Vascular smooth muscle cells (VSMC) are the main cellular component of vessel wall. The changes of VSMC functions including phenotypic transformation and apoptosis play a critical role in the pathogenesis of intracranial aneurysm (IA). Autophagy can participate in the regulation of vascular function by regulating cell function. In the initial stage of IA, the activation of autophagy can accelerate the phenotypic transformation of VSMC and inhibit VSMC apoptosis. With the progress of IA, the relationship between autophagy and apoptosis changes from antagonism to synergy or promotion, and a large number of apoptotic VSMC lead to the rupture of IA. In this review, we describe the role of autophagy regulating the function of VSMC in the occurrence, development and rupture of IA, for further understanding the pathogenesis of IA and finding molecular targets to prevent the formation and rupture of IA.
Autophagy ; Humans ; Intracranial Aneurysm ; pathology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology

Renin-angiotensin system (RAS) is involved in the regulation of vascular smooth muscle cell (VSMC) tension. Angiotensin II (Ang II) as the main effector molecule of RAS can increase the intracellular Ca concentration and cause VSMCs contraction by activating angiotensin II type 1 receptor (AT1R). The large-conductance Ca- and voltage-activated potassium (BK) channel is an essential potassium channel in VSMCs, playing an important role in maintaining membrane potential and intracellular potassium-calcium balance. The BK channel in VSMCs mainly consists of α and β1 subunits. Functional BKα subunits contain voltage-sensors and Ca binding sites. Hence, increase in the membrane potential or intracellular Ca concentration can trigger the opening of the BK channel by mediating transient K outward current in a negative regulatory manner. However, increasing evidence has shown that although Ang II can raise the intracellular Ca concentration, it also inhibits the expression and function of the BK channel by activating the PKC pathway, internalizing AT1R-BKα heterodimer, or dissociating α and β1 subunits. Under some specific conditions, Ang II can also activate the BK channel, but the underlying mechanism remains unknown. In this review, we summarize the potential mechanisms underlying the inhibitory or activating effect of Ang II on the BK channel, hoping that it could provide a theoretical basis for improving intracellular ion imbalance.
Angiotensin II ; physiology ; Calcium ; physiology ; Humans ; Large-Conductance Calcium-Activated Potassium Channels ; physiology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Renin-Angiotensin System

OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

Autophagy plays a crucial role in maintaining normal structure and vascular function in vivo. When stress-relevant stimuli are involved, the increases of autophagy can protect vascular smooth muscle cells, promote cell survival, and phenotype transformation, as well as reduce calcification. On the contrary, the decrease of autophagy can accelerate cell senescence, resulting in structural changes and dysfunction of vasomotor and vasodilation. However, excessive activation of autophagy can induce the damage of the healthy protein and essential organelles, and even lead to autophagic cell death, accelerating the progression of vascular disease. Thus, the precise targeting of autophagy opens a novel way for treatment of vascular diseases.
Autophagy ; physiology ; Cell Survival ; Cellular Senescence ; Disease Progression ; Humans ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Vascular Diseases ; pathology ; therapy

The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4-6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO, 21% O, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO, 5% O, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca]in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca]were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca].
Actins ; Animals ; Apoptosis ; Calcium ; metabolism ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Hypercapnia ; physiopathology ; Imidazoles ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; Pulmonary Artery ; cytology ; Rats ; Rats, Sprague-Dawley ; TRPC Cation Channels ; metabolism

PURPOSE: MicroRNAs are small non-coding RNAs that play important roles in vascular smooth muscle cell (VSMC) function. This study investigated the role of miR-379 on proliferation, invasion, and migration of VSMCs and explored underlying mechanisms thereof. MATERIALS AND METHODS: MicroRNA, mRNA, and protein levels were determined by quantitative real-time PCR and western blot. The proliferative, invasive, and migratory abilities of VSMCs were measured by CCK-8, invasion, and wound healing assay, respectively. Luciferase reporter assay was used to confirm the target of miR-379. RESULTS: Platelet-derived growth factor-bb was found to promote cell proliferation and suppress miR-379 expression in VSMCs. Functional assays demonstrated that miR-379 inhibited cell proliferation, cell invasion, and migration. Flow cytometry results further showed that miR-379 induced apoptosis in VSMCs. TargetScan analysis and luciferase report assay confirmed that insulin-like growth factor-1 (IGF-1) 3'UTR is a direct target of miR-379, and mRNA and protein levels of miR-379 and IGF-1 were inversely correlated. Rescue experiments showed that enforced expression of IGF-1 sufficiently overcomes the inhibitory effect of miR-379 on cell proliferation, invasion, and migration in VSMCs. CONCLUSION: Our results suggest that miR-379 plays an important role in regulating VSMCs proliferation, invasion, and migration by targeting IGF-1.
Apoptosis ; Cell Movement/*physiology ; Cell Proliferation/*physiology ; Humans ; Insulin ; Insulin-Like Growth Factor I/*physiology ; MicroRNAs/*physiology ; Muscle, Smooth, Vascular/*cytology ; Proto-Oncogene Proteins c-sis/*physiology ; RNA, Messenger/metabolism ; Real-Time Polymerase Chain Reaction ; Sincalide/physiology ; Wound Healing/physiology

OBJECTIVETo observe the effect of Pinggan Qianyang Recipe (PQR) on inhibiting angiotensin II (Ang II) induced proliferation and migration of vascular smooth muscle cells (VSMCs) and changes of DNA methylation.

METHODSVSMCs were cultured using tissue explant method, and PQR containing serum was prepared. Primarily cultured VSMCs were divided into four groups, the normal group, the model group, the folate group (folic acid intervention) , and the PQR group. The proliferation and migration of VSMCs was duplicated by Ang II. After 24-h Ang II induced culture, 40 microg/mL folic acid was added to the folate group for 48 h, while 5% PQR containing serum was added to the PQR group for 48 h. The cell growth curve of VSMCs was drawn by using Cell Counting Kit (CCK-8). The proliferative activity of VSMC was determined by MTT assay. The migration of VSMCs was measured by Millicell chamber. The general level of cytosine methylation in cell nucleus was detected via 5-mC antibodies immunofluorescence, and mRNA expression levels of DNA methyltransferase 1 (DNMT1) were measured by Real-time q-polymerase chain reaction (q-PCR).

RESULTSVSMCs were promoted by Ang II at 10(-6) mol/L for 24 h. Compared with the normal group, the proliferative activity and migration quantity of VSMCs obviously increased, and DNA methylation level obviously decreased (P < 0.05, P < 0.01). Compared with the model group, the cell growth, proliferative activity and migration quantity of VSMCs obviously decreased and the general DNA methylation level increased in the folate group and the PQR group (P < 0.05, P < 0.01). Compared with the normal group, the mRNA expression of DNMT1 decreased in the model group (P < 0.01). Compared with the model group, mRNA expression of DNMT1 in Ang II induced VSMCs was obviously enhanced in the folate group and the PQR group (P < 0.01).

CONCLUSIONSPQR could inhibit Ang II induced proliferation and migration of VSMCs, and cause high genomic DNA methylation level. Changes of DNA methylation might be associated with DNMT1 expression.

Angiotensin II ; pharmacology ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects

PURPOSE: Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels. MATERIALS AND METHODS: Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-kappaB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed. RESULTS: We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-kappaB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels. CONCLUSION: Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-kappaB pathways. These effects were inhibited by the antioxidant rutin.
Animals ; Caspase 3/metabolism ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Flavonoids/*pharmacology ; Glucose/*metabolism/pharmacology ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Kinase 1 ; Male ; Mitogen-Activated Protein Kinase 3 ; Muscle, Smooth, Vascular/cytology/*drug effects/enzymology ; Myocytes, Smooth Muscle/metabolism ; NF-kappa B/metabolism ; Phosphatidylinositol 3-Kinases ; Protein Kinase Inhibitors/*pharmacology ; Rats ; Rats, Inbred OLETF ; Rats, Long-Evans ; Reactive Oxygen Species/metabolism ; Rutin/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism

The present study attempted to test a novel hypothesis that Ca(2+) sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus (10 µmol/L) increased the frequency of Ca(2+) sparks, which could be reversed by ryanodine (10 µmol/L). Electrophysiological experiments revealed that tacrolimus (10 µmol/L) increased the large-conductance Ca(2+)-activated K(+) currents (BKCa) in rat aortic vascular smooth muscle cells (AVSMCs), which could be blocked by ryanodine (10 µmol/L). Furthermore, tacrolimus (10 and 50 µmol/L) reduced the contractile force induced by norepinephrine (NE) or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin (100 nmol/L) and ryanodine (10 µmol/L) respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca(2+) sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.
Animals ; Aorta ; cytology ; metabolism ; physiology ; Calcium Signaling ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; physiology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ryanodine ; pharmacology ; Tacrolimus ; pharmacology ; Vasoconstriction

BACKGROUNDClarifying the mechanisms underlying vascular smooth muscle cell (VSMC) proliferation is important for the prevention and treatment of vascular remodeling and the reverse of hyperplastic lesions. Previous research has shown that the gaseous signaling molecule sulfur dioxide (SO2) inhibits VSMC proliferation, but the mechanism for the inhibition of the angiotensin II (AngII)-induced VSMC proliferation by SO2has not been fully elucidated. This study was designed to investigate if SO2inhibited VSMC proliferation in mice with hypertension induced by AngII.

METHODSThirty-six male C57 mice were randomly divided into control, AngII, and AngII + SO2groups. Mice in AngII group and AngII + SO2group received a capsule-type AngII pump implanted under the skin of the back at a slow-release dose of 1000 ng·kg-1·min-1. In addition, mice in AngII + SO2received intraperitoneal injections of SO2donor. Arterial blood pressure of tail artery was determined. The thickness of the aorta was measured by elastic fiber staining, and proliferating cell nuclear antigen (PCNA) and phosphorylated-extracellular signal-regulated kinase (P-ERK) were detected in aortic tissues. The concentration of SO2 in serum and aortic tissue homogenate supernatant was measured using high-performance liquid chromatography with fluorescence determination. In the in vitro study, VSMC of A7R5 cell lines was divided into six groups: control, AngII, AngII + SO2, PD98059 (an inhibitor of ERK phosphorylation), AngII + PD98059, and AngII + SO2 + PD98059. Expression of PCNA, ERK, and P-ERK was determined by Western blotting.

RESULTSIn animal experiment, compared with the control group, AngII markedly increased blood pressure (P < 0.01) and thickened the aortic wall in mice (P < 0.05) with an increase in the expression of PCNA (P < 0.05). SO2, however, reduced the systemic hypertension and the wall thickness induced by AngII (P < 0.05). It inhibited the increased expression of PCNA and P-ERK induced by AngII (P < 0.05). In cell experiment, PD98059, an ERK phosphorylation inhibitor, blocked the inhibitory effect of SO2on VSMC proliferation (P < 0.05).

CONCLUSIONSERK signaling is involved in the mechanisms by which SO2inhibits VSMC proliferation in AngII-induced hypertensive mice via ERK signaling.

Angiotensin II ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hypertension ; chemically induced ; drug therapy ; Male ; Mice ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Signal Transduction ; drug effects ; Sulfur Dioxide ; therapeutic use