Medial Vascular Smooth Muscle Cells Research Articles

QS370. Vascular Injury Causes Early-Onset Apoptosis of Medial Vascular Smooth Muscle Cells

Objectives: This study aims to determine the mechanisms underlying the rapid and massive apoptosis of medial vascular smooth muscle cells (VSMC) induced by acute injury. Methods and Results: The rat iliac balloon injury model was used in this study. Apoptotic cells were detected using in Situ Oligo Ligation assay (ISOL) combined with immunohistochemistry (IHC) for smooth muscle cell actin (SMA). 45.3 % ± 14.6 of the medial VSMC were apoptotic (ISOL+ SMA+) at 30 min after injury and more than 95% of the medial cells were death 90 min after.

Effect of folate on neointima formation and matrix metalloproteinase-9 expression after balloon injury in hyperhomocysteinemic rabbits

BackgroundIt is known that hyperhomocysteinemia (Hhcy) is associated with the risk of restenosis after angioplasty. Folate can lower plasma homocysteine (Hcy) level and alleviate the Hhcy-induced neointima formation after balloon injury. This study aims to explore the mechanisms of folate in inhibiting neointima formation. Methods24 New Zealand White rabbits were randomly divided into three groups: Control, 2% methionine (Meth) and 2% methionine+folate (Meth+folate). At the end of 8 experimental weeks, all rabbits underwent the balloon injury in abdominal aorta. 4 weeks following this procedure, plasma Hcy concentration, aortic maximal neointimal thickness (NT), neointimal area (NA), medial area (MA), and ratio of neointimal area to medial area (NA/MA), ultrastructure of vascular smooth muscle cells (VSMCs), and matrix metalloproteinase-9 (MMP-9) expression were detected by high performance liquid chromatography, histomorphometric analysis, transmission electron microscope (TEM) and real-time PCR, respectively. ResultsIt was observed in our study that Hcy concentration, NT, NA, NA/MA and the expression of MMP-9 mRNA were higher in the Meth group than in the control. VSMCs in media exhibited typical synthetic phenotype in the Meth group compared with the transitional state between contractile phenotype and synthetic phenotype in the control group. However, in the rabbits treated with folate, Hcy concentration, NT, NA and MMP-9 mRNA expression were lower than those in the Meth group. The phenotype of VSMCs was close to that in the control group. ConclusionThis study suggested that folate could decrease the level of Hcy, reverse the Hhcy-induced exacerbation of neointima formation in rabbits following balloon injury, and the mechanisms in it may be related to the suppressive effect of folate on the expression of MMP-9 mRNA in arterial wall.

Calpain counteracts mechanosensitive apoptosis of vascular smooth muscle cellsin vitroandin vivo

Mechanical forces contribute to vascular remodeling processes. Elevated mechanical stress causes apoptosis of vascular smooth muscle cells (VSMCs) within the media. This study examined the role of the cystein protease calpain in force-induced vascular cell apoptosis and its effect on injury-induced vascular remodeling processes. VSMCs were exposed to cyclic tensile force in vitro, which resulted in increased p53 protein expression and transcriptional activity as well as a significant increase of apoptotic VSMCs. Apoptosis was prevented by the p53 inhibitor pifithrin and by p53 antisense oligonucleotides, indicating dependency of force-induced apoptosis on p53. Simultaneously, calpain activity increased by mechanical stress. Prevention of calpain activation by calpeptin or antisense oligonucleotides augmented strain-induced p53 expression and transcriptional activity, resulting in a further increase of apoptotic rate. p53 protein was directly disintegrated by activated calpain. The in vivo relevance of the findings was tested: pharmacologic inhibition of initial calpain activation augmented early apoptosis of medial VSMCs 24 h after balloon injury in a p53-dependent manner but resulted in a marked increase in late neointima formation. We conclude that calpain counteracts mechanically induced excessive VSMC apoptosis through its p53-degrading properties, which identifies calpain as a key regulator of mechanosensitive remodeling processes of the vascular wall.

Activation of the cGMP/cGKI signalling pathway in primary murine VSMCs accelerates growth

The endogenous signaling molecule nitric oxide (NO) exerts many of its actions via the cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway. Recently, the analysis of an in vivo mouse model for atherosclerosis suggested that activation of cGKI in vascular smooth muscle cells (VSMCs) promotes the phenotypic modulation of medial VSMCs and, thus, vascular lesion formation. In contrast, many in vitro studies demonstrate an anti-proliferative role for cGKI. In the current study, primary and subcultured wild-type and cGKI-deficient VSMCs in response to the membrane permeable cGMP analogue 8-Br-cGMP were compared. In agreement with the common view an anti-proliferative effect of cGKI was found in repeatedly passaged VSMCs (P5–11). In contrast, the analysis of primary VSMCs revealed that activation of cGKI in primary VSMCs strongly promotes growth. Analysing proliferation, apoptosis, cytoskeletal dynamics and various signaling pathways indicated that an increase in cell adhesion is a major mechanism for cGKI-mediated growth in primary VSMCs. This pro-adhesive effect of cGKI might be mediated via an inhibition of Rho kinase (ROCK) and enhanced integrin signaling. These processes might contribute to inhibit anoikis, the programmed cell death induced by the loss of cell/matrix interactions. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Increased apoptosis and cysteinyl aspartate specific protease-3 gene expression in human intracranial aneurysm

Objective To investigate apoptosis in vascular smooth muscle cells (VSMCs) and caspase-3 expression in ruptured intracranial aneurysm. Methods Tissue samples of 15 ruptured intracranial aneurysms, 6 abdominal aortic aneurysms (AAA) and 6 normal vessels were evaluated. Apoptosis in VSMCs was determined on transmission electron microscopy. Immunohistochemistry for alpha-SMC actin and direct cell counts (medial VSMCs per high-power field (HPF)) were employed to determine medial VSMC density. Additionally, gene expression of caspase-3 was determined using real-time RT-PCR. Results We demonstrated medial VSMCs exhibiting morphological apoptotic changes in cerebral aneurysm and AAA. Medial VSMC density was significantly decreased in intracranial aneurysm (43.9 ± 4.3 SMCs/HPF) and AAA (53.2 ± 9.4 SMCs/HPF) compared with the normal arteries (222.8 ± 12.1 SMCs/HPF; p < 0.01). An 8.94-fold and 6.73-fold increase in expression of caspase-3mRNA in intracranial aneurysm and AAA, respectively, were obtained relative to the normal vessels. Conclusions These results suggest that real time RT-PCR provides a useful tool to test gene expression in small samples, and may contribute to a better understanding of the role of apoptosis in ruptured intracranial aneurysm.

Rosuvastatin regulates vascular smooth muscle cell phenotypic modulation in vascular remodeling: Role for the urokinase receptor

The urokinase (uPA)/urokinase receptor (uPAR) multifunctional system is an important mediator of migration and proliferation of vascular smooth muscle cells (VSMC). However, whether uPA/uPAR-directed mechanisms are involved in the beneficial effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on vascular remodeling remains unexplored. In this study, we have investigated the effect of the hydrophilic statin rosuvastatin on neointimal remodeling, and the role of uPAR. Using an ex vivo organ and in vitro cell culture models we demonstrate that rosuvastatin decreases injury-induced neointima formation and proliferation of medial VSMC in porcine coronary arteries, as well as migration and proliferation of human coronary VSMC. Studies on the underlying mechanisms show that rosuvastatin impairs VSMC transition from their physiological contractile to the pathophysiologal synthetic phenotype. These effects are mediated, at least in part, via uPAR, as confirmed by means of rosuvastatin-directed uPAR expression and uPAR silencing in both models. Our findings provide evidence that rosuvastatin modulates VSMC phenotypic changes and subsequently their proliferation and migration, and indicate the important role for uPAR in these processes. This mechanism contributes to the beneficial non-lipid lowering effect of rosuvastatin on negative vascular remodeling

HMGB1 expression by activated vascular smooth muscle cells in advanced human atherosclerosis plaques

Chronic inflammation plays a key role in atherogenesis, which is followed by atheromatous plaque instability. High-mobility group box 1 is released by activated macrophages as a late-phase mediator during prolonged inflammation. However, the expression of high-mobility group box 1 and its effect on the production of C-reactive protein and matrix metalloproteinases, particularly on human vascular smooth muscle cells, still remain unknown. Immunohistochemical studies revealed that high-mobility group box 1 was abundantly expressed in vascular smooth muscle cells of carotid and coronary atheromatous plaques, but not in atrophic vascular smooth muscle cells of fibrous plaques and normal medial vascular smooth muscle cells. Receptor for advanced glycation end products was also detected in vascular smooth muscle cells positive for high-mobility group box 1. Moreover, vascular smooth muscle cells positive for high-mobility group box 1 were found to express both C-reactive protein and matrix metalloproteinases (2, 3, and 9). Administration of exogenous high-mobility group box 1 to cultured vascular smooth muscle cells caused a marked elevation of C-reactive protein mRNA by reverse transcriptase-polymerase chain reaction and of C-reactive protein levels by enzyme-linked immunosorbent assay. Conversely, C-reactive protein also triggered a significant release of high-mobility group box 1 in vascular smooth muscle cell culture medium as determined by immunoblot. Activated vascular smooth muscle cells are the source of high-mobility group box 1 in human advanced atherosclerotic lesions. High-mobility group box 1 directly stimulates the production of both C-reactive protein and matrix metalloproteinase through receptor for advanced glycation end product. These findings provide new evidence that high-mobility group box 1 produced by activated vascular smooth muscle cells may contribute to the progression and vulnerability of human atherosclerotic lesions toward rupture.

Relationship Between Hypertension and Atherosclerosis: From a Viewpoint of the Most Potent Vasoconstrictor Human Urotensin II

Human urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, and its receptor (UT) are involved in etiology of hypertension. In hypertensive patients, U-II induces vasoconstriction in forearm brachial artery infusion studies. Recent studies demonstrated elevated plasma U-II concentrations in patients with hypertension, diabetes mellitus, atherosclerosis, and coronary artery disease. U-II is expressed in endothelial cells, macrophages, macrophagederived foam cells, and myointimal and medial vascular smooth muscle cells (VSMCs) of atherosclerotic human coronary arteries. UT receptors are present in VSMCs of human coronary arteries, thoracic aorta and cardiac myocytes. Lymphocytes are the most active producers of U-II, whereas monocytes and macrophages are the major cell types expressing UT receptors, with relatively little receptor expression in foam cells, lymphocytes, and platelets. U-II accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages, and stimulates cell growth and up-regulates type 1 collagen expression in human endothelial cells. U-II also activates NADPH oxidase and plasminogen activator inhibitor-1 in human VSMCs, and stimulates VSMC proliferation with synergistic effects observed when combined with oxidized LDL, reactive oxygen species and serotonin. These findings suggest that U-II plays key roles in accelerating the development of atherosclerosis, and hence coronary artery disease.

Vascular Smooth Muscle Cells Undergo Telomere-Based Senescence in Human Atherosclerosis

Although human atherosclerosis is associated with aging, direct evidence of cellular senescence and the mechanism of senescence in vascular smooth muscle cells (VSMCs) in atherosclerotic plaques is lacking. We examined normal vessels and plaques by histochemistry, Southern blotting, and fluorescence in situ hybridization for telomere signals. VSMCs in fibrous caps expressed markers of senescence (senescence-associated beta-galactosidase [SAbetaG] and the cyclin-dependent kinase inhibitors [cdkis] p16 and p21) not seen in normal vessels. In matched samples from the same individual, plaques demonstrated markedly shorter telomeres than normal vessels. Fibrous cap VSMCs exhibited markedly shorter telomeres compared with normal medial VSMCs. Telomere shortening was closely associated with increasing severity of atherosclerosis. In vitro, plaque VSMCs demonstrated morphological features of senescence, increased SAbetaG expression, reduced proliferation, and premature senescence. VSMC senescence was mediated by changes in cyclins D/E, p16, p21, and pRB, and plaque VSMCs could reenter the cell cycle by hyperphosphorylating pRB. Both plaque and normal VSMCs expressed low levels of telomerase. However, telomerase expression alone rescued plaque VSMC senescence despite short telomeres, normalizing the cdki/pRB changes. In vivo, plaque VSMCs exhibited oxidative DNA damage, suggesting that telomere damage may be induced by oxidant stress. Furthermore, oxidants induced premature senescence in vitro, with accelerated telomere shortening and reduced telomerase activity. We conclude that human atherosclerosis is characterized by senescence of VSMCs, accelerated by oxidative stress-induced DNA damage, inhibition of telomerase and marked telomere shortening. Prevention of cellular senescence may be a novel therapeutic target in atherosclerosis.

Essential role of PKC-ζ in normal and angiotensin II-accelerated neointimal growth after vascular injury

The contribution of atypical protein kinase C (PKC)-zeta to ANG II-accelerated restenosis after endoluminal vascular injury was investigated by using the rat carotid balloon injury model. Exposure of injured arteries to ANG II resulted in an extensive neointimal thickening (1.9 times) compared with vehicle at day 14. Treatment with PKC-zeta antisense, but not scrambled, oligonucleotides reduced neointimal formation observed in the presence or absence of ANG II. Examination of early events (2 days) after injury showed an increase in cellularity in the perivascular area of the artery wall that was transferred to the adventitia and media after exposure to ANG II, events blocked by PKC-zeta antisense, but not scrambled, oligonucleotides. A positive correlation between medial cellularity at day 2 and extent of neointimal growth at day 14 was established. Immunohistochemical analysis showed that upregulation of inflammatory markers after injury, as well as infiltration of ED1(+) monocytes/macrophages from the perivascular area to the adventitia, was accelerated by ANG II. However, ANG II-stimulated medial increase in cellularity was proliferation independent, and these cells were monocyte chemoattractant protein-1(+)/vimentin(+) but ED1(-)/VCAM(-). PKC-zeta is degraded after injury, and inhibition of its neosynthesis in medial vascular smooth muscle cells or in infiltrating cells with PKC-zeta antisense attenuated medial cellularity and expression of inflammation mediators without reversing smooth muscle cell dedifferentiation. Together, these data indicate that PKC-zeta plays a critical role in normal and ANG II-accelerated neointimal growth through a mechanism involving upregulation of inflammatory mediators, leading to cell infiltration in the media of the vascular wall.

Human Urotensin II as a Link between Hypertension and Coronary Artery Disease

Hypertension is a well-known risk factor for atherosclerosis, but the molecular mechanisms that link elevated blood pressure to the progression of atherosclerosis remain unclear. Human urotensin II (U-II), the most potent endogenous vasoconstrictor peptide identified to date, and its receptor (UT receptor) are involved in the etiology of essential hypertension. In patients with essential hypertension, U-II infused into the forearm brachial artery has been shown to induce vasoconstriction. Recent studies have demonstrated elevated plasma U-II concentrations in patients with essential hypertension, diabetes mellitus, atherosclerosis, and coronary artery disease. U-II is expressed in endothelial cells, macrophages, macrophage-derived foam cells, and myointimal and medial vascular smooth muscle cells (VSMCs) of atherosclerotic human coronary arteries. UT receptors are present in VSMCs of human coronary arteries, the thoracic aorta and cardiac myocytes. Lymphocytes are the most active producers of U-II, whereas monocytes and macrophages are the major cell types expressing UT receptors, with relatively little receptor expression in foam cells, lymphocytes, and platelets. U-II accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages. In human endothelial cells, U-II promotes cell proliferation and up-regulates type 1 collagen expression. U-II also activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and plasminogen activator inhibitor-1 in human VSMCs, and stimulates VSMC proliferation with synergistic effects observed when combined with oxidized low-density lipoprotein, lysophosphatidylcholine, reactive oxygen species or serotonin. These findings suggest that U-II plays key roles in accelerating the development of atherosclerosis, thereby leading to coronary artery disease.

Potential role of Chinese medicinal herbs in the prevention of coronary artery restenosis

Potential role of Chinese medicinal herbs in the prevention of coronary artery restenosis

Inhibition of the ubiquitin-proteasome system: a new avenue for atherosclerosis

The ubiquitin-proteasome system (UPS) is thought to be functionally active in atherosclerosis (AS) lesions. Aspirin was found to be a potent inhibitor of the UPS in some tumour studies; however, its effect on AS remains to be demonstrated in vivo. New Zealand rabbits were placed on a normal diet (N) or on a normal diet with aspirin (NI) or on an atherogenic diet without (H) or with aspirin (HI) for 12 weeks. Proteasome activity, concentrations of plasma lipids and levels of peroxidation were determined. Ubiquitin/ubiquitin-conjugates (Ub), IkappaBalpha, phosphorylated IkappaB (pIkappaBalpha) and p65 were investigated by Western blotting or immunochemistry. Concentrations of plasma lipids and peroxidation levels were higher in H or HI vs. N or NI. Histological analysis showed that atheroma was increased in H. Ub and IkappaBalpha were mainly localised in subendothelium and media vascular smooth muscle cells. Western blots revealed that Ub, IkappaBalpha, and pIkappaBalpha were increased, whereas p65 was lower in HI vs. H. The activity of the 20S proteasome was functionally active in H vs. N, NI or HI, while the 26S proteasome was not affected in any of the groups. Aspirin can attenuate the pathogenesis of atheroma formation, the degradation of IkappaBalpha and pIkappaBalpha, and lower the expression of p65, indicating that its therapeutic effects on AS may be via inhibition of the UPS.

Role of the Media in Vascular Injury: Atherosclerosis and Dissection

Although a metabolic role for endothelium in toxic injury has been well established, a similar role has not been as thoroughly explored for the vascular media. In this study two forms of vascular medial involvement in toxic injury are examined. Early atherosclerotic plaques are studied by immunohistochemistry for an a class glutathione-S-transferase (GST) isozyme known as hGSTA4-4, which has preferential metabolic activity for a, ss -unsaturated aldehydes derived from lipid peroxidation, especially 4-hydroxy-2-nonenal. Findings in human plaque indicate that hGST A4-4 is highly upregulated in vascular smooth muscle cells (VSMCs) within the plaque and in the medial VSMCs underlying plaque. Endothelial cells, while not expressing hGST A4-4 distant from plaques, were found to express the isozyme in cytoplasm overlying plaque. In a series of second experiments, we illustrate a developmental model of dissecting aortic aneurysm (DAA) obtained by administering semicarbazide, an inhibitor of the little-studied VSMC enzyme semicarbazide-sensitive amine oxidase (SSAO), to pregnant rats during the last trimester of development. Newborn rats consistently developed DAA which is characterized by splitting of media of ascending thoracic aorta and extensive blood collections surrounding the vessel. These experimental examples emphasize the potential role of the media in toxic insults to blood vessels. Also, the potential importance of toxic injury to developing blood vessels by in utero exposure to xenobiotic substances is illustrated.

Increased Neointima Formation in Cysteine-Rich Protein 2–Deficient Mice in Response to Vascular Injury

In response to arterial injury, medial vascular smooth muscle cells (VSMCs) proliferate and migrate into the intima, contributing to the development of occlusive vascular disease. The LIM protein cysteine-rich protein (CRP) 2 associates with the actin cytoskeleton and may maintain the cytoarchitecture. CRP2 also interacts with transcription factors in the nucleus to mediate SMC gene expression. To test the hypothesis that CRP2 may be an important regulator of vascular development or function we generated Csrp2 (gene symbol of the mouse CRP2 gene)-deficient (Csrp2(-/-)) mice by targeted mutation. Csrp2(-/-) mice did not have any gross vascular defects or altered expression levels of SM alpha-actin, SM22alpha, or calponin. Following femoral artery injury, CRP2 expression persisted in the vessel wall at 4 days and then decreased by 14 days. Intimal thickening was enhanced 3.4-fold in Csrp2(-/-) compared with wild-type (WT) mice 14 days following injury. Cellular proliferation was similar between WT and Csrp2(-/-) VSMC both in vivo and in vitro. Interestingly, Csrp2(-/-) VSMC migrated more rapidly in response to PDGF-BB and had increased Rac1 activation. Our data demonstrate that CRP2 is not required for vascular development. However, an absence of CRP2 enhanced VSMC migration and increased neointima formation following arterial injury.

Uterine Artery Remodeling in Pseudopregnancy Is Comparable to That in Early Pregnancy1

During pregnancy, the lumenal diameter and wall mass of the uterine artery (UA) increase, most likely in response to the increased hemodynamic strain resulting from the chronically elevated uterine blood flow (UBF). In this remodeling process, the phenotype of vascular smooth-muscle cells (VSMC) is transiently altered to enable VSMC proliferation. These phenomena are already seen during early pregnancy, when the rise in UBF is still modest. This raises the question whether the newly instituted endocrine environment of pregnancy is involved in the onset of the pregnancy-related UA remodeling. We tested the hypothesis that the conceptus is not essential for the onset of UA remodeling of pregnancy. Six control and 18 pseudopregnant (Postcopulation Days 5, 11, and 17; n = 6 per subgroup) C57Bl/6 mice were killed and UAs were dissected and processed for either morphometric analysis or immunohistochemistry. The latter consisted of staining UA cross sections for the differentiation markers smooth muscle alpha-actin and smoothelin, and for the proliferation marker MKI67. We analyzed the UA changes in response to pseudopregnancy by ANOVA. Data are presented as mean +/- SD. By Day 11 of pseudopregnancy, the UA lumen was 25% wider and the media cross-sectional area 71% larger than in control mice. These differences were accompanied by reduced smoothelin expression and increased proliferation of UA medial VSMC. All UA morphological differences had returned or were in the process of returning to baseline values by Day 17 of pseudopregnancy. The structural and cellular aspects of UA remodeling as seen at midpregnancy are also seen in pseudopregnancy. These results support the concept that the conceptus does not contribute to the initiation of UA remodeling. We suggest that ovarian hormones trigger the onset of UA remodeling.

Statin-induced Vascular Smooth Muscle Cell Apoptosis: A Possible Role in the Prevention of Restenosis?

Growing evidence suggests that statins are more than simple lipid-lowering drugs. The so called pleiotropic effects of statins include multiple actions on cells of the vasculature. A large number of studies have confirmed that these compounds exert beneficial effects by mechanisms unrelated to cholesterol metabolism. For example, statins have been shown to inhibit the migration and proliferation of vascular smooth muscle cells (VSMC), and to induce apoptosis in this cell type. It is not yet clear if the induction of apoptosis in VSMC by statins is beneficial or detrimental. In the context of post-angioplasty restenosis, recurrent plaque growth after intervention, the inhibition of neointimal proliferation as well as a reduction of neointimal cell numbers by apoptosis is appealing. Multiple animal studies and clinical trials have therefore been undertaken to investigate effects of statin treatment on the development of restenosis, with very controversial results. Conversely, in advanced atherosclerotic lesions VSMC in the intima may stabilize the plaque and prevent plaque rupture by synthesizing collagen. VSMC in media adjacent to plaque areas or restenotic lesions should not be exposed to apoptosis promoting agents. In this context, recent evidence suggests that pravastatin protects such lesions by inhibiting inflammation and macrophage activation Our recent findings together with observations from other groups suggest that neointima cells are more sensitive to the induction of apoptosis than media VSMC. Importantly, statins were found to preferentially induce apoptosis in neointimal VSMC in our study. The purpose of the present review is to summarize statin effects on proliferation and apoptosis in VSMC in vitro and in vivo. Furthermore, the development of drug-coated stents may help to deliver high local doses of statins to enhance their effectiveness in the treatment of post-angioplasty restenosis.

Altered AP-1/Ref-1 redox pathway and reduced proliferative response in iNOS-deficient vascular smooth muscle cells

We previously reported that injury-induced medial vascular smooth muscle cell (VSMC) proliferation and neointima formation in carotid arteries of inducible nitric oxide synthase knockout (iNOS KO) mice were significantly reduced compared with wild type (WT). However, the molecular pathway underlying such differences is not known. In this in vitro study, we discovered that the AP-1/Ref-1/thioredoxin signaling pathway is altered in aortic VSMC from iNOS KO mice, which leads to reduced growth response when compared with aortic VSMC from WT mice. After equal initial seeding, the cell number after 7 days in serum medium was less in iNOS KO cells compared with WT VSMC (1.2 +/- 0.6 x 10(5) vs 3.2 +/- 1.1 x 10(5); p < 0.05). Significantly more iNOS KO cells remained in the G0/G1 phase compared with WT cells after 24-h serum treatment (82.6 +/- 13.7% vs 62.3 +/- 14.6%; p < 0.05) by cell-cycle analysis. Nuclear PCNA expression was also less in the iNOS KO cells, which was not affected by exogenous NO or superoxide. Superoxide generation after 24-h serum stimulation was less in the iNOS KO cells compared with WT cells. After 30-min serum stimulation, AP-1 DNA binding was reduced and a lack of increase in nuclear c-Jun protein was observed in iNOS KO VSMC. RT-PCR analysis confirmed a lack of inducible c-Jun mRNA after serum stimulation in the KO cells. In addition, KO cells had less nuclear reducing factor-1 (Ref-1) and serum-inducible thioredoxin protein expression. Reduced proliferative response of iNOS KO VSMC to serum treatment is associated with altered AP-1 /Ref-1 /thioredoxin pathway activation.

Involvement of clusterin in 15-deoxy-$Delta;12,14-prostaglandin J2-induced vascular smooth muscle cell differentiation*1

To establish an in vitro model of vascular smooth muscle cell (VSMC) differentiation, we examined the effect of 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) on the expression of VSMC differentiation markers. After the addition of 15d-PGJ 2 to confluent human umbilical artery smooth muscle cells synchronized in the G 0 phase, cells showed a “hill and valley” appearance and thereafter aggregated and formed macroscopic nodules. Cells forming nodules expressed high levels of SM2, the most specific VSMC differentiation marker, comparable to medial VSMCs in vivo. 15d-PGJ 2 significantly increased the mRNA and protein expression levels of clusterin , a secreted glycoprotein reported to induce nodule formation and differentiation of VSMCs. Moreover, addition of an anti-clusterin antibody completely inhibited the nodule formation induced by 15d-PGJ 2 and induced apoptosis. Our results suggested that clusterin is involved in 15d-PGJ 2 -induced nodule formation and cell differentiation in VSMCs.

Involvement of clusterin in 15-deoxy-Δ 12,14-prostaglandin J 2-induced vascular smooth muscle cell differentiation

To establish an in vitro model of vascular smooth muscle cell (VSMC) differentiation, we examined the effect of 15-deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2) on the expression of VSMC differentiation markers. After the addition of 15d-PGJ 2 to confluent human umbilical artery smooth muscle cells synchronized in the G 0 phase, cells showed a “hill and valley” appearance and thereafter aggregated and formed macroscopic nodules. Cells forming nodules expressed high levels of SM2, the most specific VSMC differentiation marker, comparable to medial VSMCs in vivo. 15d-PGJ 2 significantly increased the mRNA and protein expression levels of clusterin, a secreted glycoprotein reported to induce nodule formation and differentiation of VSMCs. Moreover, addition of an anti-clusterin antibody completely inhibited the nodule formation induced by 15d-PGJ 2 and induced apoptosis. Our results suggested that clusterin is involved in 15d-PGJ 2-induced nodule formation and cell differentiation in VSMCs.