Genipin Inhibits TNF-α-Induced Vascular Smooth Muscle Cell Proliferation and Migration via Induction of HO-1

Vascular smooth muscle cell (VSMC) proliferation and migration triggered by inflammatory stimuli contributes importantly to the pathogenesis of atherosclerosis and restenosis. On the other hand, genipin, an aglycon of geniposide, exhibits diverse pharmacological functions such as antitumor and anti-inflammatory effects. The protective effects of genipin on the cardiovascular system have also been reported. However, the molecular mechanism involved remains unknown. This study aimed to elucidate the precise function of genipin in VSMCs, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. We found that pretreatment of genipin induced HO-1 mRNA and protein levels, as well as its activity in VSMCs. Genipin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. At the molecular level, genipin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Genipin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 siRNA partially abolished the beneficial effects of genipin on VSMCs. These results suggest that genipin may serve as a novel drug in the treatment of these pathologies by inducing HO-1 expression/activity and subsequently decreasing VSMC proliferation and migration.     

Tungstate-Targeting of BKαβ1 Channels Tunes ERK Phosphorylation and Cell Proliferation in Human Vascular Smooth Muscle

Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca²⁺-dependent large-conductance BKαβ₁ potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BKαβ₁ channels in the tungstate-induced ERK phosphorylation and its relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BKαβ₁ channels potentiates the tungstate-induced ERK1/2 phosphorylation in a G_i/o protein-dependent manner. Tungstate activated BKαβ₁ channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDPβS or pertussis toxin. Moreover, analysis of G_i/o protein activation measuring the FRET among heterologously expressed G_i protein subunits suggested that tungstate-targeting of BKαβ₁ channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and β₁-knockout mice indicated that the presence of the regulatory β₁ subunit was essential for the tungstate-mediated activation of BK channels in VSMCs. Moreover, the specific BK channel blocker iberiotoxin lowered tungstate-induced ERK phosphorylation by 55% and partially reverted (by 51%) the tungstate-produced reduction of platelet-derived growth factor (PDGF)-induced proliferation in human VSMCs. Our observations indicate that tungstate-targeting of BKαβ₁ channels promotes activation of PTX-sensitive G_i proteins to enhance the tungstate-induced phosphorylation of ERK, and inhibits PDGF-stimulated cell proliferation in human vascular smooth muscle.     

Transforming Growth Factor β Inhibits Platelet Derived Growth Factor-Induced Vascular Smooth Muscle Cell Proliferation via Akt-Independent,Smad-Mediated Cyclin D1 Downregulation

In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor β (TGFβ) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFβ and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFβ completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFβ. TGFβ had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFβ on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFβ. siRNA against Smad4 completely reversed the inhibitory effect of TGFβ on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFβ-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFβ via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.     

PGC-1α Is a Key Regulator of Glucose-Induced Proliferation and Migration in Vascular Smooth Muscle Cells

Background

Atherosclerosis is a complex pathological condition caused by a number of mechanisms including the accelerated proliferation of vascular smooth muscle cells (VSMCs). Diabetes is likely to be an important risk factor for atherosclerosis, as hyperglycemia induces vascular smooth muscle cell (VSMC) proliferation and migration and may thus contribute to the formation of atherosclerotic lesions. This study was performed to investigate whether PGC-1α, a PPARγ coactivator and metabolic master regulator, plays a role in regulating VSMC proliferation and migration induced by high glucose.

Methodology/Principal Findings

PGC-1α mRNA levels are decreased in blood vessel media of STZ-treated diabetic rats. In cultured rat VSMCs, high glucose dose-dependently inhibits PGC-1α mRNA expression. Overexpression of PGC-1α either by infection with adenovirus, or by stimulation with palmitic acid, significantly reduces high glucose-induced VSMC proliferation and migration. In contrast, suppression of PGC-1α by siRNA mimics the effects of glucose on VSMCs. Finally, mechanistic studies suggest that PGC-1α-mediated inhibition of VSMC proliferation and migration is regulated through preventing ERK1/2 phosphorylation.

Conclusions/Significance

These results indicate that PGC-1α is a key regulator of high glucose-induced proliferation and migration in VSMCs, and suggest that elevation of PGC-1α in VSMC could be a useful strategy in preventing the development of diabetic atherosclerosis.     

Rhinovirus and dsRNA Induce RIG-I-Like Receptors and Expression of Interferon β and λ1 in Human Bronchial Smooth Muscle Cells

Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-β and IFN-λ₁ production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1–10 µg/ml dsRNA, 0.1–1 µg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05–0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-β and IFN-λ₁ mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-β and IFN-λ₁ protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-β, and IFN-λ₁ mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-β and IFN-λ₁.     

Binding of Extracellular Maspin to ��1 Integrins Inhibits Vascular Smooth Muscle Cell Migration

Maspin is a serpin that has multiple effects on cell behavior, including inhibition of migration. How maspin mediates these diverse effects remains unclear, as it is devoid of protease inhibitory activity. We have previously shown that maspin rapidly inhibits the migration of vascular smooth muscle cells (VSMC), suggesting the involvement of direct interactions with cell surface proteins. Here, using immunofluorescence microscopy, we demonstrate that maspin binds specifically to the surface of VSMC in the dedifferentiated, but not the differentiated, phenotype. Ligand blotting of VSMC lysates revealed the presence of several maspin-binding proteins, with a protein of 150 kDa differentially expressed between the two VSMC phenotypes. Western blotting suggested that this protein was the β1 integrin subunit, and subsequently both α3β1 and α5β1, but not αvβ3, were shown to associate with maspin by coimmunoprecipitation. Specific binding of these integrins was also observed using maspin-affinity chromatography, using HT1080 cell lysates. Direct binding of maspin to α5β1 was confirmed using a recombinant α5β1-Fc fusion protein. Using conformation-dependent anti-β1 antibodies, maspin binding to VSMC was found to lead to a decrease in the activation status of the integrin. The functional involvement of α5β1 in mediating the effect of maspin was established by the inhibition of migration of CHO cells overexpressing human α5 integrin, but not those lacking α5 expression. Our observations suggest that maspin engages in specific interactions with a limited number of integrins on VSMC, leading to their inactivation, and that these interactions are responsible for the effects of maspin in the pericellular environment.Maspin is a member of the serpin family of serine protease inhibitors (SERPINB5).² It was originally identified as a gene down-regulated in invasive breast cancer and proposed as a class II tumor suppressor (1), and has since been shown to have many effects on cellular behavior that are consistent with this activity. It has been shown to decrease the proliferation, migration, and metastasis of tumor cells in vivo (1, 2) and their invasion in vitro (3, 4), and to increase apoptosis of endothelial cells (5) and inhibit angiogenesis (6). However, the cellular effects of maspin are not restricted to tumor cells, and we have demonstrated that maspin can inhibit the migration of vascular smooth muscle cells (7).VSMC migration is a key event in the development of atherosclerosis (8), and contributes significantly to restenosis after angioplasty (9) and transplant arteriosclerosis (10). VSMC are not terminally differentiated and acquire migratory capacity as part of a phenotypic switch from a contractile, quiescent state to a dedifferentiated phenotype, characterized by proliferation and increased extracellular matrix synthesis, in addition to motility (11). This allows VSMC to respond to environmental cues following vascular injury. The phenotypic plasticity of VSMC is regulated by an array of signals, among which integrin-mediated association with surrounding extracellular matrix and changes in the expression of matrix-degrading proteases are prominent (12–14).How maspin mediates its various cellular effects is unclear. Maspin has been reported to be an inhibitor of plasminogen activation (3, 15, 16), but we have shown that maspin is unable to inhibit either uPA- or tPA-catalyzed plasminogen activation under conditions in which the serpin PAI-1 was completely inhibitory (7). The anti-proteolytic inhibitory mechanism of serpins is dependent on characteristics of the reactive center loop (RCL) allowing it to adopt the necessary canonical conformation and rearrangements subsequent to protease binding (17). The RCL of maspin does not have the required characteristics (7, 18), and the conclusion that maspin is a non-inhibitory serpin is fully supported by its crystal structure (19, 20).Another confounding factor in understanding the mechanisms underlying the cellular effects of maspin is that, in common with the serpin PAI-2, it lacks an authentic secretion signal sequence. Nevertheless it has been shown to enter secretory vesicles (21) and is found extracellularly, in the cytoplasm and also in the nucleus (21, 22). Cytoplasmic and nuclear binding proteins for maspin have been identified (23–25), and may be responsible for its effects on proliferation and apoptosis. How secreted, extracellular maspin exerts its effects is unclear, but a function as a cell signaling ligand has been proposed (26–28). However, the characteristics of the maspin inhibitory effect on VSMC migration point to a more direct effect of maspin.To determine the mechanism of the maspin effect on VSMC migration, we have now attempted to identify maspin-binding proteins on the surface of these cells. In this report we provide biochemical, cellular, and functional evidence that the effect of maspin on cell migration is mediated by specific binding to cell adhesion receptors of the integrin family. We find that maspin binds specifically to β1 integrins on the surface of dedifferentiated VSMC, which leads to a reduction in the activation status of the integrin, and that the binding of maspin to α5β1 is sufficient for its inhibitory effects on cell migration and may represent a more general mechanism underlying its diverse biological effects.     

Essential Role of TGF-β/Smad Pathway on Statin Dependent Vascular Smooth Muscle Cell Regulation

Background

The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-β (TGF-β) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-β/Smad pathway in atherosclerosis and vascular cells.

Methodology

In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-β. In addition, statins upregulated TGF-β receptor type II (TRII), and increased TGF-β synthesis and TGF-β/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-β induced apoptosis and increased TGF-β-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-β/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected.

Conclusions

Statins enhance TGF-β/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-β/Smad pathway is essential for statins-dependent actions in VSMCs.     

Prostacyclin and PPARα Agonists Control Vascular Smooth Muscle Cell Apoptosis and Phenotypic Switch through Distinct 14-3-3 Isoforms

We hypothesized that prostacyclin (PGI₂) protects vascular smooth muscle cell (VSMC) against apoptosis and phenotypic switch through peroxisome proliferator-activated receptor-α (PPARα) activation and 14-3-3 upregulation. Here we showed that transfection of rat aortic VSMC, A-10, with PGI₂-producing vectors, Ad-COPI, resulted in attenuated H₂O₂-induced apoptosis accompanied by a selective increase in 14-3-3β and 14-3-3θ expression. Carbaprostacyclin (cPGI₂) and Wy14,643 exerted a similar effect. The effects of PGI₂ were abrogated by MK886, a PPARα antagonist, but not GSK3787, a PPARδ antagonist. PPARα transfection upregulated 14-3-3β and θ expression and attenuated H₂O₂-induced apoptosis. H₂O₂-induced 14-3-3β but not 14-3-3θ degradation was blocked by a caspase 3 inhibitor. Furthermore, 14-3-3β but not 14-3-3θ overexpression reduced, while 14-3-3β siRNA aggravated apoptosis. VSMC contractile proteins and serum response factor (SRF) were reduced in H₂O₂-treated A-10 cells which were concurrently prevented by caspase 3 inhibitor. By contrast, PGI₂ prevented H₂O₂-induced SM22α and Calponin-1 degradation without influencing SRF. cPGI₂ and Wy14,643 also effectively blocked VSMC phenotypic switch induced by growth factors (GFs). GFs suppressed 14-3-3β, θ, ε and η isoforms and cPGI₂ prevented the decline of β, θ and η, but not ε. 14-3-3θ siRNA abrogated the protective effect of cPGI₂ on SM22α and Calponin-1 while 14-3-3 θ or 14-3-3β overexpression partially restored SM22α. These results indicated that PGI₂ protects VSMCs via PPARα by upregulating 14-3-3β and 14-3-3θ. 14-3-3β upregulation confers resistance to apoptosis whereas 14-3-3θ and β upregulation protects SM22α and Calponin-1 from degradation.     

Pathway of Programmed Cell Death and Oxidative Stress Induced by β-Hydroxybutyrate in Dairy Cow Abomasum Smooth Muscle Cells and in Mouse Gastric Smooth Muscle

The administration of exogenous β-hydroxybutyrate (β-HB), as well as fasting and caloric restriction, is a condition associated with β-HB abundance and decreased appetite in animals. Increased β-HB and decreased appetite exist simultaneously in some diseases, such as bovine left displaced abomasums (LDA) and human chronic gastritis. However, the effects of β-HB on stomach injuries have not been explored. To elucidate the possible effects of exogenous β-HB on the stomach, mice were injected intraperitoneally with β-HB, and bovine abomasum smooth muscle cells (BSMCs) were treated with different concentrations of β-HB. We found that β-HB induced BSMCs endoplasmic reticulum- and mitochondria-mediated apoptotic cell death. β-HB promoted Bax expression and caspase-12, -9, and -3 activation while blocking Bcl-2 expression. β-HB also promoted AIF, EndoG release and p53 expression. β-HB acted on key molecules in the apoptotic cell death pathway and increased p38 and c-June NH2-terminal kinase phosphorylation while inhibiting ERK phosphorylation and PCNA expression. β-HB upregulated P27 and P21 mRNA levels while downregulating cyclin and CDK mRNA levels, arresting the cell cycle. These results suggest that BSMCs treated with β-HB can induce oxidative stress, which can be prevented by intracellular calcium chelators BAPTA/AM but not antioxidant NAC. Additionally, these results suggest that β-HB causes ROS generation through a Ca²⁺-dependent mechanism and that intracellular Ca²⁺ levels play a critical role in β-HB -induced apoptotic cell death. The impact of β-HB on programmed cell death and oxidative stress in vivo was confirmed in murine experiments. For the first time, we show oxidative stress effects of β-HB on smooth muscle. We propose that β-HB is a possible cause of some stomach diseases, including bovine LDA.     

Vascular Smooth Muscle Cell Motility Is Mediated by a Physical and Functional Interaction of Ca2+/Calmodulin-dependent Protein Kinase IIδ2 and Fyn

In vascular smooth muscle (VSM) cells, Ca²⁺/calmodulin-dependent protein kinase IIδ₂ (CaMKIIδ₂) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ₂ was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ₂ and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ₂ and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ₂ and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ₂ and Fyn, implicating CaMKIIδ₂ as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ₂ and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ₂ and Fyn may coordinately regulate VSM cell motility.