Grape Seed Procyanidin B2 Inhibits Human Aortic Smooth Muscle Cell Proliferation and Migration Induced by Advanced Glycation End Products

Advanced glycation end product (AGE)-induced vascular smooth muscle cell (VSMC) proliferation is vital to the progression of diabetic vasculopathy. A grape seed procyanidin extract has been reported to possess anti-oxidative and anti-inflammatory properties and to display a significant cardiovascular protective effect, but little is know about the underlying mechanism. The objective of this present study was to determine whether GSPB2 (grape seed procyanidin B2), which is a dimeric procyanidin and more biologically active, could inhibit AGE-induced VSMC proliferation by affecting the production of ubiquitin COOH-terminal hydrolase 1 (UCH-L1), the degradation of IκB-α and nuclear translocation of NF-κB in human aortic smooth muscle cells (HASMCs). Our data show that GSPB2 preincubation markedly inhibited AGE-induced proliferation and migration of HASMCs in a dose-dependent manner and upregulated the protein level of UCH-L1. Further studies revealed that the GSPB2 pretreatment markedly attenuated the degradation of IκB-α and nuclear translocation of NF-κB by modulating ubiquitination of IκB-α in AGE-exposed HASMCs. These results collectively suggest that AGE-induced HASMC proliferation and migration was suppressed by GSPB2 through regulating UCH-L1 and ubiquitination of IκB-α. GSPB2 may therefore have therapeutic potential in preventing and treating vascular complications of diabetes mellitus.     

Grape seed procyanidin b2 inhibits human aortic smooth muscle cell proliferation and migration induced by advanced glycation end products

Advanced glycation end product (AGE)-induced vascular smooth muscle cell (VSMC) proliferation is vital to the progression of diabetic vasculopathy. A grape seed procyanidin extract has been reported to possess anti-oxidative and anti-inflammatory properties and to display a significant cardiovascular protective effect, but little is know about the underlying mechanism. The objective of this present study was to determine whether GSPB2 (grape seed procyanidin B2), which is a dimeric procyanidin and more biologically active, could inhibit AGE-induced VSMC proliferation by affecting the production of ubiquitin COOH-terminal hydrolase 1 (UCH-L1), the degradation of IκB-α and nuclear translocation of NF-κB in human aortic smooth muscle cells (HASMCs). Our data show that GSPB2 preincubation markedly inhibited AGE-induced proliferation and migration of HASMCs in a dose-dependent manner and upregulated the protein level of UCH-L1. Further studies revealed that the GSPB2 pretreatment markedly attenuated the degradation of IκB-α and nuclear translocation of NF-κB by modulating ubiquitination of IκB-α in AGE-exposed HASMCs. These results collectively suggest that AGE-induced HASMC proliferation and migration was suppressed by GSPB2 through regulating UCH-L1 and ubiquitination of IκB-α. GSPB2 may therefore have therapeutic potential in preventing and treating vascular complications of diabetes mellitus.     

Long non‐coding RNA SNHG16 regulates human aortic smooth muscle cell proliferation and migration via sponging miR‐205 and modulating Smad2

The present study investigated the role of long non‐coding RNA (lncRNA) small nucleolar RNA host gene 16 (SNHG16) in the human aortic smooth muscle cell (HASMC) proliferation and migration and explored the potential link between SNHG16 and atherosclerosis. Our results showed that platelet‐derived growth factor (PDGF)‐bb treatment promoted cell proliferation and migration with concurrent up‐regulation of SNHG16 in HASMCs. Small nucleolar RNA host gene 16 overexpression promoted HASMC proliferation and migration, while SNHG16 knockdown suppressed cell proliferation and migration in PDGF‐bb‐stimulated HASMCs. The bioinformatic analyses showed that SNHG16 possessed the complementary binding sequence with miR‐205, where the interaction was confirmed by luciferase reporter assay and RNA pull‐down assay in HASMCs, and SNHG16 inversely regulated miR‐205 expression. MiR‐205 overexpression attenuated the enhanced effects of PDGF‐bb treatment on HASMC proliferation and migration. Moreover, Smad2 was targeted and inversely regulated by miR‐205, while being positively regulated by SNHG16 in HASMCs. Smad2 knockdown attenuated PDGF‐bb‐mediated actions on HASMC proliferation and migration. Both miR‐205 overexpression and Smad2 knockdown partially reversed the effects of SNHG16 overexpression on HASMC proliferation and migration. Moreover, SNHG16 and Smad2 mRNA were up‐regulated, while miR‐205 was down‐regulated in the plasma from patients with atherosclerosis. Small nucleolar RNA host gene 16 expression was inversely correlated with miR‐205 expression and positively correlated with Smad2 expression in the plasma from atherosclerotic patients. In conclusion, our data showed the up‐regulation of SNHG16 in pathogenic‐stimulated HASMCs and clinical samples from atherosclerotic patients. Small nucleolar RNA host gene 16 regulated HASMC proliferation and migration possibly via regulating Smad2 expression by acting as a competing endogenous RNA for miR‐205.     

LncRNA TNK2‐AS1 regulated ox‐LDL‐stimulated HASMC proliferation and migration via modulating VEGFA and FGF1 expression by sponging miR‐150‐5p

Long non‐coding RNAs (lncRNAs) have been indicated for the regulatory roles in cardiovascular diseases. This study determined the expression of lncRNA TNK2 antisense RNA 1 (TNK2‐AS1) in oxidized low‐density lipoprotein (ox‐LDL)‐stimulated human aortic smooth muscle cells (HASMCs) and examined the mechanistic role of TNK2‐AS1 in the proliferation and migration of HASMCs. Our results demonstrated that ox‐LDL promoted HASMC proliferation and migration, and the enhanced proliferation and migration in ox‐LDL‐treated HASMCs were accompanied by the up‐regulation of TNK2‐AS1. In vitro functional studies showed that TNK2‐AS1 knockdown suppressed cell proliferation and migration of ox‐LDL‐stimulated HASMCs, while TNK2‐AS1 overexpression enhanced HASMC proliferation and migration. Additionally, TNK2‐AS1 inversely regulated miR‐150‐5p expression via acting as a competing endogenous RNA (ceRNA), and the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by miR‐150‐5p overexpression. Moreover, miR‐150‐5p could target the 3’ untranslated regions of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 1 (FGF1) to regulate FGF1 and VEGFA expression in HASMCs, and the inhibitory effects of miR‐150‐5p overexpression in ox‐LDL‐stimulated HASMCs were attenuated by enforced expression of VEGFA and FGF1. Enforced expression of VEGFA and FGF1 also partially restored the suppressed cell proliferation and migration induced by TNK2‐AS1 knockdown in ox‐LDL‐stimulated HASMCs, while the enhanced effects of TNK2‐AS1 overexpression on HASMC proliferation and migration were attenuated by the knockdown of VEGFA and FGF1. Collectively, our findings showed that TNK2‐AS1 exerted its action in ox‐LDL‐stimulated HASMCs via regulating VEGFA and FGF1 expression by acting as a ceRNA for miR‐150‐5p.     

Comparison of doxycycline and minocycline in the inhibition of VEGF-induced smooth muscle cell migration

Smooth muscle cell migration plays an important role during angiogenesis and vascular remodeling. In this study, we examined the effects of doxycycline and minocycline on vascular endothelial growth factor (VEGF)-induced human aortic smooth muscle cell (HASMCs) migration, and explored the mechanisms in which doxycycline or minocycline inhibit HASMC migration. We demonstrated that both doxycycline and minocycline attain consistent anti-angiogenic effects in the inhibition of HASMC migration via a different signal pathway (p<0.05). This effect is through attenuating VEGF-induced matrix metalloproteinase-9 (MMP-9) activity (p<0.05). Doxycycline could increase tissue inhibitors of metalloproteinases-1 (TIMP-1) expression while minocycline down-regulated PI3K/Akt phosphorylation in HASMC. Our study suggests that doxycycline has a stronger ability to inhibit MMP secretion in HASMC by up-regulating endogenous MMPs inhibitor TIMP-1, while minocycline implements anti-angiogenic effect through inhibiting HASMC migration by down-regulating PI3K/Akt pathway.     

Screening and functional prediction of differentially expressed circRNAs in proliferative human aortic smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation is the pathological base of vascular remodelling diseases. Circular RNAs (circRNAs) are important regulators involved in various biological processes. However, the function of circRNAs in VSMC proliferation regulation remains largely unknown. This study was conducted to identify the key differentially expressed circRNAs (DEcircRNAs) and predict their functions in human aortic smooth muscle cell (HASMC) proliferation. To achieve this, DEcircRNAs between proliferative and quiescent HASMCs were detected using a microarray, followed by quantitative real‐time RT‐PCR validation. A DEcircRNA‐miRNA‐DEmRNA network was constructed, and functional annotation was performed using Gene Ontology (GO) and KEGG pathway analysis. The function of hsa_circ_0002579 in HASMC proliferation was analysed by Western blot. The functional annotation of the DEcircRNA‐miRNA‐DEmRNA network indicated that the four DEcircRNAs might play roles in the TGF‐β receptor signalling pathway, Ras signalling pathway, AMPK signalling pathway and Wnt signalling pathway. Twenty‐seven DEcircRNAs with coding potential were screened. Hsa_circ_0002579 might be a pro‐proliferation factor of HASMC. Overall, our study identified the key DEcircRNAs between proliferative and quiescent HASMCs, which might provide new important clues for exploring the functions of circRNAs in vascular remodelling diseases.     

Cyclin D1 Is a Bona Fide Target Gene of NFATc1 and Is Sufficient in the Mediation of Injury-induced Vascular Wall Remodeling

Platelet-derived growth factor BB induced cyclin D1 expression in a time- and nuclear factor of activated T cells (NFAT)-dependent manner in human aortic smooth muscle cells (HASMCs), and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G₁ to S phase. Selective inhibition of NFATc1 by its small interfering RNA also blocked HASMC proliferation and migration. Characterization of the cyclin D1 promoter revealed the presence of several NFAT binding sites, and the site at nucleotide −1333 was found to be sufficient in mediating platelet-derived growth factor BB-induced cyclin D1 promoter-luciferase reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in an NFATc1-dependent manner. Balloon injury-induced cyclin D1-CDK4 activity requires NFAT activation, and adenovirus-mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury-induced vascular wall remodeling events, including smooth muscle cell migration from the medial to luminal region, their proliferation in the intimal region, and neointima formation. Together, these results provide more mechanistic evidence for the role of NFATs, particularly NFATc1, in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the renarrowing of artery after angioplasty.     

Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation,Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a delayed time-dependent manner. MCP1 also stimulated F-actin stress fiber formation in a delayed manner in HASMCs, as well as the migration and proliferation of these cells. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 induced cyclin D1 expression as well as CDK6 and CDK4 activities, and these effects were dependent on activation of NFATc1. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak1 phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. MCP1 also activated Rac1 in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. In addition, smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities, resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1-dependent, NFATc1-mediated cyclin D1 expression and CDK6 activity mediate this effect. In addition, smooth muscle-specific deletion of NFATc1 prevented the capacity of vascular smooth muscle cells for MCP-1-induced activation of the cyclin D1-CDK6-CDK4-Pak1 signaling axis, affecting their migration and proliferation in vitro and injury-induced neointima formation in vivo.     

Pigment epithelium-derived factor reduces the PDGF-induced migration and proliferation of human aortic smooth muscle cells through PPARγ activation

Our previous study demonstrated that pigment epithelium-derived factor (PEDF) plays an important role in the proliferation and migration of human aortic smooth muscle cells (HASMCs). In the present study, we examined whether PEDF inhibited platelet-derived growth factor (PDGF)-stimulated HASMC migration and proliferation. PEDF dose-dependently reduced PDGF-induced HASMC migration and proliferation in vitro and also arrested cell cycle progression in the G0/G1 phase, and this was associated with decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and p21(Cip1) and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1). The antiproliferative and antimigratory effects of PEDF were partially blocked by the PPARγ antagonist GW9662, but not by the PPARα antagonist MK886. In in vivo studies, the femoral artery of C57BL/6 mice was endothelial-denuded and the mice injected intravenously with PEDF or vehicle. After 2 weeks, both the neointima/media area ratio and cell proliferation (proliferating cell nuclear antigen-positive cells) in the neointima were significantly reduced and again these effects were partially reversed by GW9662 pretreatment. Our data show that PEDF increases PPARγ activation, preventing entry of HASMCs into the cell cycle in vitro and reducing the neointimal area and cell proliferation in the neointima in vivo. Thus, PEDF may represent a safe and effective novel target for the prevention and treatment of vascular proliferative diseases.     

Tanshinone IIA from Salvia miltiorrhiza BUNGE inhibits human aortic smooth muscle cell migration and MMP-9 activity through AKT signaling pathway

Smooth muscle cell (SMC) migration plays an important role in normal angiogenesis and is relevant to disease-related vascular remodeling in conditions such as brain arteriovenous malformations, pulmonary hypertension, arteriosclerosis, and restenosis after angioplasty. In this present study, we showed that tanshinone IIA, the major lipid-soluble pharmacological constituent of Salvia miltiorrhiza BUNGE, inhibits human aortic smooth muscle cell (HASMC) migration and MMP-9 activity. Tanshinone IIA significantly inhibited IkappaBalpha phosphorylation and p65 nuclear translocation through inhibition of AKT phosphorylation. Tanshinone IIA inhibited TNF-alpha-induced ERK and c-jun phosphorylation, but not other MAPKs such as JNK and p38. Tanshinone IIA also inhibited NF-kappaB and AP-1 DNA-binding. Moreover, tanshinone IIA inhibited the migration of TNF-alpha-induced HASMCs. Our results provide evidence that tanshinone IIA has multiple effects in the inhibition of HASMC migration and may offer a therapeutic approach to block HASMC migration.     

Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22(phox) and gp91(phox), membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22(PHOX) and gp91(PHOX). This led to blockade of the AGE-elicited Ras activation and NF-kappaB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy.     

Purinergic signaling modulates the cerebral inflammatory response in experimentally infected fish with <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis>: an attempt to improve the immune response

Epigallocatechin gallate (EGCG), a bioactive ingredient of green tea, plays a protective role in the cardiovascular system. Homocysteine (Hcy) is a major risk factor for chronic kidney disease and cardiovascular disease. The present study aimed to investigate the role of EGCG in Hcy-induced proliferation of vascular smooth muscle cells (VSMCs) and its underlying mechanism. We also explored the roles of rennin-angiotensin system (RAS), extracellular signal-regulated kinases (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) in this process. Human aortic smooth muscle cells (HASMCs) were treated with different drugs for different periods. The proliferation rate of HASMCs was detected using the CCK-8 and BrdU labeling assays. The Western blot assay was used to determine the expression levels of angiotensin II type 1 receptor (AT-1R), ERK1/2, and p38 MAPK. Compared with the control group, the HASMCs treated with Hcy at different doses (100, 200, 500, and 1000 µM) showed significantly increased proliferation. Hcy increased the expression of AT-1R, whereas EGCG decreased the protein expression of AT-1R. Furthermore, we found that Hcy-induced expression of p-ERK1/2 and p-p38MAPK was dependent on AT-1R. Compared with Hcy (500 µM)-treated cells, EGCG (20 µM)-treated cells showed decreased proliferation as well as expression of AT-1R, p-ERK1/2, and p-p38MAPK. In addition, HASMC proliferation was suppressed by the addition of an AT-1R blocker (olmesartan), an ERK1/2 inhibitor (PD98059), and a p38MAPK inhibitor (SB202190). EGCG can inhibit AT-1R and affect ERK1/2 and p38MAPK signaling pathways, resulting in the decrease of VSMC proliferation induced by Hcy.     

BMP-2 inhibits proliferation of human aortic smooth muscle cells via p21Cip1/Waf1

Bone-morphogenetic proteins (BMP)-2 and -7, multifunctional members of the transforming growth factor (TGF)-beta superfamily with powerful osteoinductive effects, cause cell cycle arrest in a variety of transformed cell lines by activating signaling cascades that involve several cyclin-dependent kinase inhibitors (CDKIs). CDKIs in the cip/kip family, p21(Cip1/Waf1) and p27(Kip1), have been shown to negatively regulate the G1 cyclins and their partner cyclin-dependent kinase proteins, resulting in BMP-mediated growth arrest. Bone morphogens have also been associated with antiproliferative effects in vascular tissue by unknown mechanisms. We now show that BMP-2-mediated inhibition of platelet-derived growth factor (PDGF)-stimulated human aortic smooth muscle cell (HASMC) proliferation is accompanied by increased levels of p21 protein. Antisense oligodeoxynucleotides specific for p21 attenuate BMP-2-induced inhibition of proliferation when transfected into HASMCs, demonstrating that BMP-2 inhibits PDGF-stimulated proliferation of HASMCs through induction of p21. Whether p21-mediated induction of cell cycle arrest by BMP-2 sets the stage for osteogenic differentiation of vascular smooth muscle cells, ultimately leading to vascular mineralization, remains to be investigated.     

Epigallocatechin-3-gallate inhibits proliferation of human aortic smooth muscle cells via up-regulating expression of mitofusin 2

Previous studies have shown that epigallocatechin-3-gallate (EGCG) inhibits the proliferation of vascular smooth muscle cells (VSMCs) via the extracellular-signal-regulated kinase (ERK1/2) and mitogen activated protein kinases (MAPKs) pathway. Mitofusin 2 (Mfn-2) also suppresses VSMC proliferation through Ras-Raf-ERK/MAPK, suggesting a possible link between EGCG, Mfn-2 and ERK/MAPK. However, the effect of EGCG on Mfn-2 remains unknown. In this study, we investigated the role of Mfn-2 in the regulation of VSMC proliferation by EGCG, and assessed the underlying mechanisms. The effects of EGCG on the proliferation of cultured human aortic smooth muscle cells (HASMCs) were observed by 5-ethynl-2-deoxyuridine (EdU) incorporation assay. Mfn-2 gene and protein levels, and Ras, p-c-Raf and p-ERK1/2 protein levels were determined by quantitative real-time polymerase chain reaction and western blotting, respectively. Mfn-2 gene silencing was achieved by RNA interference. EGCG 50 μmol/L profoundly inhibited the proliferation of HASMCs in culture, up-regulated Mfn-2, and down-regulated the expression of p-c-Raf and p-ERK1/2. Furthermore, RNA interference-mediated gene knockdown of Mfn-2 antagonized EGCG-induced anti-proliferation and down-regulation of Ras, p-c-Raf and p-ERK1/2. These results suggest that EGCG inhibits the proliferation of HASMCs in vitro largely via Mfn-2-mediated suppression of the Ras-Raf-ERK/MAPK signaling pathway.     

Pure atmospheric pressure promotes an expression of osteopontin in human aortic smooth muscle cells

Although the types of pathophysiological stimulation that initiate an overexpression of OPN have yet to be determined, we hypothesized that mechanical stress is one of the candidates which initiates OPN expression in vascular smooth muscle cells. Cell proliferation assay indicated that a pure atmospheric pressure of 160 mmHg activated cell proliferation by 11% in human aortic smooth muscle cells (HASMC) compared to nonpressurized controls. Immunoblot analysis probed with an anti-OPN antibody demonstrated a 50% increase in OPN. Dual-luciferase reporter assay demonstrated that OPN promoter, corresponding to the -771 through -1 region of OPN gene, was highly responsive to pure atmospheric pressure by ten times that of the control. From these observations, we concluded that pure atmospheric pressure directly promotes an expression of OPN in HASMC, with these results also suggesting that high blood pressure-mediated mechanical compression is involved in the process of atherosclerosis and remodeling via OPN expression in HASMC.