Dominant negative insulin-like growth factor-1 receptor inhibits neointimal formation through suppression of vascular smooth muscle cell migration and proliferation, and induction of apoptosis

Blocking of the IGF-1 signaling pathway targeting the IGF-1 receptor (IGF-1R) provides a potential treatment strategy for restenosis. In this study, we have examined the effects of a dominant negative IGF-1R (IGF-1Rt) on primary rat VSMCs in vitro and on injured rat carotid artery in vivo. Ad/IGF-1Rt infection inhibited VSMC migration and proliferation, and it also induced apoptosis by inhibiting phosphorylation of Akt and phosphorylation of ERK1/2. Consistent with the anti-proliferative and apoptotic effects in vitro, the Ad/IGF-1Rt infection markedly reduced neointimal formation in carotid injury model. Ad/IGF-1Rt treated carotid arteries exhibited a suppressed proliferation index, PCNA expression, and also were stained positive for TUNEL assay. These results indicate that a dominant negative IGF-1R has the potential to reduce neointimal formation of injured rats' carotid arteries. The delivery of dominant negative IGF-1R by adenoviral or other vectors may provide a useful strategy for inhibiting restenosis after angioplasty.     

<Emphasis Type="Italic">Cordyceps bassiana</Emphasis> inhibits smooth muscle cell proliferation via the ERK1/2 MAPK signaling pathway

Cordyceps belongs to a genus of acormycete fungi and is known to exhibit various pharmacological effects. The aim of this study was to investigate the effect of Cordyceps species on the proliferation of vascular smooth muscle cells (VSMC) and their underlying molecular mechanism. A cell proliferation assay showed that Cordyceps bassiana ethanol extract (CBEE) significantly inhibited VSMC proliferation. In addition, neointimal formation was significantly reduced by treatment with CBEE in the carotid artery of balloon-injured rats. We also investigated the effects of CBEE on the extracellular signal-regulated kinase (ERK) signal pathway. Western blot analysis revealed increased ERK 1/2 phosphorylation in VSMCs treated with CBEE. Pretreatment with U0126 completely abrogated CBEE-induced ERK 1/2 phosphorylation. In conclusion, CBEE exhibited anti-proliferative properties that affected VSMCs through the ERK1/2 MAPK signaling pathway. Our data may elucidate the inhibitory mechanism of this natural product.     

Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells

Angiogenic factor AGGF1 (AngioGenic factor with G-patch and FHA (Forkhead-Associated) domain 1) blocks neointimal formation (formation of a new or thickened layer of arterial intima) after vascular injury by regulating phenotypic switching of vascular smooth muscle cells (VSMCs). However, the AGGF1 receptor on VSMCs and the underlying molecular mechanisms of its action are unknown. In this study, we used functional analysis of serial AGGF1 deletions to reveal the critical AGGF1 domain involved in VSMC phenotypic switching. This domain was required for VSMC phenotypic switching, proliferation, cell cycle regulation, and migration, as well as the regulation of cell cycle inhibitors cyclin D, p27, and p21. This domain also contains an RDDAPAS motif via which AGGF1 interacts with integrin α7 (ITGA7), but not α8. In addition, we show that AGGF1 enhanced the expression of contractile markers MYH11, α-SMA, and SM22 and inhibited MEK1/2, ERK1/2, and ELK phosphorylation in VSMCs, and that these effects were inhibited by knockdown of ITGA7, but not by knockdown of ITGA8. In vivo, deletion of the VSMC phenotypic switching domain in mice with vascular injury inhibited the functions of AGGF1 in upregulating α-SMA and SM22, inhibiting MEK1/2, ERK1/2, and ELK phosphorylation, in VSMC proliferation, and in blocking neointimal formation. Finally, we show the inhibitory effect of AGGF1 on neointimal formation was blocked by lentivirus-delivered shRNA targeting ITGA7. Our data demonstrate that AGGF1 interacts with its receptor integrin α7 on VSMCs, and this interaction is required for AGGF1 signaling in VSMCs and for attenuation of neointimal formation after vascular injury.     

MicroRNA-31 regulated by the extracellular regulated kinase is involved in vascular smooth muscle cell growth via large tumor suppressor homolog 2

Aberrant growth of vascular smooth muscle cells (VSMCs) is a major cellular event in the pathogenesis of many proliferative vascular diseases. Recently, microRNA-31 (miR-31) has been found to play a critical role in cancer cell proliferation. However, the biological role of miR-31 in VSMC growth and the mechanisms involved are currently unknown. In the present study, the expression of rat mature miR-31 (rno-miR-31) was determined in cultured VSMCs and in rat carotid arteries. We identified that rno-miR-31 is an abundant miRNA in VSMCs, and its expression was significantly increased in proliferative VSMCs and in vascular walls with neointimal growth. The up-regulation of rno-miR-31 in proliferative VSMCs was inhibited by the inhibitor of mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK). By both gain-of-function and loss-of-function approaches, we demonstrated that rno-miR-31 had a proproliferative effect on VSMCs. We further identified that LATS2 (large tumor suppressor homolog 2) is a downstream target gene product of rno-miR-31 that is involved in rno-miR-31-mediated effect on VSMC proliferation. The LATS2 as a target gene protein of rno-miR-31 is verified in vivo in balloon-injured rat carotid arteries. The results suggest that MAPK/ERK/miR-31/LATS2 may represent a novel signaling pathway in VSMC growth. miR-31 is able to enhance VSMC proliferation via its downstream target gene product, LATS2.     

Insulin-like growth factor-I (IGF-I) induces epidermal growth factor receptor transactivation and cell proliferation through reactive oxygen species

Insulin-like growth factor-I (IGF-I) plays an important role in proliferation of vascular smooth muscle cells (VSMCs). However, the mechanism that IGF-I induces VSMCs proliferation is not completely understood. In this study, we determined (a) whether and how IGF-I induces transactivation of epidermal growth factor receptor (EGFR) in primary rat aortic VSMCs, (b) the contribution of EGFR to IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK) and cell proliferation, and (c) the role of reactive oxygen species (ROS) in the cellular function. We showed that IGF-I induced phosphorylation of EGFR and ERK1/2 in VSMCs. AG1478, an EGFR inhibitor, inhibited IGF-I-induced phoshorylation of EGFR and ERK1/2. IGF-I stimulated ROS production and Src activation. Antioxidants inhibited IGF-I-induced ROS generation and activation of EGFR, ERK, and Src. Src kinase inhibitor PP1 and Src siRNA blocked IGF-I-induced activation of EGFR and ERK1/2. Inhibition of IGF-I-stimulated EGFR activation inhibited IGF-I-induced VSMC proliferation. These results suggest that (1) IGF-I induces EGFR activation through production of ROS and ROS-mediated Src activation in VSMCs, and (2) EGFR transactivation is required for IGF-I-induced VSMC proliferation.     

PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.     

Anti-proliferative effect of rosiglitazone on angiotensin II-induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway

VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.     

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.     

Isoeugenodilol inhibits smooth muscle cell proliferation and neointimal thickening after balloon injury via inactivation of ERK1/2 pathway

The purpose of this study was to determine the efficacy and the possible mechanism of action of the synthesized drug isoeugenodilol (a new third-generation β-adrenoceptor blocker) on the growth factor-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a rat carotid arterial balloon injury model. Isoeugenodilol significantly inhibited 10% FBS, 20 ng/ml PDGF-BB, and 20 ng/ml vascular endothelial growth factor (VEGF)-induced proliferation. In accordance with these findings, isoeugenodilol revealed blocking of the FBS-inducible progression through the G₀/G₁ to the S phase of the cell cycle in synchronized cells. Neointimal formation, measured 14 days after injury, was reduced by the oral administration of isoeugenodilol (10 mg/kg/day). In an in vitro assay, isoeugenodilol inhibited the migration of VSMCs stimulated by PDGF-BB. These findings indicate that isoeugenodilol shows an inhibitory potency on neointimal formation due to inhibition of both migration and proliferation of VSMCs. In addition, isoeugenodilol in concentration-dependent manner decreased the levels of phosphorylated ERK1/2 in both VSMCs and balloon-injured carotid arteries. The levels of phosphorylated MEK1/2 and Pyk2 as well as intracellular Ca²⁺ and reactive oxygen species (ROS) were in concentration-dependent manner reduced by isoeugenodilol. Taken together, these results indicate that isoeugenodilol may suppress mitogen-stimulated proliferation and migration partially through inhibiting cellular ROS and calcium, and hence, through activation of the Pyk2-ERK1/2 signal pathway. This suggests that isoeugenodilol has potential for the prevention of atherosclerosis and restenosis.     

YS 49, 1-(alpha-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, regulates angiotensin II-stimulated ROS production, JNK phosphorylation and vascular smooth muscle cell proliferation via the induction of heme oxygenase-1

Overexpression of the gene for heme oxygenase (HO)-1 leads to a reduction in pressor responsiveness to angiotensin II (Ang II) in experimental animals. Using rat vascular smooth muscle cells (VSMCs), we tested whether YS 49 [1-(alpha-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] inhibits Ang II-stimulated proliferation of VSMCs via induction of HO-1. YS 49 induced HO-1 protein production in a dose-and time-dependent manner in VSMCs. Treatment with YS 49 significantly and dose-dependently inhibited Ang II-induced VSMC proliferation, ROS production, and phosphorylation of JNK, but not P38 MAP kinase or ERK1/2. The antiproliferation effect of YS 49 was reversed by pretreatment with the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX), or with hemoglobin, a carbon monoxide (CO) scavenger. Similarly, VSMC proliferation, ROS production and phosphorylation of JNK by Ang II were significantly inhibited in VSMCs transfected with the HO-1 gene. Thus, HO-1 and the HO-1 product CO play, at least in part, a crucial role in Ang II-stimulated VSMC proliferation through the regulation of ROS production and JNK phosphorylation. Therefore, YS 49 has potential as a therapeutic strategy for the pathogenesis of Ang II-related vascular diseases such as hypertension and atherosclerosis, via the induction of HO-1 gene activity.     

Effect of sinomenine on vascular smooth muscle cell dedifferentiation and neointima formation after vascular injury in mice

Sinomenine, a pure alkaloid extract from Sinomenium acutum, has anti-inflammatory and immunoregulatory functions. This study investigated the efficiency and the signalling pathways involved in the effect of sinomenine on vascular smooth muscle cell (VSMC) dedifferentiation in response to platelet-derived growth factor (PDGF)-BB stimulation and vascular injury. VSMCs were isolated from rat aorta and preincubated with sinomenine before being stimulated with PDGF-BB. WST and BrdU incorporation assays were used to evaluate VSMC proliferation. Flow cytometric analysis was performed for testing the cell cycle progression. The cell migration of VSMCs were analysed using a Transwell system. The expression of VSMC specific genes and signalling proteins were tested by Western blot. For the animal study, C57/BL6 mice were fed either normal rodent chow diets or sinomenine chow diets that supplemented with 0.09 % sinomenine (w/w) in the normal chows for 14 days before carotid artery wire injury. PDGF-BB activated the dedifferentiation of VSMCs characterised by decreased expression of SMA, Smoothelin and SM22α. However, sinomenine treatment preserved the dedifferentiation in response to PDGF-BB. The activations of mitogen-activated protein kinase extracellular signal-regulated kinases, Akt, GSK3β and STAT3 induced by PDGF-BB were also inhibited in sinomenine-treated VSMCs. In vivo evidence with wire-injured mice exhibited a reduction in neointimal area and an increase in smooth muscle-specific gene expression in the sinomenine-treated group. In this study, we found that sinomenine-suppressed VSMC phenotype switching induced by PDGF-BB in vitro and neointimal formation in vivo. Therefore, sinomenine is a potential candidate to be used in the treatment of vascular proliferative disease.     

Sphingolipids stimulate cell growth via MAP kinase activation in osteoblastic cells

Ceramide, ceramide-1-phosphate (C1P) sphingosine (SPH) and sphingosine-1-phosphate (S1P) effects on proliferation and extracellular-signal regulated kinases, ERKs (also known as MAPKs), activation were investigated in human and rat osteoblastic cells. MAPK activation was sphingolipid-specific in cells from both species. In human osteoblastic cells, S1P and C1P markedly stimulated ERK2 phosphorylation with a slight increase in phosphorylation of ERK1. SPH nor ceramide induced phosphorylation of either ERK isoform. In rat osteoblastic cells, SIP, ceramide and SPH stimulated phosphorylation of both isoforms. C1P did not induce phosphorylation of ERK1 but produced a mild increase in phosphorylation of ERK2. In human cells, only S1P significantly (P<0.05) increased osteoblastic cell proliferation, while in the rat cells all four sphingolipids significantly (P<0.05) induced proliferation. The calcium channel blocker verapamil blocked (P<0.05) these effects in both cell types. The MAPK inhibitor, PD98059, inhibited (P<0.05) the mitogenic effect of SIP in human cells. In rat cells, PD98059 effects were less substantial but significant for S1P and C1P. This study demonstrates that sphingolipids are mitogens for both human and rat osteoblastic cells with the MAPK pathway and calcium mediating in part these effects in a species specific manner.     

PLD1 promotes reactive oxygen species production in vascular smooth muscle cells and injury-induced neointima formation

Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1^?/? VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1^?/? VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty.     

The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1α expression

Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 alpha (PGC-1α) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1α in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1α expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1α mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1α expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1α by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1α had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1α decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPARγ activation by a PPARγ antagonist GW9662 abolished the suppressive effects of PGC-1α on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1α were enhanced by a PPARγ agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1α expression. PGC-1α suppresses PDGF-induced VSMC migration through PPARγ coactivation and, consequently, p38 MAPK inhibition.     

Carotid artery ligation induced intimal thickening and proliferation is unaffected by ageing

Following interventions to treat atherosclerosis, such as coronary artery bypass graft surgery, restenosis occurs in approximately 40% of patients. Identification of proteins regulating intimal thickening could represent targets to prevent restenosis. Our group previously demonstrated that in a murine model of vascular occlusion, Wnt4 protein expression and β-catenin signalling was upregulated which promoted vascular smooth muscle cell (VSMC) proliferation and intimal thickening. In this study, the effect of age on VSMC proliferation, intimal hyperplasia and Wnt4 expression was investigated. In vitro proliferation of VSMCs isolated from young (2 month) or old (18–20 month) C57BL6/J mice was assessed by immunocytochemistry for EdU incorporation. As previously reported, 400 ng/mL recombinant Wnt4 protein increased proliferation of VSMCs from young mice. However, this response was absent in VSMCs from old mice. As our group previously reported reduced intimal hyperplasia in Wnt4^+/? mice compared to wildtype controls, we hypothesised that impaired Wnt4 signalling with age may result in reduced neointimal formation. To investigate this, carotid artery ligation was performed in young and old mice and neointimal area was assessed 21 days later. Surprisingly, neointimal area and percentage lumen occlusion were not significantly affected by age. Furthermore, neointimal cell density and proliferation were also unchanged. These data suggest that although Wnt4-mediated proliferation was impaired with age in primary VSMCs, carotid artery ligation induced neointimal formation and proliferation were unchanged in old mice. These results imply that Wnt4-mediated proliferation is unaffected by age in vivo, suggesting that therapeutic Wnt4 inhibition could inhibit restenosis in patients of all ages.     

Studies on microRNAs that are correlated with the cancer stem cells in chronic myeloid leukemia

Vascular remodeling is characterized by the aggregation of vascular smooth muscle cells (VSMCs) in intima. Previous studies have demonstrated that dehydroepiandrosterone (DHEA), a steroid hormone, can reverse vascular remodeling. However, it is still far clear that whether and how DHEA participates in the modulation of VSMCs activation and vascular remodeling. VSMCs were obtained from the thoracic aorta of SD rats. Cell proliferation was evaluated by CCK-8 assay and BrdU assay. To measure VSMCs migration activity, a transwell chamber assay was performed. Quantitative real-time RT-PCR and western blot were used to explore the molecular mechanisms. ROS generation by VSMCs was measured by DCF fluorescence. NADPH oxidase activity and SOD activity were measured by the corresponding kits. NF-κB activity was detected by NF-κB luciferase reporter gene assay. A rat carotid artery balloon injury model was built to evaluate the neointimal formation, and plasma PGF2 was measured by ELISA. Our results showed that DHEA significantly inhibited VSMCs proliferation after angiotensin (Ang II) stimulation by down-regulation of NADPH oxidase activity and ERK1/2 phosphorylation. Ang II can increase IL-6 and MCP-1 expression, but DHEA reverses these changes via inhibiting p38-MAPK/NF-κB (p65) signaling pathway. DHEA has no significant effects on VSMCs phenotype transition, but can reduce the neointimal to media area ratio after balloon injury. DHEA can alleviate oxidative stress and inflammation in VSMCs via ERK1/2 and NF-κB signaling pathway, but has no effect on VSMCs phenotype transition. Furthermore, DHEA attenuates VSMCs activation and neointimal formation after carotid injury in vivo. Taken together, DHEA might be a promising treatment for vascular injury under pathological condition.     

Dysregulation of HSG triggers vascular proliferative disorders

Vascular proliferative disorders, such as atherosclerosis and restenosis, are the most common causes of severe cardiovascular diseases, but a common molecular mechanism remains elusive. Here, we identify and characterize a novel hyperplasia suppressor gene, named HSG (later re-named rat mitofusin-2). HSG expression was markedly reduced in hyper-proliferative vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rat arteries, balloon-injured Wistar Kyoto rat arteries, or ApoE-knockout mouse atherosclerotic arteries. Overexpression of HSG overtly suppressed serum-evoked VSMC proliferation in culture, and blocked balloon injury induced neointimal VSMC proliferation and restenosis in rat carotid arteries. The HSG anti-proliferative effect was mediated by inhibition of ERK/MAPK signalling and subsequent cell-cycle arrest. Deletion of the p21(ras) signature motif, but not the mitochondrial targeting domain, abolished HSG-induced growth arrest, indicating that rHSG-induced anti-proliferation was independent of mitochondrial fusion. Thus, rHSG functions as a cell proliferation suppressor, whereas dysregulation of rHSG results in proliferative disorders.