Glioblastoma cell death induced by asiatic acid

Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca²⁺. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca²⁺ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca²⁺-mediated necrotic cell death predominating.     

Expression and mechanism of spleen tyrosine kinase activation by angiotensin II and its implication in protein synthesis in rat vascular smooth muscle cells

Syk, a 72-kDa tyrosine kinase, is involved in development, differentiation, and signal transduction of hematopoietic and some non-hematopoietic cells. This study determined if Syk is expressed in vascular smooth muscle cells (VSMC) and contributes to angiotensin II (Ang II) signaling and protein synthesis. Syk was found in VSMC and was phosphorylated by Ang II through AT1 receptor. Ang II-induced Syk phosphorylation was inhibited by piceatannol and dominant negative but not wild type Syk mutant. Syk phosphorylation by Ang II was attenuated by cytosolic phospholipase A(2) (cPLA(2)) inhibitor pyrrolidine-1 and retrovirus carrying small interfering RNAs (shRNAs) of this enzyme. Arachidonic acid (AA) increased Syk phosphorylation, and AA- and Ang II-induced phosphorylation was diminished by inhibitors of AA metabolism (5,8,11,14-eicosatetraynoic acid) and lipoxygenase (LO; baicalein) but not cyclooxygenase (indomethacin). AA metabolites formed via LO, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids, which activate p38 MAPK, increased Syk phosphorylation. p38 MAPK inhibitor SB202190, and dominant negative p38 MAPK mutant attenuated Ang II- and AA-induced Syk phosphorylation. Adenovirus dominant negative c-Src mutant abolished Ang II - and AA-induced Syk phosphorylation and SB202190, and dominant negative p38 MAPK mutant inhibited Ang II-induced c-Src phosphorylation. Syk dominant negative mutant but not epidermal growth factor receptor blocker AG1478 also inhibited Ang II-induced VSMC protein synthesis. These data suggest that Syk expressed in VSMC is activated by Ang II through p38 MAPK-activated c-Src subsequent to cytosolic phospholipase A(2) and generation of AA metabolites via LO, and it mediates Ang II-induced protein synthesis independent of epidermal growth factor receptor transactivation (Ang II --> cPLA(2) --> AA metabolites of LO --> p38 MAPK --> c-Src --> Syk --> protein synthesis).     

Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility

In addition to their role in many vital cellular functions, arachidonic acid (AA) and its eicosanoid metabolites are involved in the pathogenesis of several diseases, including atherosclerosis and cancer. To understand the potential mechanisms by which these lipid molecules could influence the disease processes, particularly cardiovascular diseases, we studied AA's effects on vascular smooth muscle cell (VSMC) motility and the role of cAMP-response element binding protein-1 (CREB-1) in this process. AA exerted differential effects on VSMC motility; at lower doses, it stimulated motility, whereas at higher doses, it was inhibitory. AA-induced VSMC motility requires its conversion via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. AA stimulated the phosphorylation of extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38MAPK) in a time-dependent manner, and blockade of these serine/threonine kinases significantly attenuated AA-induced VSMC motility. In addition, AA stimulated CREB-1 phosphorylation and activity in a manner that was also dependent on its metabolic conversion via the LOX and COX pathways and the activation of ERKs and p38MAPK but not JNKs. Furthermore, suppression of CREB-1 activation inhibited AA-induced VSMC motility. 15(S)-Hydroxyeicosatetraenoic acid and prostaglandin F2alpha, the 15-LOX and COX metabolites of AA, respectively, that are produced by VSMC at lower doses, were also found to stimulate motility in these cells. Together, these results suggest that AA induces VSMC motility by complex mechanisms involving its metabolism via the LOX and COX pathways as well as the ERK- and p38MAPK-dependent and JNK-independent activation of CREB-1.     

Angiotensin II type 1 receptor-associated protein regulates carotid intimal hyperplasia through controlling apoptosis of vascular smooth muscle cells

Intimal hyperplasia is the main cause of restenosis after carotid artery injury, and the underlying mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). Angiotensin II Type 1 Receptor-Associated Protein (ATRAP) has been reported to withstand intimal hyperplasia by inhibiting VSMCs proliferation and migration; however, whether the beneficial effect of ATRAP associates with VSMCs apoptosis remains unclarified. We demonstrated that the adenoviral-mediated overexpression of ATRAP induced VSMC apoptosis, alleviating the balloon injury-induced neointima formation in rats. Under the condition of Angiotensin-II stimulation, ATRAP overexpression induced the apoptosis of rat VSMCs by depressing the PI3K-Akt signaling; whereas up-regulation of Akt by PTEN inhibitor abolished the apoptotic death. Thus, ATRAP regulates carotid intimal hyperplasia through controlling the PI3K-Akt signal-mediated VSMCs apoptosis.     

Inhibition of cell cycle progression and migration of vascular smooth muscle cells by prostaglandin D2 synthase: resistance in diabetic Goto-Kakizaki rats

The regulation of vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis plays a clear role in the atherosclerotic process. Recently, we reported on the inhibition of the exaggerated growth phenotype of VSMCs isolated from hypertensive rats by lipocalin-type prostaglandin D₂ synthase (L-PGDS). In the present study, we report the differential effects of L-PGDS on VSMC cell cycle progression, migration, and apoptosis in wild-type VSMCs vs. those from a type 2 diabetic model. In wild-type VSMCs, exogenously added L-PGDS delayed serum-induced cell cycle progression from the G₁ to S phase, as determined by gene array analysis and the decreased protein expressions of cyclin-dependent kinase-2, p21^Cip1, and cyclin D1. Cyclin D3 protein expression was unaffected by L-PGDS, although its gene expression was stimulated by L-PGDS in wild-type cells. In addition, platelet-derived growth factor-induced VSMC migration was inhibited by L-PGDS in wild-type cells. Type 2 diabetic VSMCs, however, were resistant to the L-PGDS effects on cell cycle progression and migration. L-PGDS did suppress the hyperproliferation of diabetic cells, albeit through a different mechanism, presumably involving the 2.5-fold increase in apoptosis and the concomitant 10-fold increase of L-PGDS uptake we observed in these cells. We propose that in wild-type VSMCs, L-PGDS retards cell cycle progression and migration, precluding hyperplasia of the tunica media, and that diabetic cells appear resistant to the inhibitory effects of L-PGDS, which consequently may help explain the increased atherosclerosis observed in diabetes. apoptosis; atherosclerosis; insulin resistance     

Involvement of clusterin in 15-deoxy-delta12,14-prostaglandin J2-induced vascular smooth muscle cell differentiation

To establish an in vitro model of vascular smooth muscle cell (VSMC) differentiation, we examined the effect of 15-deoxy-delta12,14-prostaglandin J(2) (15d-PGJ(2)) on the expression of VSMC differentiation markers. After the addition of 15d-PGJ(2) to confluent human umbilical artery smooth muscle cells synchronized in the G(0) phase, cells showed a "hill and valley" appearance and thereafter aggregated and formed macroscopic nodules. Cells forming nodules expressed high levels of SM2, the most specific VSMC differentiation marker, comparable to medial VSMCs in vivo. 15d-PGJ(2) significantly increased the mRNA and protein expression levels of clusterin, a secreted glycoprotein reported to induce nodule formation and differentiation of VSMCs. Moreover, addition of an anti-clusterin antibody completely inhibited the nodule formation induced by 15d-PGJ(2) and induced apoptosis. Our results suggested that clusterin is involved in 15d-PGJ(2)-induced nodule formation and cell differentiation in VSMCs.     

Evidence for two-pore domain potassium channels in rat cerebral arteries

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.     

Protective effect of p53 in vascular smooth muscle cells against nitric oxide-induced apoptosis is mediated by up-regulation of heme oxygenase-2

The tumor suppressor gene p53 regulates apoptotic cell death and the cell cycle. In this study, we investigated the role of p53 in nitric oxide (NO)-induced apoptosis in vascular smooth muscle cells (VSMCs). We found that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased apoptotic cell death in p53-deficient VSMCs compared with wild-type cells. The heme oxygenase (HO) inhibitor tin protoporphyrin IX reduced the resistance of wild-type VSMCs to SNAP-induced cell death. SNAP promoted HO-1 expression in both cell types. HO-2 protein was increased only in wild-type VSMCs following SNAP treatment; however, similar levels of HO-2 mRNA were detected in both cell types. SNAP significantly increased the levels of non-heme-iron and dinitrosyl iron-sulfur clusters in wild-type VSMCs compared with p53-deficient VSMCs. Moreover, pretreatment with FeSO4 and the carbon monoxide donor CORM-2, but not biliverdin, significantly protected p53-deficient cells from SNAP-induced cell death compared with normal cells. These results suggest that wild-type VSMCs are more resistant to NO-mediated apoptosis than p53-deficient VSMCs through p53-dependent up-regulation of HO-2.     

Inhibition of proliferation and apoptosis of vascular smooth muscle cells by ghrelin

To evaluate the possible role of ghrelin in the development of atherosclerosis, its effects on tumor necrosis factor (TNF)-alpha-induced proliferation and apoptosis of vascular smooth muscle cells (VSMCs) were investigated. Rat VSMCs were pretreated with different concentrations of ghrelin and then with TNF-alpha. VSMC proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry method. Apoptosis was detected using propidium iodide and Annexin-V labeling method. Exogenous ghrelin (10-1000 ng/ml) significantly inhibited TNF-alpha-induced proliferation of VSMCs in a concentration-dependent manner. Treatment with 1000 ng/ml ghrelin was most effective at inhibiting VSMC proliferation rate and the expression of proliferating cell nuclear antigen. However, treatment with des-acyl ghrelin affected neither proliferation nor PCNA expression. In contrast, TNF-alpha-induced apoptosis of VSMCs was inhibited by both ghrelin and des-acyl ghrelin in concentration-dependent manners, with maximal inhibition observed for both compounds at 1000 ng/ml. Taken together, our results suggested that ghrelin inhibited both the proliferation and apoptosis of rat VSMCs. Furthermore, the former effect is probably mediated by the growth hormone secretagogue receptor type 1a receptor, while the latter effect may be mediated through other receptors.     

Glutamate dehydrogenase requirement for apoptosis induced by aristolochic acid in renal tubular epithelial cells

Ingestion of aristolochic acids (AA) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adduct formation, is well-documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. Epithelial cell death is a critical characteristic of these pathological conditions. To elucidate the mechanisms of AA-induced cytotoxicity, we explored AA-interacting proteins in tubular epithelial cells (TEC). We found that AA interacts with a mitochondrial enzyme glutamate dehydrogenase (GDH) and moderately inhibits its activity. We report that AA induces cell death in GDH-knockdown TEC preferentially via non-apoptotic means, whereas in GDH-positive cells, death was executed by both the non-apoptotic and apoptotic mechanisms. Apoptosis is an energy-reliant process and demands higher adenosine 5′-triphosphate (ATP) consumption than does the non-apoptotic cell death. We found that, after AAI treatment, the ATP depletion is more pronounced in GDH-knockdown cells. When we reduced ATP in TEC cells by inhibition of glycolysis and mitochondrial respiration, cell death mode switched from apoptosis and necrosis to necrosis only. In addition, in cells incubated at low glucose and no glutamine conditions, oxaloacetate and pyruvate reduced AAI-induced apoptosis our data suggest that AAI-GDH interactions in TEC are critical for the induction of apoptosis by direct inhibition of GDH activity. AA binding may also induce changes in GDH conformation and promote interactions with other molecules or impair signaling by GDH metabolic products, leading to apoptosis.     

Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2(+)-dependent phospholipase A2

Angiotensin II (ANG II) promotes vascular smooth muscle cell (VSMC) growth, stimulates Ca(2+)-calmodulin (CaM)-dependent kinase II (CaMKII), and activates cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2), which releases arachidonic acid (AA). ANG II also generates H2O2 and activates Akt, which have been implicated in ANG II actions in VSMC. This study was conducted to investigate the relationship of these signaling molecules to Akt activation in rat aortic VSMC. ANG II increased Akt activity, as measured by its phosphorylation at serine-473. ANG II (200 nM)-induced Akt phosphorylation was decreased by extracellular Ca2+ depletion and calcium chelator EGTA and inhibitors of CaM [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and CaMKII [(2-[N-(2-hydroxyethyl)]-N-(4-me-thoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine)]. cPLA2 inhibitor pyrrolidine-1, antisense oligonucleotide, and retroviral small interfering RNA also attenuated ANG II-induced Akt phosphorylation. AA increased Akt phosphorylation, and AA metabolism inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) blocked ANG II- and AA-induced Akt phosphorylation (199.03 +/- 27.91% with ANG II and 110.18 +/- 22.40% with ETYA + ANG II; 405.00 +/- 86.22% with AA and 153.97 +/- 63.26% with ETYA + AA). Inhibitors of lipoxygenase (cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate) and cytochrome P-450 (ketoconazole and 17-octadecynoic acid), but not cyclooxygenase (indomethacin), attenuated ANG II- and AA-induced Akt phosphorylation. Furthermore, 5(S)-, 12(S)-, 15(S)-, and 20-hydroxyeicosatetraenoic acids and 5,6-, 11,12-, and 14,15-epoxyeicosatrienoic acids increased Akt phosphorylation. Catalase inhibited ANG II-increased H2O2 production but not Akt phosphorylation. Oleic acid, which also increased H2O2 production, did not cause Akt phosphorylation. These data suggest that ANG II-induced Akt activation in VSMC is mediated by AA metabolites, most likely generated via lipoxygenase and cytochrome P-450 consequent to AA released by CaMKII-activated cPLA2 and independent of H2O2 production.     

Arachidonic acid cytotoxicity in leukocytes: implications of oxidative stress and eicosanoid synthesis

Arachidonic acid (AA)-induced cytotoxicity was evaluated in leukocytes: the human leukemia cell lines HL-60, Jurkat and Raji and in rat lymphocytes. Such cytotoxicity was dose- and time-dependent. At concentrations below 5 microM, AA was not toxic; at 10-400 microM, AA induced apoptosis and at concentrations beyond 400 microM, necrosis. The minimum exposure time to trigger cell death was of around 1 h, but the effect was increased by longer exposure times until 6-24 h. Apoptosis was morphologically characterized by a decrease in cell and nuclear volume, chromatin condensation and DNA fragmentation and the presence of lipid bodies, without changes in organelle integrity. Biochemically, AA-induced apoptosis was associated with internucleosomal fragmentation and caspase activation, evaluated by PARP cleavage and the use of a caspase inhibitor. Necrosis was characterized by increased cell volume, presence of loose chromatin, appearance of vacuoles, loss of membrane integrity and of the definition of organelles. The apoptotic effect of AA was studied as to oxidative-reductive imbalance and the participation of eicosanoids. Apoptotic AA treatment was accompanied by an increase in the quantity of thiobarbituric acid reactive substances (TBARS), low-level chemiluminescence and in the glutathione disulfide/reduced glutathione ratio, indicating oxidative stress. The addition of tocopherol, ascorbate, prostaglandin E2 and lipoxygenase inhibitors delayed cell death, whereas the inhibition of cyclooxygenase promoted AA-induced cell death. Cell treatment with AA was accompanied by increased cellular production of LTB4. AA, therefore, is cytotoxic at physiological and supraphysiological concentrations, causing apoptosis and necrosis. Cell treatment with apoptotic concentrations of AA involves oxidative stress and changes in eicosanoid biosynthesis.     

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.     

Essential role of Pin1 via STAT3 signalling and mitochondria‐dependent pathways in restenosis in type 2 diabetes

Type 2 diabetes (T2D) is associated with accelerated restenosis rates after angioplasty. We have previously proved that Pin1 played an important role in vascular smooth muscle cell (VSMC) cycle and apoptosis. But neither the role of Pin1 in restenosis by T2D, nor the molecular mechanism of Pin1 in these processes has been elucidated. A mouse model of T2D was generated by the combination of high‐fat diet (HFD) and streptozotocin (STZ) injections. Both Immunohistochemistry and Western blot revealed that Pin1 expression was up‐regulated in the arterial wall in T2D mice and in VSMCs in culture conditions mimicking T2D. Next, increased activity of Pin1 was observed in neointimal hyperplasia after arterial injury in T2D mice. Further analysis confirmed that 10% serum of T2D mice and Pin1‐forced expression stimulated proliferation, inhibited apoptosis, enhanced cell cycle progression and migration of VSMCs, whereas Pin1 knockdown resulted in the converse effects. We demonstrated that STAT3 signalling and mitochondria‐dependent pathways played critical roles in the involvement of Pin1 in cell cycle regulation and apoptosis of VSMCs in T2D. In addition, VEGF expression was stimulated by Pin1, which unveiled part of the mechanism of Pin1 in regulating VSMC migration in T2D. Finally, the administration of juglone via pluronic gel onto injured common femoral artery resulted in a significant inhibition of the neointima/media ratio. Our findings demonstrated the vital effect of Pin1 on the VSMC proliferation, cell cycle progression, apoptosis and migration that underlie neointima formation in T2D and implicated Pin1 as a potential therapeutic target to prevent restenosis in T2D.     

Procyanidin B2 and a cocoa polyphenolic extract inhibit acrylamide-induced apoptosis in human Caco-2 cells by preventing oxidative stress and activation of JNK pathway

Humans are exposed to dietary acrylamide (AA) during their lifetime; it is therefore necessary to investigate the mechanisms associated with AA induced toxic effects. Accumulating evidence indicates that oxidative stress may contribute to AA cytotoxicity, but the link between oxidative stress and AA cytotoxicity in the gastrointestinal tract, the primary organ in contact with dietary AA, has not been described. In this study, we evaluate the alterations of the redox balance induced by AA in Caco-2 intestinal cells as well as the potential protective role of natural antioxidants such as a well-standardized cocoa polyphenolic extract (CPE) and its main polyphenol components epicatechin (EC) and procyanidin B2 (PB2). We found that AA-induced oxidative stress in Caco-2 cells is evidenced by glutathione (GSH) depletion and reactive oxygen species (ROS) overproduction. AA also activated the extracellular-regulated kinases and the c-Jun N-amino terminal kinases (JNKs) leading to an increase in caspase-3 activity and cell death. Studies with appropriate inhibitors confirmed the implication of oxidative stress and JNKs activation in AA-induced apoptosis. Additionally, AA cytotoxicity was counteracted by CPE or PB2 by inhibiting GSH consumption and ROS generation, increasing the levels of gamma-glutamyl cysteine synthase and glutathione-S-transferase and blocking the apoptotic pathways activated by AA. Therefore, AA-induced cytotoxicity and apoptosis are closely related to oxidative stress in Caco-2 cells. Interestingly, natural dietary antioxidant such as PB2 and CPE were able to suppress AA toxicity by improving the redox status of Caco-2 cells and by blocking the apoptotic pathway activated by AA.     

Thymoquinone suppresses platelet‐derived growth factor‐BB–induced vascular smooth muscle cell proliferation,migration and neointimal formation

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are mainly responsible for vascular occlusion diseases, such as pulmonary arterial hypertension and restenosis. Our previous study demonstrated thymoquinone (TQ) attenuated monocrotaline‐induced pulmonary arterial hypertension. The aim of the present study is to systematically examine inhibitory effects of TQ on platelet‐derived growth factor‐BB (PDGF‐BB)–induced proliferation and migration of VSMCs in vitro and neointimal formation in vivo and elucidate the potential mechanisms. Vascular smooth muscle cells were isolated from the aorta in rats. Cell viability and proliferation were measured in VSMCs using the MTT assay. Cell migration was detected by wound healing assay and Transwell assay. Alpha‐smooth muscle actin (α‐SMA) and Ki‐67‐positive cells were examined by immunofluorescence staining. Reactive oxygen species (ROS) generation and apoptosis were measured by flow cytometry and terminal deoxyribonucleotide transferase–mediated dUTP nick end labelling (TUNEL) staining, respectively. Molecules including the mitochondria‐dependent apoptosis factors, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), PTEN/AKT and mitogen‐activated protein kinases (MAPKs) were determined by Western blot. Neointimal formation was induced by ligation in male Sprague Dawley rats and evaluated by HE staining. Thymoquinone inhibited PDGF‐BB–induced VSMC proliferation and the increase in α‐SMA and Ki‐67‐positive cells. Thymoquinone also induced apoptosis via mitochondria‐dependent apoptosis pathway and p38MAPK. Thymoquinone blocked VSMC migration by inhibiting MMP2. Finally, TQ reversed neointimal formation induced by ligation in rats. Thus, TQ is a potential candidate for the prevention and treatment of occlusive vascular diseases.     

Apatinib attenuates phenotypic switching of arterial smooth muscle cells in vascular remodelling by targeting the PDGF Receptor‐β

Apatinib (YN968D1) is a small‐molecule tyrosine kinase inhibitor（TKI）which can inhibit the activity of vascular endothelial growth factor receptor‐2 (VEGFR‐2). It has been reported that apatinib has anti‐tumour effect of inhibiting proliferation and inducing apoptosis of a variety of solid tumour cells, whereas its effect on vascular smooth muscle cells (VSMC) remains unclear. This study investigated the effect of apatinib on phenotypic switching of arterial smooth muscle cells in vascular remodelling. Compared to the vehicle groups, mice that were performed carotid artery ligation injury and treated with apatinib produced a reduction in abnormal neointimal area. For in vitro experiment, apatinib administration inhibited VSMC proliferation, migration and reversed VSMC dedifferentiation with the stimulation of platelet‐derived growth factor type BB (PDGF‐BB).In terms of mechanism, with the preincubation of apatinib, the activations of PDGF receptor‐β (PDGFR‐β) and phosphoinositide‐specific phospholipase C‐γ1 (PLC‐γ1) induced by PDGF‐BB were inhibited in VSMCs. With the preincubation of apatinib, the phosphorylation of PDGFR‐β, extracellular signal‐related kinases (ERK1/2) and Jun amino‐terminal kinases (JNK) induced by PDGF‐BB were also inhibited in rat vascular smooth muscle cell line A7r5. Herein, we found that apatinib attenuates phenotypic switching of arterial smooth muscle cells induced by PDGF‐BB in vitro and vascular remodelling in vivo. Therefore, apatinib is a potential candidate to treat vascular proliferative diseases.