DNA Synthesis In Smooth Muscle Cells Research Articles

Histone deacetylase and GATA-binding factor 6 regulate arterial remodeling in angiotensin II-induced hypertension.

Histone deacetylase (HDAC) inhibitors have been reported to improve essential and secondary hypertension. However, the specific HDAC that might serve as a therapeutic target and the associated upstream and downstream molecules involved in regulating hypertension remain unknown. Our study was aimed at investigating whether a selective inhibitor of class II HDAC (MC1568) modulates hypertension, elucidating the underlying mechanism. Hypertension was established by administering angiotensin II (Ang II) to mice before treatment with MC1568. SBP was measured. Treatment with MC1568 reduced elevated SBP; attenuated arterial remodeling in the kidney's small arteries and thoracic aorta; and inhibited cell cycle regulatory gene expression, vascular smooth muscle cell (VSMC) proliferation, DNA synthesis, and VSMC hypertrophy in vivo and in vitro. Ang II enhanced the expression of phosphorylated HDAC4 and GATA-binding factor 6 (GATA6) proteins, which were specifically localized in the cytoplasm of cells in the arteries of kidneys and in aortas. Forced expression and knockdown of HDAC4 increased and decreased, respectively, the proliferation and expression of cell cycle genes in VSMCs. GATA6, a newly described binding partner of HDAC4, markedly enhanced the size and number of VSMCs. Calcium/calmodulin-dependent kinase IIα (CaMKIIα), but not HDAC4, translocated from the nucleus to the cytoplasm in response to Ang II. CaMKIIα and protein kinase D1 were associated with VSMC hypertrophy and hyperplasia via direct interaction with HDAC4. MC1568 treatment weakened the association between HDAC4 and CaMKIIα. These results suggest that class II HDAC inhibition attenuates hypertension by negatively regulating VSMC hypertrophy and hyperplasia via the CaMKIIα/protein kinase D1/HDAC4/GATA6 pathway.

CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice--brief report.

To ascertain the importance of a single regulatory element in the control of Cnn1 expression using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing. The CRISPR/Cas9 system was used to produce 3 of 18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell-restricted Cnn1 gene. Each founder was bred for germline transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1(ΔCArG/ΔCArG)) mice. Quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal immunofluorescence microscopy showed dramatic reductions in Cnn1 mRNA and CNN1 protein expression in Cnn1(ΔCArG/ΔCArG) mice with no change in other smooth muscle cell-restricted genes and little evidence of off-target edits elsewhere in the genome. In vivo chromatin immunoprecipitation assay revealed a sharp decrease in binding of serum response factor to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall. CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in smooth muscle cell DNA synthesis. This facile genome editing system paves the way for a new generation of studies designed to test the importance of individual regulatory elements in living animals, including regulatory variants in conserved sequence blocks linked to human disease.

UAP56 is an important mediator of Angiotensin II/platelet derived growth factor induced vascular smooth muscle cell DNA synthesis and proliferation

Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reported that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease.

Inhibition of smooth muscle cell proliferation by adiponectin requires proteolytic conversion to its globular form

Accelerated atherosclerosis is the primary cardiovascular manifestation of diabetes and correlates inversely with levels of circulating adiponectin, an anti-atherosclerotic adipokine that declines in diabetes. We therefore initiated a study to examine the mechanisms by which adiponectin, a hormone released from adipose tissue, influences the proliferation of vascular smooth muscle cells (SMCs). Addition of adiponectin to quiescent porcine coronary artery SMCs increased both protein and DNA synthesis and concurrently activated ERK1/2 and Akt. By contrast, globular adiponectin, a truncated form of this protein, exhibited anti-mitogenic properties as indicated by the inhibition of protein and DNA synthesis in SMCs stimulated with platelet-derived growth factor (PDGF). Whereas globular adiponectin did not stimulate growth-related signal transduction pathways, it was able to block the PDGF-dependent phosphorylation of eukaryotic elongation factor 2 kinase, a regulator of protein synthesis. Proteolysis of adiponectin with trypsin, which produces globular adiponectin, reversed the growth-stimulating actions of the undigested protein. As the existence of globular adiponectin remains controversial, western blotting was used to establish its presence in rat serum. We found that globular adiponectin was detectable in rat serum, but this result was not obtained with all antibodies. The contrasting properties of adiponectin and its globular form with respect to SMC proliferation suggest that protection against atherosclerosis may therefore be mediated, in part, by the level of globular adiponectin.

UAP56 is a novel interacting partner of Bcr in regulating vascular smooth muscle cell DNA synthesis

Bcr is a serine/threonine kinase that is a critical regulator of vascular smooth muscle cell inflammation and proliferation. We have previously demonstrated that Bcr acts in part via phosphorylation and inhibition of PPARγ. We have identified the RNA helicase UAP56 as another substrate of Bcr. In this report we demonstrate that knockdown of UAP56 blocks Bcr induced DNA synthesis in vascular smooth muscle cells (VSMC). We also found that over expression of Bcr increased the expression of cyclin E and decreased the expression of p27. Knockdown of UAP56 reversed the effect of Bcr on cyclin E and p27 expression. Furthermore, we found that Bcr binds to UAP56 and demonstrate that binding of UAP56 to Bcr is critical for Bcr induced DNA synthesis in VSMC. Our data identify UAP56 as an important binding partner of Bcr and a novel target for inhibiting vascular smooth muscle cell proliferation.

A novel function of FoxO transcription factors in thrombin-stimulated vascular smooth muscle cell proliferation

Thrombin exerts coagulation-independent effects on the proliferation and migration of vascular smooth muscle cells (SMC). Forkhead box-O (FoxO) transcription factors regulate cell proliferation, apoptosis and cell cycle arrest, but a possible functional interaction between thrombin and FoxO factors has not been identified to date. In human cultured vascular SMC, thrombin induced a time-dependent phosphorylation of FoxO1 and FoxO3 but not FoxO4. This effect was mimicked by an activating-peptide (AP) for protease-activated receptor (PAR)-1, and abolished by a PAR-1 antagonist (SCH79797). APs for other PARs were without effect. FoxO1 and FoxO3 phosphorylation were prevented by the PI3 kinase (PI3K) inhibitor LY294002 while inhibitors of ERK1/2 (PD98059) or p38MAPK (SB203580) were ineffective. LY294002 moreover prevented thrombin-stimulated SMC mitogenesis and proliferation. FoxO1 and FoxO3 siRNA augmented basal DNA synthesis and proliferation of SMC. Nuclear content of FoxO proteins decreased time-dependently in response to thrombin, coincided with suppressed expression of the cell cycle regulating genes p21CIP1 and p27kip1 by thrombin. FoxO1 siRNA reduced basal p21CIP1 while FoxO3 siRNA attenuated p27kip1 expression; thrombin did not show additive effects. LY294002 restored p21CIP1 and p27kip1 protein expression. Immunohistochemistry revealed that human native and failed saphenous vein grafts were characterised by the cytosolic presence of p-FoxO factors in co-localisation of p21CIP1 and p27kip1 with SMC. In conclusion, thrombin and FoxO factors functionally interact through PI3K/Akt-dependent FoxO phosphorylation leading to expression of cell cycle regulating genes and ultimately SMC proliferation. This may contribute to remodelling and failure of saphenous vein bypass grafts.

Signaling Mechanisms Mediating Uridine Adenosine Tetraphosphate–induced Proliferation of Human Vascular Smooth Muscle Cells

Proliferation of vascular smooth muscle cells (SMCs) plays an important role in the development of atherosclerosis and restenosis. Extracellular mononucleotides, such as adenosine triphosphate and uridine-5'-triphosphate stimulate SMC proliferation. However, the effects of dinucleotides on SMC proliferation and their underlying signaling mechanisms are less well defined. Recently, increasing evidence suggests that the dinucleotide, uridine adenosine tetraphosphate (Up4A) plays a role in the regulation of cardiovascular function. We have previously demonstrated that Up4A stimulates DNA synthesis and proliferation of human SMCs. This study investigated the signaling mechanisms underlying the proliferative effect of Up4A. Up4A-induced increase in bromodeoxyuridine incorporation was blocked by the mammalian target of rapamycin inhibitor, rapamycin, and the MEK inhibitor, PD98059. Up4A-stimulated phosphorylation and kinase activity of S6 kinase (S6K) and Erk1/2 were inhibited by PD98059, whereas phosphorylation and kinase activity of S6K, but not Erk1/2, were inhibited by rapamycin. Up4A also increased the phosphorylation of Akt, which was blocked by the PI3-kinase inhibitor, LY294002. Up4A-stimulated activation of S6K, but not Erk1/2, was also prevented by LY294002. Furthermore, Up4A-stimulated phosphorylation and kinase activity of S6K and Erk1/2 were inhibited by the P2 receptor antagonist, suramin, but not by the P2X receptor antagonist, Ip5I. Up4A also stimulated an increase in the protein expression of cycle-dependent kinase 2, which was prevented by rapamycin, PD98059, and suramin. These results suggest that the signaling mechanisms underlying the Up4A-stimulated proliferation of SMCs are mediated by P2Y receptors and involve the PI3-K/Akt and mitogen-activated protein kinase pathways, leading to the independent activation of S6K and an increase in cycle-dependent kinase 2 expression. This work stresses the concept that dinucleotides, like mononucleotides, play potentially important roles in the regulation of vascular function.

Different effects of epidermal growth factor on smooth muscle cells derived from human myometrium and from leiomyoma

To determine different effects of epidermal growth factor (EGF) on cultured smooth muscle cells (SMCs) derived from human myometrium and leiomyoma. EGF effects on DNA synthesis and intracellular signal transduction were studied in cultured SMCs from leiomyoma and its matched myometrium. Research laboratories. Patients 35-50 years old with uterine leiomyomas. Hysterectomy. Signal transduction from EGF receptor. As analyzed by laser scanning cytometry (LSC), EGF treatment stimulated DNA synthesis and induced polyploidization of leiomyomal, but not myometrial, SMCs. EGF stimulation was inhibited by AG1478, an EGF receptor (EGFR) inhibitor and PD98059, a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor. Both leiomyomal and myometrial SMCs had similar expression levels of EGFR, but EGF treatment induced transient phosphorylation activation of EGFR and Akt in leiomyomal SMCs. Consequently, EGF triggered transient phosphorylation activation of p44/42 MAPK in leiomyomal SMCs, followed by down-regulation of p27. In myometrial SMCs, however, EGF induced sustained activation of EGFR, Akt, and p44/42 MAPK with up-regulation of p27. EGF stimulates DNA synthesis and polyploidization in leiomyomal SMCs through transient activation of the EGFR-MAPK pathway. Given that polyploidization plays a role in tumorigenesis, our results shed new light on the pathogenesis of human uterine leiomyoma.

Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival

Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. Laminar FSS of arterial level (14 dynes/cm(2)) was applied to SMC cultures for 24 hours in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology, and results further verified by real time polymerase chain reaction (RT-PCR) and immunoblotting. Tissue factor pathway inhibitor-2 (TFPI-2) and caspase-3 protein expression was studied in the rat carotid artery after balloon-injury, and the effect of TFPI-2 on SMC DNA synthesis and apoptosis was examined in vitro. Microarrays identified TFPI-2 as one of the most differentially expressed gene by FSS in cultured SMCs (P < .001). Gene set enrichment analysis revealed significant regulation of genes linked to proliferation, apoptosis, and cell cycle regulation. TFPI-2 induction was confirmed by RT-PCR and immunoblotting demonstrating a more than 400-fold (P < .001) increase in TFPI-2 mRNA in SMCs exposed to FSS compared with static controls, and a consistent protein upregulation. Functionally, SMC proliferation was decreased by FSS (P < .001), and recombinant TFPI-2 was found to inhibit SMC proliferation (P < .001) and induce SMC apoptosis as indicated by activation of caspase-3 (P < .01). In vivo, TFPI-2 expression was found to be upregulated 5, 10, and 20 hours (P < .01) after rat carotid balloon injury, and immunohistochemistry demonstrated TFPI-2 protein in FSS-exposed luminal SMCs, co-localized with caspase-3 in the rat carotid neointima. FSS influenced gene expression associated with cell growth and apoptosis in cultured SMCs and strongly induced expression of TFPI-2 mRNA and protein. TFPI-2 was expressed in luminal, FSS-exposed SMCs together with caspase-3 in the rat carotid neointima after balloon injury. Functionally, TFPI-2 may play a role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 controls SMC function.

Cell-Shape Regulation of Smooth Muscle Cell Proliferation

Vascular smooth muscle cells (SMCs) play an important role in vascular remodeling. Heterogeneity and phenotypic changes in SMCs are usually accompanied by a morphological difference, i.e., elongated/spindle-like versus spread-out or epithelioid/rhomboid cell shapes. However, it is not known whether the cell shape directly regulates SMC proliferation, and what the underlying mechanisms are. In this study, microgrooves and micropatterned matrix islands were used to engineer the cell shape and investigate the associated biophysical and biological mechanisms. Compared to spread-out SMCs on nonpatterned surfaces, SMCs on micropatterned surfaces demonstrated elongated morphology, significantly lower cell and nucleus shape indexes, less spreading, a lower proliferation rate, and a similar response (but to a lesser extent) to platelet-derived growth factor, transforming growth factor-β, and mechanical stretching. DNA microarray profiling revealed a lower expression of neuron-derived orphan receptor-1 (NOR-1) in elongated SMCs. Knocking down NOR-1 suppressed DNA synthesis in SMCs, suggesting that NOR-1 is a mediator of cell elongation effects. Regulation of DNA synthesis in SMCs by the cell shape alone and a decrease in DNA synthesis in the case of small cell spreading area were achieved by micropatterning SMCs on matrix islands of different shapes and spreading areas. Changes in the cell shape also affected the nucleus shape, whereas variations in the cell spreading area modulated the nucleus volume, indicating a possible link between nucleus morphology (both shape and volume) and DNA synthesis. The findings of this investigation provide insight into cell shape effects on cell structure and proliferation, and have direct implications for vascular pathophysiology.

E013 Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells

Smooth muscle cell (SMC) proliferation is a major feature in atherosclerosis, since it contributes to the formation of the fibrous cap, thus to plaque stability, but also to arterial stenosis and post-angioplasty restenosis. Among the various mitogenic signaling pathways involved in SMC proliferation, the mTOR pathway regulates both the cell cycle and cell growth. Resveratrol, a polyphenolic compound from grapes and red wine, has potential anti-atherogenic and anti-cancer properties. This work was designed to investigate the activation of the mTOR pathway by the proatherogenic oxidized LDL (oxLDL) in SMC, and the potential inhibitory effect of resveratrol. mTOR and its downstream target p70S6 kinase are phosphorylated and activated by mitogenic concentrations of oxLDL (50mug/ml), and are involved in SMC proliferation, as assessed by the inhibitory effect of the mTOR inhibitor rapamycin. The activation of mTOR signaling by oxLDL, requires the upstream activation of PI3K and Akt, as assessed by the inhibitory effect of the PI3K inhibitor Ly294002 on mTOR activation and DNA synthesis. Resveratrol blocked the oxLDL-induced phosphorylation and activation of the PI3K/Akt/mTOR/p70S6K pathway and strongly inhibited both the DNA synthesis and proliferation of SMC. This activity is independent of the anti-oxidant effect and of AMPK activation by resveratrol. These data indicate that the mTOR pathway is activated by oxLDL via PI3K/PDK1/Akt, and is required for SMC proliferation. Resveratrol blocks specifically this pathway, thereby inhibiting oxLDL-induced SMC proliferation. These data highlight a new property for resveratrol that could contribute to the general anti-atherogenic properties of this polyphenol.

Medroxyprogesterone Abrogates the Inhibitory Effects of Estradiol on Vascular Smooth Muscle Cells by Preventing Estradiol Metabolism

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA.

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.

Adventitial Microvessel Formation After Coronary Stenting and the Effects of SU11218, a Tyrosine Kinase Inhibitor

The aim of this study was to delineate the temporal profile of adventitial microvessel (Ad-MV) formation after stenting, its relationship to arterial wall hypoxia, and the effects of a tyrosine kinase inhibitor (TKI), SU11218, on Ad-MV and in-stent intimal hyperplasia (IH). Adventitial microvessels have been reported after arterial injury; however, the underlying stimulus for this response and its relationship to IH is unknown. Coronary stenting was performed in 40 pigs randomized to SU11218 (n = 20) or placebo (n = 20). Vessel wall hypoxia was assessed by pimonidazole adducts and hypoxia-inducible factor (HIF)-1 alpha expression. Adventitial microvessels were quantified by three-dimensional microscopic computed tomography (3D micro CT). Intimal hyperplasia was measured by intravascular ultrasound (IVUS), 3D micro CT, and morphometry. The effects of SU11218 were assessed in vitro on smooth muscle cell (SMC) and endothelial cell (EC) functions and in vivo on Ad-MV and IH. Hypoxia was evident in the vessel wall at 48 h and persisted for four weeks. Adventitial microvessels increased significantly at one week (24 +/- 7 microvessels/segment) and four weeks (23 +/- 7 microvessels/segment) compared with uninjured arteries (16 +/- 2 microvessels/segment; p < 0.001) and correlated with IH (r = 0.77, p < 0.001). The TKI SU11218 inhibited platelet-derived growth factor receptor-beta phosphorylation, EC and SMC DNA synthesis, and migration in a dose-dependent manner in vitro and significantly inhibited Ad-MV (16 +/- 5 vs. 23 +/- 7 microvessels/segment in placebo, p < 0.001) and produced approximately 80% reduction in IH (0.52 +/- 0.51 mm2 vs. 2.47 +/- 1.66 mm2 in placebo, p < 0.001) at four weeks in vivo. Arterial stenting causes arterial wall hypoxia followed by Ad-MV formation. The TKI SU11218 inhibits both Ad-MV formation and IH and represents a promising therapeutic agent to prevent in-stent restenosis.

Increased Expression and Function of Vascular 1D-Adrenoceptors May Mediate The Prohypertensive Effects Of Angiotensin II

The peptide Angiotensin II (Ang II), part of the renin-angiotensin system (RAS), participates in the control of systemic arterial pressure. Ang II participates in increasing smooth muscle tone, and its positive effects on smooth muscle cell DNA synthesis are inhibited by treatment with prazosin, an alpha(1)-adrenoceptor agonist. Ang II also induces the expression of alpha(1)-adrenoceptor, especially the alpha(1D) subtype. Other findings suggest that the molecular signals activated by Ang II and by alpha(1D)-adrenoceptor might interweave, thus leading to the augmentation of smooth muscle tone and hypertension.

A quantitative proteomic analysis of growth factor-induced compositional changes in lipid rafts of human smooth muscle cells

Signals that promote proliferation and migration of smooth muscle cells (SMC) have been implicated in pathologic growth of hollow organs. Members of the platelet-derived growth factor (PDGF) family, potent mitogens and motility factors for SMC, have been shown to signal through cholesterol-enriched lipid rafts. We recently demonstrated that PDGF-stimulated DNA synthesis in urinary tract SMC was dependent on the integrity of lipid rafts. Despite its known ability to rapidly alter discrete proteins within rafts, the effect of PDGF on overall raft protein composition is unknown. In this study, we employed isotope coded affinity tag (ICAT) analysis to evaluate PDGF-induced protein changes in lipid rafts of primary culture human SMC. Following acute (i.e., 15 min) exposure of SMC to PDGF, 23 proteins increased in rafts >20%. In contrast, raft localization of only three proteins increased after 12 h of PDGF treatment. Among the proteins that increased at 15 min were the glycophosphatidylinositol-anchored proteins Thy-1, 5'-nucleotidase, and CD55, the cytoskeletal proteins actin, actinin, tropomyosin-3 and -4, and the endocytosis-related proteins clathrin and beta-adaptin. In addition, eight Rho family members were localized to rafts by ICAT analysis. Collectively, these observations suggest a role for lipid rafts in regulation of PDGF-stimulated changes in the cytoskeleton.

Thrombin induces expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells

To understand the mechanisms by which thrombin induces vascular smooth muscle cell (VSMC) DNA synthesis and motility, we have studied the role of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-S6K1 signaling. Thrombin stimulated the phosphorylation of Akt and S6K1 in VSMC in a sustained manner. Blockade of PI3K-Akt-mTOR-S6K1 signaling by LY-294002, and rapamycin suppressed both thrombin-induced VSMC DNA synthesis and migration. Adenovirus-mediated expression of dominant-negative Akt also inhibited thrombin-induced VSMC DNA synthesis and migration. Furthermore, thrombin induced the expression of Fra-1 in a sustained PI3K-Akt-dependent and mTOR-independent manner in VSMC. Suppression of Fra-1 by its small interfering RNA attenuated both thrombin-induced VSMC DNA synthesis and migration. Thrombin also induced the expression of FGF-2 in a PI3K-Akt-Fra-1-dependent and mTOR-independent manner, and neutralizing anti-FGF-2 antibodies inhibited thrombin-stimulated VSMC DNA synthesis and motility. In addition, thrombin stimulated the tyrosine phosphorylation of EGF receptor (EGFR), and inhibition of its kinase activity significantly blocked Akt and S6K1 phosphorylation, Fra-1 and FGF-2 expression, DNA synthesis, and motility induced by thrombin in VSMC. Together these observations suggest that thrombin induces both VSMC DNA synthesis and motility via EGFR-dependent stimulation of PI3K/Akt signaling targeting in parallel the Fra-1-mediated FGF-2 expression and mTOR-S6K1 activation.

Plasmin-induced smooth muscle cell proliferation requires epidermal growth factor activation through an extracellular pathway

Plasminogen activators are used routinely for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. We have shown recently that plasmin induces extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated cell proliferation. Plasmin can also activate metalloproteinases on the cell surface, which can release the tethered ligand heparin-binding epidermal growth factor (HB-EGF), which can in turn activate the epidermal growth factor receptor (EGFR). Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis and cell proliferation, EGFR phosphorylation, and ERK1/2 activation were examined in response to plasmin in the presence and absence of the plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), matrix metalloproteinase (MMP) inhibitor GM6001, HB-EGF inhibitor CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies, and the EGFR inhibitor AG1478. Plasmin-induced smooth muscle cell DNA synthesis, which was blocked by EGFR and HB-EGF inhibition. Plasmin-induced time-dependent EGFR phosphorylation and ERK1/2 activation, which were inhibited by AG1478. This response was dependent on the proteolytic activity of plasmin since both plasmin inhibitors blocked the response. EGFR phosphorylation by plasmin was blocked by inhibition of MMP activity and the ligand HB-EGF. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs, or HB-EGF. Direct blockade of the EGFR prevented activation by both plasmin and EGF. Plasmin can induce smooth muscle cell proliferation through activation of EGFR by an extracellular MMP-mediated, HB-EGF-dependent process.

Nitric oxide-independent regulation of the vascular injury response

A plethora of cellular and molecular signaling pathways are elicited in response to vascular trauma that eventuates in formation of an invasive neointima and adaptive remodeling of the vessel wall. The pivotal role of nitric oxide (NO) and downstream soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) in mediating vasoprotection under both homeostatic and inimical conditions has been extensively studied in clinical medicine and basic science research. Recently, the parallel system of sGC-activating heme oxygenase (HO) and the diatomic gas carbon monoxide (CO) has emerged as a viable alternate pathway in mediating vascular protection following injury. Potential candidate factors involved in this regulation include inducible HO (HO-1) and CO and the novel exogenous sGC/cGMP-sensitizing agents YC-1 and BAY 41-2272. The overall goal of these studies is to elucidate potential protective influence of these NOindependent systems in regulating the neointimal and vascular remodeling responses to experimental carotid artery balloon injury in laboratory rats. Results show that HO-1 and CO independently attenuate neointima development 2 weeks following injury through inhibition of acute vascular smooth muscle cell (SMC) DNA synthesis and proliferation, decreased acute expression of growthpromoting TGF-β1 and the G1 cyclins E and A, and enhanced SMC apoptosis. Similar experiments utilizing the sGC-sensitizing benzyl indazole compound YC-1 found vessel wall cGMP to correlate with reduced vascular SMC growth and platelet function and diminished neointima formation. Promising early results suggest the NO-independent sGC activator BAY 41-2272 reduces the neointimal response to injury through anti-proliferative and anti-migratory actions on vascular SMCs. In addition, the anti-proliferative effects of BAY 41-2272 were significantly potentiated in the presence of the HO-1 inducer and substrate hemin. These cumulative results strongly suggest that the HO-1/CO system and novel NO-independent routes of sGC/cGMP activation exert salutary influence upon compromised blood vessels via regulation of various cellular growth-promoting functions and may represent attractive therapeutic strategies aimed at reducing vascular lesion formation. from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005

Plasmin Induces Smooth Muscle Cell Proliferation 1,2

Plasminogen activators are routinely used for thrombolysis. They lead to the generation of the protease, plasmin, which can induce smooth muscle cell proliferation and may thus promote further intimal hyperplasia in the thrombolysed vessel. The signaling pathways used by plasmin are not understood. Murine aortic smooth muscle cells were cultured in vitro. Assays of DNA synthesis, cell proliferation, MAPKK and MAPK activation were examined in response to plasmin alone and in the presence of plasmin inhibitors (epsilon-aminocaproic acid and aprotinin), pertussis toxin (Galphai inhibitor, PTx), GP-2A (Galphaq inhibitor), wortmannin (PI3-K inhibitor, Wn), LY294002, (PI3-K inhibitor, LY), PD98059 (MEK inhibitor, PD), and SB203580 (p38MAPK inhibitor, SB). Plasmin produced concentration dependent smooth muscle cells DNA synthesis and proliferation and induced ERK1/2 and p38MAPK phosphorylation. Inhibition of the proteolytic activity of plasmin prevented these responses. The ERK1/2 inhibitor, PD, but not the p38MAPK inhibitors, SB, blocked cell proliferation. The activation of the MEK1/2 and ERK1/2 pathway was both Galphai dependent (PTx-sensitive) and Galphaq dependent (GP-2A-sensitive). It was blocked by the PI3-K inhibitors, Wn and LY. PI3-K activation as measured by akt phosphorylation was dependent on Galphai, but was independent of Galphaq. Plasmin induces smooth muscle cell proliferation. Plasmin induced ERK1/2 phosphorylation occurs through two pathways: one which is Galphai mediated/PI3-K dependent and a second which is Galphaq mediated/PI3K independent. p38MAPK appears not to be involved in plasmin-mediated cell proliferation. This pattern of activation is distinct from that seen with urokinase plasminogen activator.