Ohioensin F suppresses TNF-α-induced adhesion molecule expression by inactivation of the MAPK, Akt and NF-κB pathways in vascular smooth muscle cells

AimsThe expression of cell adhesion molecules on vascular smooth muscle cells is central to leukocyte recruitment and progression of atherosclerotic disease. Ohioensin F, a chemical compound of the Antarctic moss Polyerichastrum alpinum, exhibited inhibitory activity against protein tyrosine phosphatase 1B and antioxidant activity. However, published scientific information regarding other biological activities and pharmacological function of ohioensin F is scarce. In the present study, we aimed to examine the in vitro effects of ohioensin F on the ability to suppress TNF-α-induced adhesion molecule expression in vascular smooth muscle cells (VSMCs).Main methodsThe inhibitory effect of ohioensin F on TNF-α-induced upregulation in expression of adhesion molecules was investigated by enzyme-linked immunosorbent assay, cell adhesion assay, RT-PCR, western blot analysis, immunofluorescence, and transfection and reporter assay, respectively.Key findingsPretreatment of VSMCs with ohioensin F at nontoxic concentrations of 0.1–10 μg/ml dose-dependently inhibited TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In addition, ohioensin F suppressed adhesion of THP-1 monocytes to TNF-α-stimulated VSMCs. Ohioensin F reduced TNF-α-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Finally, ohioensin F inhibited TNF-α-induced CAM mRNA expression and NK-κB translocation.SignificanceThese results suggest a new mechanism of ohioensin F's anti-inflammatory action, owing to the negative regulation of TNF-α-induced adhesion molecule expression, monocyte adhesion and ROS production in vascular smooth muscle cells. Our finding also supports ohioensin F as a potential pharmacological, anti-inflammatory molecule that has a protective effect on the atherosclerotic lesion.     

Inhibition of vascular smooth muscle cell calcification by vasorin through interference with TGFβ1 signaling

Elevated transforming growth factor β1 (TGFβ1) levels are frequently observed in chronic kidney disease (CKD) patients. TGFβ1 contributes to development of medial vascular calcification during hyperphosphatemia, a pathological process promoted by osteo−/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Vasorin is a transmembrane glycoprotein highly expressed in VSMCs, which is able to bind TGFβ to inhibit TGFβ signaling. Thus, the present study explored the effects of vasorin on osteo−/chondrogenic transdifferentiation and calcification of VSMCs. Primary human aortic smooth muscle cells (HAoSMCs) were treated with recombinant human TGFβ1 or β-glycerophosphate without or with recombinant human vasorin or vasorin gene silencing by siRNA. As a result, TGFβ1 down-regulated vasorin mRNA expression in HAoSMCs. Vasorin supplementation inhibited TGFβ1-induced pathway activation, SMAD2 phosphorylation and downstream target genes expression in HAoSMCs. Furthermore, treatment with exogenous vasorin blunted, while vasorin knockdown augmented TGFβ1-induced osteo−/chondrogenic transdifferentiation of HAoSMCs. In addition, phosphate down-regulated vasorin mRNA expression in HAoSMCs. Phosphate-induced TGFβ1 expression was not affected by addition of exogenous vasorin. Nonetheless, the phosphate-induced TGFβ1 signaling, osteo−/chondrogenic transdifferentiation and calcification of HAoSMCs were all blunted by vasorin. Conversely, silencing of vasorin aggravated osteoinduction in HAoSMCs during high phosphate conditions. Aortic vasorin expression was reduced in the hyperphosphatemic klotho-hypomorphic mouse model of CKD-related vascular calcification. In conclusion, vasorin, which suppresses TGFβ1 signaling and protects against osteo−/chondrogenic transdifferentiation and calcification of VSMCs, is reduced by pro-calcifying conditions. Thus, vasorin is a novel key regulator of VSMC calcification and may represent a potential therapeutic target for vascular calcification during CKD.     

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.     

Enhanced proliferation and migration of vascular smooth muscle cells in response to vascular injury under hyperglycemic conditions is controlled by β3 integrin signaling

Atheroma formation and restenosis following percutaneous vascular intervention involve the growth and migration of vascular smooth muscle cells (SMCs) into neointimal lesions, in part due to changes in the extracellular matrix. While some clinical studies have suggested that, in comparison to non-diabetics, β3 integrin inhibition in diabetic patients confers protection from restenosis, little is known regarding the role of β3 integrin inhibition on SMC responses in this context. To understand the molecular mechanisms underlying integrin-mediated regulation of SMC function in diabetes, we examined SMC responses in diabetic mice deficient in integrin β3 and observed that the integrin was required for enhanced proliferation, migration and extracellular regulated kinase (ERK) activation. Hyperglycemia-enhanced membrane recruitment and catalytic activity of PKCβ in an integrin β3-dependent manner. Hyperglycemia also promoted SMC filopodia formation and cell migration, both of which required αVβ3, PKCβ, and ERK activity. Furthermore, the integrin–kinase association was regulated by the αVβ3 integrin ligand thrombospondin and the integrin modulator Rap1 under conditions of hyperglycemia. These results suggest that there are differences in SMC responses to vascular injury depending on the presence or absence of hyperglycemia and that SMC response under hyperglycemic conditions is largely mediated through β3 integrin signaling.     

α-MSH inhibits TNF-α-induced maturation of human dendritic cells in vitro through the up-regulation of ANXA1

α-Melanocyte-stimulating hormone (α-MSH), an anti-inflammatory and immunomodulatory neuropeptide, has been shown to be effective in the experimental treatment of autoimmune diseases and allograft rejection. However, its regulatory mechanism is still unclear. Mature dendritic cells (DCs) are pivotal initiators of immune response and inflammation. We hypothesized that the regulatory role of α-MSH in DC maturation would contribute to the effects of α-MSH in immune-response-mediated disease models. It was found that α-MSH inhibited tumor necrosis factor-alpha (TNF-α)-induced maturation of human peripheral-monocyte-derived DCs (MoDCs), both phenotypically and functionally. This occurred through the down-regulation of the expression of co-stimulatory molecules CD83 and CD86, the production of IL-12, the promotion of IL-10 secretion, and the MoDC phagocytic activity, suggesting that the inhibition of DC maturation by α-MSH could contribute to the anti-inflammatory effect of this neuropeptide. Furthermore, increased expression of annexin A1 (ANXA1) was found to be responsible for the α-MSH inhibiting effect on TNF-α-induced MoDC maturation, which could be abolished by the treatment of MoDCs with specific, small interfering RNAs targeting ANXA1 (ANXA1-siRNA), suggesting that α-MSH-induced ANXA1 mediates the inhibition. Therefore, α-MSH inhibits TNF-α-induced maturation of human DCs through α-MSH-up-regulated ANXA1, suggesting that inhibition of the maturation of DCs by α-MSH could mediate the anti-inflammatory effect of the neuropeptide. Furthermore, ANXA1 could be identified as a new therapeutic drug target based on the role of DCs in immune-mediated inflammatory diseases.     

Involvement of mitochondrial fission in calcium sensing receptor-mediated vascular smooth muscle cells proliferation during hypertension

Hyperproliferation of vascular smooth muscle cells (VSMC) is a major risk factor for cardiovascular diseases. Proper mitochondrial fission and fusion is involved with VSMC function. However, the role and mechanism of mitochondrial morphological changes in VSMC proliferation are not well understood. Here, we found that calcium sensing receptor (CaSR) was increased in the aortas from spontaneous hypertensive rats (SHRs) compared with age-matched Wistar Kyoto (WKY) rats. There was also an increase in mitochondrial fission and VSMC proliferation, which was attenuated by Calhex231. In primary rat VMSC, angiotensin II (Ang II) stimulation induced cytosolic [Ca²⁺]_i increase, mitochondrial shortening and proliferation, all of which could be attenuated by pretreatment with mitochondrial division inhibitor-1 (Mdivi-1) and Calhex231. Our data indicate that CaSR-mediated mitochondrial fission could be a therapeutic target for hyperproliferative disorders.     

PPARδ modulates oxLDL-induced apoptosis of vascular smooth muscle cells through a TGF-β/FAK signaling axis

The peroxisome proliferator-activated receptor delta (PPARδ) has been implicated in the modulation of vascular homeostasis. However, its roles in the apoptotic cell death of vascular smooth muscle cells (VSMCs) are poorly understood. Here, we demonstrate that PPARδ modulates oxidized low-density lipoprotein (oxLDL)-induced apoptosis of VSMCs through the transforming growth factor-β (TGF-β) and focal adhesion kinase (FAK) signaling pathways. Activation of PPARδ by GW501516, which is a specific ligand, significantly inhibited oxLDL-induced cell death and generation of reactive oxygen species in VSMCs. These inhibitory effects were significantly reversed in the presence of small interfering (si)RNA against PPARδ, or by blockade of the TGF-β or FAK signaling pathways. Furthermore, PPARδ-mediated recovery of FAK phosphorylation suppressed by oxLDL was reversed by SB431542, a specific ALK5 receptor inhibitor, indicating that a TGF-β/FAK signaling axis is involved in the action of PPARδ. Among the protein kinases activated by oxLDL, p38 mitogen-activated protein kinase was suppressed by ligand-activated PPARδ. In addition, oxLDL-induced expression and translocation of pro-apoptotic or anti-apoptotic factors were markedly affected in the presence of GW501516. Those effects were reversed by PPARδ siRNA, or inhibitors of TGF-β or FAK, which also suggests that PPARδ exerts its anti-apoptotic effect via a TGF-β/FAK signaling axis. Taken together, these findings indicate that PPARδ plays an important role in the pathophysiology of disease associated with apoptosis of VSMC, such as atherosclerosis and restanosis.     

TGF-β1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells

Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-β1 (TGF-β1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-β1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-β1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-β1 and IL-6 signalling and suggested another mechanism of how defects in TGF-β signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.     

Puerarin attenuates high-glucose-and diabetes-induced vascular smooth muscle cell proliferation by blocking PKCβ2/Rac1-dependent signaling

Oxidative stress has been implicated in several steps leading to the development of diabetic vascular complications. The purpose of this study was to determine the efficacy and the possible mechanism of puerarin on high-glucose (HG; 25 mM)-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a carotid arterial balloon injury model of obese Zucker rats. Our data demonstrated that puerarin significantly inhibited rat VSMC proliferation as well as reactive oxygen species (ROS) generation and NADPH oxidase activity induced by HG treatment. Further studies revealed that HG treatment resulted in phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits, leading to NADPH oxidase activation. Puerarin treatment remarkably disrupted the phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits. Blocking PKCβ2 by infection with AdDNPKCβ2 also abolished HG-induced phosphorylation and membrane translocation of Rac1, p47phox, and p67phox subunits as well as ROS production and NADPH oxidase activation in VSMCs. In vivo neointimal formation of obese Zucker rats evoked by balloon injury was evidently attenuated by the administration of puerarin. These results demonstrate that puerarin may exert inhibitory effects on HG-induced VSMC proliferation via interfering with PKCβ2/Rac1-dependent ROS pathways, thus resulting in the attenuation of neointimal formation in the context of hyperglycemia in diabetes mellitus.     

Divergent effects of 17-β-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-α-induced neointima formation

Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α). TNF-α can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protective effects of estradiol (E2). We therefore investigated the effects of TNF-α on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-α (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-α, an effect that was abolished by co-culture with E2. TNF-α increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-α alone. SV-EC migration was significantly impaired by TNF-α (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-α increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-α potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there was no modulation by E2 in either cell-type. In conclusion, TNF-α induced SV neointima formation, increased SMC proliferation and migration, impaired SV-EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.     

TGF-β and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells

Transforming growth factor-β (TGF-β) is upregulated at the time of arterial injury; however, the mechanism through which TGF-β enhances the development of intimal hyperplasia is not clear. Recent studies from our laboratory suggest that in the presence of elevated levels of Smad3, TGF-β stimulates smooth muscle cell (SMC) proliferation. This is a novel phenomenon in that TGF-β has traditionally been known as a potent inhibitor of cellular proliferation. In these studies we explore the signaling pathways through which TGF-β mediates its proliferative effect in vascular SMCs. We found that TGF-β phosphorylates and activates Akt in a time-dependent manner, and this effect is significantly enhanced by overexpression of Smad3. Furthermore, both chemical and molecular inhibition of Smad3 can reverse the effect of TGF-β on Akt. Although we found numerous signaling pathways that might function as intermediates between Smad3 and Akt, p38 appeared the most promising. Overexpression of Smad3 enhanced p38 phosphorylation and inhibition of p38 with a chemical inhibitor or a small interfering RNA blocked TGF-β-induced Akt phosphorylation. Moreover, TGF-β/Smad3 enhancement of SMC proliferation was blocked by inhibition of p38. Phosphorylation of Akt by TGF-β/Smad3 was not dependent on gene expression or protein synthesis, and immunoprecipitation studies revealed a physical association among p38, Akt, and Smad3 suggesting that activation requires a direct protein-protein interaction. Our findings were confirmed in vivo where overexpression of Smad3 in a rat carotid injury model led to enhancement of p-p38, p-Akt, as well as SMC proliferation. Furthermore, inhibition of p38 in vivo led to decreased Akt phosphorylation and SMC proliferation. In summary, our studies reveal a novel pathway whereby TGF-β/Smad3 stimulates SMC proliferation through p38 and Akt. These findings provide a potential mechanism for the substantial effect of TGF-β on intimal hyperplasia and suggest new targets for chemical or molecular prevention of vascular restenosis.     

Essential role of NF-κB and AP-1 transcription factors in TNF-α-induced TSLP expression in human airway smooth muscle cells

Human airway smooth muscle (HASM) cells are a rich source of inflammatory mediators that may propagate the airway inflammatory responses. Recent studies from our laboratory and others demonstrate that HASM cells express the proallergic cytokine thymic stromal lymphopoietin (TSLP) in vitro and in vivo. Compelling evidence from in vitro studies and animal models suggest that the TSLP is a critical factor sufficient and necessary to induce or maintain the allergic airway inflammation. Despite of an immense interest in pathophysiology of TSLP in allergic inflammation, the triggers and mechanisms of TSLP expression remain inadequately understood. In this study, we found that TNF-α upregulates the TSLP mRNA and induces high levels of TSLP protein release in primary human ASM cells. Interestingly, TNF-α induced the TSLP promoter activity (P < 0.05; n = 4) in HASM that was mediated by upstream NF-κB and activator protein-1 (AP-1) binding sites. Mutation in NF-κB and AP-1 binding sites completely abrogated the effect of TNF-α-mediated TSLP promoter activity and so did the expression of a dominant-negative mutant construct of IκB kinase. Furthermore, the peptide inhibitors of IκB kinase or NF-κB inhibited the TNF-α-induced TSLP protein release (P < 0.05; n = 3) in HASM. Collectively, our data suggest a novel important biological role for NF-κB pathway in TNF-α-induced TSLP expression in HASM and recommend this as a prime target for anti-inflammatory drugs.