitor, do not affect proliferation in HVSM cells. Structure-activity relationships demonstrate that the addition of an acid group to the dodecyl carbon chain, changing the cyclohexyl group to an adamantyl group, and shortening the carbon chain to two carbons all abolish the antiproliferative effect. Our finding that a highly selective urea-based inhibitor of sEH can alter biology independently of its putative target enzyme suggests that there may be other useful properties of this class of compounds unrelated to their influence on epoxyeicosanoids. In addition, our results show that caution should be used when attempting to infer conclusions of EET biology based solely on the effects these inhibitors in tissue culture models, especially when used at micromolar concentrations.

Rapamycin, but Not FK-506, Increases Endothelial Tissue Factor Expression

Drugs released from stents affect the biology of vascular cells. We examined the effect of rapamycin and FK-506 on tissue factor (TF) expression in human aortic endothelial cells (HAECs) and vascular smooth muscle cells (HAVSMCs). Rapamycin enhanced thrombin- and tumor necrosis factor (TNF)-alpha-induced endothelial TF expression in a concentration-dependent manner. The maximal increase was 2.5-fold more pronounced than that by thrombin or TNF-alpha alone and was paralleled by a 1.4-fold higher TF surface activity compared with thrombin alone. Rapamycin by itself increased basal TF levels by 40%. In HAVSMCs, rapamycin did not affect thrombin- or TNF-alpha-induced TF expression. In contrast to rapamycin, FK-506 did not enhance thrombin- or TNF-alpha-induced endothelial TF expression. Thrombin induced a transient dephosphorylation of the mammalian target of rapamycin downstream target p70S6 kinase. Rapamycin completely abrogated p70S6 kinase phosphorylation, but FK-506 did not. FK-506 antagonized the effect of rapamycin on thrombin-induced TF expression. Rapamycin did not alter the pattern of p38, extracellular signal-regulated kinase, or c-Jun NH2-terminal kinase phosphorylation. Real-time polymerase chain reaction analysis revealed that rapamycin had no influence on thrombin-induced TF mRNA levels for up to 2 hours but led to an additional increase after 3 and 5 hours. Rapamycin, but not FK-506, enhances TF expression in HAECs but not in HAVSMCs. This effect requires binding to FK binding protein-12, is mediated through inhibition of the mammalian target of rapamycin, and partly occurs at the posttranscriptional level. These findings may be clinically relevant for patients receiving drug-eluting stents, particularly when antithrombotic drugs are withdrawn or ineffective, and may open novel perspectives for the design of such stents.

Nicotinic and PDGF‐receptor function are essential for nicotine‐stimulated mitogenesis in human vascular smooth muscle cells

Cigarette smoking is implicated in the formation of occlusive vascular diseases. Nicotine's role in this process is incompletely understood. Nicotine's effect on human aortic vascular smooth muscle cells (HaVSMC) and the role of the nicotinic receptor (nAChR), platelet-derived growth factor (PDGF), and the PDGF-receptor (PDGF-R) in this response were studied. Nicotine's mitogenic effect was characterized by three methods: thymidine incorporation, a viability/proliferation assay based on metabolic conversion of tetrazolium salt to formazan dye and cell counting. Nicotine administration (10(-6) M) stimulated cell cycle entry marked by increased DNA synthesis, PCNA and cyclin D1 production, and increased cell division. Nicotinic receptor blockade with d-tubocurarine, a nicotinic AchR blocker, decreased nicotine-induced DNA synthesis, and cell division (0.33 +/- 0.04, 0.77 +/- 0.31-fold decrease, respectively). Nicotine increased cellular PDGF-BB transcript levels and protein release (ELISA: 1.6 +/- 0.5-fold increase) but not PDGF-AA or PDGF-AB release. Nicotine increased PDGFbeta-receptor protein content. PDGF inactivation with anti-PDGF antibody abolished nicotine-induced DNA synthesis (1.9 +/- 0.08-fold decrease). PDGF-R blockade with the PDGF-R antagonist tyrphostin AG 1295 decreased nicotine-induced DNA synthesis and cell division (0.25 +/- 0.01, 0.44 +/- 0.2-fold decrease, respectively). PDGF-R blockade reversed nicotine-stimulated increases in PDGF release, PDGF-BB transcripts, and PDGF-receptor levels (0.68 +/- 0.34; 0.46 +/- 0.01; 0.28 +/- 0.01-fold decrease, respectively). In conclusion, nicotine-mediated activation of nAChRs increases PDGF-BB transcription and protein production as well as PDGF beta-receptor levels. PDGF-BB/PDGF-R interaction is vital in nicotine's mitogenic actions on human aortic smooth muscle cells.

Histamine Induces Tissue Factor Expression

Histamine can induce coronary vasospasm, leading to variant angina and acute myocardial infarction. However, the role of histamine in thrombus formation is ill defined. Hence, this study investigates whether histamine induces tissue factor (TF) expression in vascular cells. Histamine (10(-8) to 10(-5) mol/L) induced TF expression in a concentration-dependent manner in human aortic endothelial and vascular smooth muscle cells, whereas TF pathway inhibitor expression remained unaffected. RT-PCR and Northern blotting revealed that histamine stimulated TF mRNA transcription, peaking at 1 hour. Protein expression increased 18-fold (P<0.02) with a maximum at 5 hours, which was paralleled by a 4-fold augmentation in surface activity (P<0.01). These effects were completely prevented by pretreatment with the H1 receptor antagonists mepyramine (P<0.0001), chlorpheniramine, and diphenhydramine but not the H2 receptor antagonist cimetidine (P=NS). Histamine induced a time-dependent, H1 receptor-mediated activation of p38 MAP kinase (p38), p44/42 MAP kinase (ERK), and c-jun terminal NH2 kinase (JNK). Blocking of p38, ERK, or JNK with SB203580 (P<0.0001), PD98059 (P<0.0001), or SP600125 (P<0.0001), respectively, impaired histamine-induced TF expression in a concentration-dependent manner. In contrast, histamine-stimulated TF expression was increased by phosphatidylinositol 3-kinase inhibition with LY294002 or wortmannin, whereas it was not affected by Rho-kinase inhibition with Y-27632 or hydroxyfasudil. Histamine induces expression of TF, but not TF pathway inhibitor, in vascular cells via activation of the H1, but not H2, receptor. This effect is mediated by the MAP kinases p38, ERK, and JNK. This observation may open novel perspectives in the treatment of variant angina and acute coronary syndromes.

Association of Osteoprotegerin With Human Abdominal Aortic Aneurysm Progression

Abdominal aortic aneurysm (AAA) is characterized by destruction of the arterial media associated with loss of vascular smooth muscle cells, infiltration of mononuclear cells, and high concentration of metalloproteinases (MMPs) and cytokines. Osteoprotegerin (OPG) has recently been identified in atherosclerosis. The presence and functional importance of OPG in human AAA was investigated. In 146 men with small AAA followed up by ultrasound for 3 years, serum OPG was weakly correlated with aneurysm growth rate. Western analysis showed 3-, 8-, and 12-fold-greater OPG concentrations in human AAA biopsies compared with biopsies of atherosclerotic narrowed aorta (1.4+/-0.1 versus 0.5+/-0.1 ng/mg tissue; P=0.002), postmortem nondiseased abdominal aorta (1.4+/-0.1 versus 0.2+/-0.1 ng/mg tissue; P<0.001), and nondiseased thoracic aorta (1.4+/-0.1 versus 0.1+/-0.06 ng/mg tissue; P<0.001). Healthy human aortic vascular smooth muscle cells incubated with recombinant human (rh)OPG (0 to 20 ng rhOPG/10(5) cells per 1 mL per 24 hours) developed an aneurysmal phenotype defined by impaired cell proliferation (P<0.001), increased apoptosis (P<0.01), and increased MMP-9 (92 kDa) expression (P<0.001). Incubation of monocytic THP-1 cells with 1 ng rhOPG/10(5) cells per 1 mL per 24 hours induced a 2-fold increase in MMP-9 expression (P<0.001), a 1.5-fold increase in MMP-2 activity (P=0.005), and a 2-fold stimulation of IL-6 production in these cells (P=0.02). Finally, secretion of OPG from human AAA explant was abrogated by treatment with the angiotensin II blocker irbesartan, with the reduction in secreted levels averaging 63.0+/-0.9 ng/mg tissue per 48-hour period. These findings support a role for OPG in the growth of human AAA and suggest a potential benefit for angiotensin II blockade in slowing aneurysm expansion.

Angiotensin II: A Devious Activator of Mineralocorticoid Receptor-Dependent Gene Expression

See related article, pages 643–650 In this issue of Circulation Research Jaffe and Mendelsohn1 show that angiotensin II (Ang II) directly activates the mineralocorticoid receptor (MR). Using cultured human coronary and aortic vascular smooth muscle cells (VSMC) they show strong evidence for expression of both the MR and the cortisol inactivating enzyme 11-β-hydroxysteroid-dehydrogenase-2 necessary for mineralocorticoid action. Importantly they show that Ang II activation of MR is independent of aldosterone generation by VSMC, suggesting a direct effect. Finally they used microarray analysis to define genes induced by aldosterone-dependent MR transcription. The microarray results yielded an “All Star” parade of candidates for vascular aging as they included genes involved in vascular fibrosis, inflammation and calcification. These data provide further support for the evolving role of the MR as a signal effector for Ang II and provide a new MR-dependent mechanism by which Ang II promotes cardiovascular disease. The links between Ang II and aldosterone have become increasingly numerous over the past 10 years. Because Ang II stimulates the synthesis and release of aldosterone, it has been suggested that vascular effects of Ang II are mediated in part by aldosterone. Support for this concept includes findings that Ang II receptors are up-regulated by aldosterone,2 data that spironolactone decreased cardiac hypertrophy, inflammation and fibrosis induced by Ang II,3 and …