Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.     

垂体腺苷酸环化酶激活肽对血管壁细胞成分的作用及其机制的实验研究——PACAP与动脉粥样硬化关系的探讨

以培养的猪主动脉内皮细胞（ＥＣ）和肺动脉平滑肌细胞（ＳＭＣ）为主要实验对象,研究了垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）对正常及高脂环境下培养的ＥＣ、ＳＭＣ形态和功能的影响,并对其作用机制进行了初步探讨。结果显示：ＰＡＣＡＰ可部分对抗高脂因素造成的ＥＣ、ＳＭＣ形态的损伤;能提高ＥＣ抗动脉粥样硬化（ＡＳ）物质的产生;抑制ＳＭＣ的增殖;并具有抗脂质过氧化作用。本研究表明,ＰＡＣＡＰ对ＥＣ、ＳＭＣ具有一定程度的细胞保护作用,因此提示ＰＡＣＡＰ可能具有一定的抗ＡＳ作用。     

垂体腺苷酸环化酶激活肽对血管壁细胞成分的作用及其机制的实验研究

以培养的猪主动脉内皮细胞（ＥＣ）和肺动脉平滑肌细胞（ＳＭＣ）为主要实验对象, 垂体腺苷酸环化酶激活肽（ＰＡＣＡＰ）对正常及高脂环境下培养的ＥＣ、ＳＭＣ形态和功能的影响,并对其作用机制进行了初步探讨。结果显示：ＰＡＣＡＰ可部分对抗高脂因素造砀ＥＣ、ＳＭＣ形态的损伤;能提高ＥＣ抗动脉粥样硬化（ＡＳ）物质的产生;抑制ＳＭＣ的增殖;并具有抗脂质氧化作用。本研究表明,ＰＡＣＡＰ对ＥＣ、ＳＭＣ具有一定程度的细     

Heparanase and platelet factor-4 induce smooth muscle cell proliferation and migration via bFGF release from the ECM

Basic fibroblast growth factor (bFGF) has been shown to play an instrumental role in the cascade of events leading to restenosis; however, the mechanisms of bFGF activation following vascular injury have remained elusive. We have demonstrated that heparanase and platelet factor-4 (PF4), released from activated platelets at the site of injury, liberate bFGF from the extracellular matrix (ECM) of vascular smooth muscle cells (SMC), resulting in the induction of SMC proliferation and migration. Increases in proliferation and migration were inhibited by treatment with a bFGF-neutralizing antibody, suggesting that proliferation and migration in response to heparanase or PF4 are mediated by bFGF activation. When platelets were seeded on top of SMCs, degranulation products were found to release bFGF from the ECM, increasing cell proliferation and cell migration. Again, these increases in SMC proliferation and migration were inhibited by treatment with an anti-bFGF antibody. Furthermore, these increases in proliferation were completely inhibited by treatment with an anti-heparanase antibody. Platelet degranulation products, such as heparanase and PF4, may liberate bFGF from extracellular sequestration, activating the growth factor and inducing the SMC proliferation and migration that contribute to the wound healing response following vascular injury.     

Tempol therapy attenuates medial smooth muscle cell apoptosis and neointima formation after balloon catheter injury in carotid artery of diabetic rats

Accumulating data support the hypothesis that reactive oxygen species (ROS) play a critical role in the vascular complications observed in diabetes. However, the mechanisms of ROS-mediated vascular complications in diabetes are not clear. We tested the hypothesis that ROS-mediated increase in proapoptotic factor Bax expression leads to medial smooth muscle cell (SMC) apoptosis that is associated with neointima formation. We used a fructose-rich diet for 4 wk to model Type 2 diabetes in rats. SOD mimetic membrane-permeable 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempol, 1 mM) was administered in drinking water to scavenge superoxide starting 1 day before surgery and continued during the duration of the experiment. Vascular injury resulted in a significant increase in medial SMC apoptosis that was associated with neointima formation. The number of medial SMC positive for Bax immunostaining significantly increased in injured arteries compared with uninjured arteries. Superoxide scavenging by Tempol treatment inhibited both the Bax-positive index as well as the apoptotic index of medial SMC in response to vascular injury. Tempol treatment inhibited apoptotic loss of medial SMC, thus increasing their density in the injured arteries. These alterations in the media were associated with a marked decrease in neointima formation in injured arteries. We conclude that Bax expression may play an important role in vascular SMC apoptosis and, finally, that this regulatory mechanism is redox sensitive.     

Suppression of growth factor expression and human vascular smooth muscle cell growth by small interfering RNA targeting EGR-1

Smooth muscle cell (SMC) proliferation and migration are key processes that occur in the reparative response to injury after percutaneous coronary intervention and in failed bypass grafts for the treatment of atherosclerosis. In the present study, we generated novel synthetic small interfering RNA (siRNA) molecules targeting the coding region of human early growth response-1 (EGR-1) mRNA that attenuate the expression of EGR-1 and that of fibroblast growth factor-2 (FGF-2) and granulocyte-colony stimulating factor (G-CSF). These agents suppressed SMC proliferation in a dose-dependent and non-toxic manner and blocked SMC regrowth from the wound edge following mechanical injury in vitro. In contrast, the scrambled counterpart did not inhibit SMC proliferation, EGR-1 protein expression or SMC regrowth after injury. These findings demonstrate that EGR-1 siRNA can serve as inhibitors of SMC proliferation and wound repair suggesting that these agents may potentially be useful in the control of vascular proliferative disorders.     

The kinase inhibitor fasudil (HA-1077) reduces intimal hyperplasia through inhibiting migration and enhancing cell loss of vascular smooth muscle cells.

Smooth muscle cell (SMC) migration plays an important role in restenosis after angioplasty. Myosin phosphorylation is necessary for cell migration. Fasudil is an inhibitor of protein kinases, including myosin light chain kinase and Rho associated kinase, thereby inhibiting myosin phosphorylation, and it has been clinically used to prevent vasospasm following subarachnoid hemorrage. Based on these findings, we examined the anti-migrative action of fasudil. In SMC (SM-3), fasudil (1-100 microM) inhibited SMC migration in a dose-dependent manner (p < 0.001). Fasudil suppressed actin stress fiber formation dose dependently. In rabbit carotid artery, fasudil (10 mg/kg/day) markedly reduced intimal hyperplasia 14 days following balloon injury. Cell kinetic study showed that fasudil did not affect proliferation but enhanced cell loss in the media after injury. We concluded that fasudil reduced neointimal formation after balloon injury through both inhibiting migration and enhancing cell loss of medial SMC.     

YC-1 enhances the responsiveness of tolerant vascular smooth muscle to glyceryl trinitrate

A major limitation of the use of organic nitrates in cardiovascular medicine is the development of tolerance, which has been attributed, in part, to a decrease in their metabolic activation in the vascular smooth muscle cell. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was shown to potentiate vascular smooth muscle responsiveness to glyceryl trinitrate (GTN), sodium nitroprusside, and the nitric oxide donor NOC 18, in organic nitrate-naive vascular smooth muscle. We used GTN-tolerant rabbit aortic rings (RARs) to test the hypothesis that a non-vasorelaxant concentration of YC-1 enhances the ability of the prototypical organic nitrate GTN to relax vascular smooth muscle and elevate intravascular cGMP under conditions of GTN tolerance. Treatment with YC-1 (3 microM) produced a left shift of the GTN concentration-response curve and decreased the EC50 value for GTN-induced relaxation in both GTN-tolerant and non-tolerant RARs (P < 0.05). Intravascular cGMP elevation induced by GTN was enhanced in the presence of YC-1 in GTN-tolerant and non-tolerant RARs (P < 0.05). These observations indicate that YC-1, or similarly acting drugs, may be useful in overcoming the tolerance that develops during sustained GTN therapy, and that its mechanism may involve enhanced cGMP formation.     

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.     

Vascular smooth cell proliferation in perfusion culture of porcine carotid arteries

Objective of this study was to develop a novel in vitro artery culture system to study vascular smooth muscle cell (SMC) proliferation of porcine carotid arteries in response to injury, basic fibroblast growth factor (FGF2), and FGF2 conjugated with cytotoxin saporin (SAP). Perfusion-cultured porcine carotid arteries remained contractile in response to norepinephrine and relaxant to acetylcholine for up to 96 h. SMC proliferation of cultured arteries was detected by bromodeoxyuridine incorporation in both non-injured and balloon-injured arteries. In the inner layer of the vessel wall near the lumen, SMC proliferation were less than 10% in uninjured vessels, 66% in injured vessels, 80% in injured vessels with FGF2 treatment, and 5% in injured vessels with treatment of FGF2-SAP. Thus, the cultured porcine carotid arteries were viable; and the injury stimulated SMC proliferation, which was significantly enhanced by FGF2 and inhibited by FGF2-SAP.     

Tyrosine Kinase Inhibitors Inhibit Multiple Steps of the Cell Cycle of Vascular Smooth Muscle Cells

Protein tyrosine kinase (PTK) inhibitors have been reported to inhibit proliferation of vascular smooth muscle cells (SMC). To elucidate the made of this inhibition, the effects on the cell cycle of cultured vascular SMC of three PTK inhibitors with different modes of action (methyl 2,5-dihydroxyeinnamate, genistein, and herbimycin A) were studied. Rat aortic SMC were synchronized to the G0 phase of the cell cycle and then released to proceed through the cell cycle by the addition of platelet-derived growth factor (PDGF), and [³H]thymidine incorporation into DNA was measured. The three PTK inhibitors all inhibited PDGF-induced DNA synthesis in a dose-dependent fashion, with IC₅₀ values of 4.7 ± 1.4 μM for methyl 2,5.dihydroxycinnamate, 6.7 ± 2.5 μM for genistein, and 0.17 ± 0.07 μM for herbimycin A. Time course studies suggested that the agents inhibited early G1 phase but not the G0-G1 transition. The lack of effect on the G0-G1 transition was also supported by the finding that the agents did not inhibit the ligand-induced autophosphorylation of PDGF receptor nor the induction of c-fos mRNA at concentrations which were sufficient to inhibit DNA synthesis. PTK inhibitors inhibited progression of the S phase when they were added to SMC that had been arrested at the G1-S border with hydroxyurea. Methyl 2,5-dihydroxyeinnamate also blocked the M phase when it was added to SMC cultured in the presence of 10% fetal calf serum, while genistein and herbimycin A did not inhibit the M phase under the same experimental conditions. In accordance with our previous observation, methyl 2,5-dihydroxycinnamate impaired microtubule networks and formation of the mitotic spindle during the M phase. Our findings indicated that PTK inhibitors inhibit multiple steps of the vascular SMC cell cycle.     

纤维蛋白（原）在动脉粥样硬化发病中的作用及机制

在高胆固醇饮食诱发的家兔动脉粥样硬化模型上,发现ＡＳ早期即出现高纤维蛋白原血症,血浆Ｆｇ和胆固醇水平呈正相关。组织免疫化学检查发现,Ｆｇ密布于血管内皮下和平滑肌细胞间质。     

Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures

In culture, vascular smooth muscle cells (SMC) grow in a "hill-and-valley" (multilayered) pattern of organization. We have studied the growth, behavioral organization, and biosynthetic phenotype of rat aortic SMC exposed to purified platelet-derived growth regulatory molecules. We show that multilayered growth is not a constitutive feature of cultured SMC, and that beta-type transforming growth factor (TGF-beta) is the primary determinant of multilayered growth and the hill-and-valley pattern of organization diagnostic for SMC in culture. TGF-beta inhibited, in a dose-dependent manner, the serum- or platelet-derived growth factor-mediated proliferation of these cells in two-dimensional culture, but only when cells were plated at subconfluent densities. The ability of TGF-beta to inhibit SMC growth was inversely correlated to plating cell density. When SMC were plated at monolayer density (5 X 10(4) cells/cm2) to allow maximal cell-to-cell contact, TGF-beta potentiated cell growth. This differential response of SMC to TGF-beta may contribute to the hill-and-valley pattern of organization. Unlike its effect on other cell types, TGF-beta did not enhance the synthesis of fibronectin or its incorporation into the extracellular matrix. However, the synthesis of a number of other secreted proteins was altered by TGF-beta treatment. SMC treated with TGF-beta for 4 or 8 h secreted markedly enhanced amounts of an Mr 38,000-D protein doublet whose synthesis is known to be increased by heparin (another inhibitor of SMC growth), suggesting metabolic similarities between heparin- and TGF-beta-mediated SMC growth inhibition. The data suggest that TGF-beta may play an important and complex regulatory role in SMC proliferation and organization during development and after vascular injury.     

Heme oxygenase-1 protects against vascular constriction and proliferation

Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.     

Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation

We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC.     

Functional significance of human vascular smooth muscle cell-derived interleukin 1 in paracrine and autocrine regulation pathways.

Interleukin 1 (IL1), a key mediator in the cytokine network, alters many pathophysiologically important functions of blood vessel wall cells. Vascular cells, such as endothelial cells and smooth muscle cells (SMC) can themselves transcribe IL1 genes, raising the possibility that IL1 regulates blood vessel wall functions by local autocrine or paracrine mechanisms. However, IL1 lacks a recognizable signal sequence and it is still unclear how vascular cells might release IL1 or if IL1 derived from vascular cells can actually produce autocrine or paracrine effects. We explored these issues in human vascular SMC, the most numerous cell type in arteries and veins, using cultured SMC and short term organoid cultures. SMC treated with lipopolysaccharide recombinant tumor necrosis factor (recTNF), or recIL1 itself ("activated SMC") elaborated thymocyte costimulatory activity, a biological activity traditionally ascribed to IL1. However, neutralization experiments with monospecific antibodies disclosed that the more recently recognized cytokine IL6 rather than IL1 accounted for most of the soluble thymocyte costimulatory activity released by activated SMC. Using the D10S assay that distinguishes IL1 from IL6 and TNF we found that the culture supernatant of activated SMC contained little or no IL1, but that the cytosol and surface of these cells did exhibit this activity. Antiserum to recIL1 alpha inhibited stimulation of D10S cells by surface-associated IL1 of activated SMC, while treatment with acid to elute receptor- or nonspecifically bound IL1 did not abrogate this D10S proliferation. Short term organoid cultures of both normal veins and human arteriosclerotic plaque also expressed tissue-associated IL1 activity upon stimulation with LPS but did not release significant soluble IL1 activity. To establish further the biological functions of cell-associated IL1, we incubated stimulated or unstimulated SMC that were fixed with paraformaldehyde and washed extensively (fixed SMC) with overlayered viable SMC of the same donor (responder SMC). Contact with fixed SMC that bore surface IL1 following TNF or IL1 stimulation evoked up to 20-fold higher IL6 release from responder SMC than did exposure to unstimulated SMC (57 vs 1052 ng/ml/day). Addition of anti-IL1 antibody inhibited the release of IL6 from the responder SMC. These results demonstrate that cytokine-activated SMC express biologically active IL1 on their cell surface and illustrate how these cells might actually participate in autocrine and paracrine signaling in the vessel wall. The requirement for direct intercellular contact for IL1 effects could facilitate local information exchange among vascular wall cells and/or infiltrating leukocytes and permit costimulation while limiting undue propagation of inflammatory stimuli.