Vascular Muscle Cells Research Articles

Role of CGRP modified mesenchymal stem cells in restenosis in rat model with carotid angioplasty

To explore the role of calcitonin gene-related peptide (CGRP) in the proliferation of vascular smooth muscle cells (VSMCs) and restenosis. The high-expression CGRP lentivirus [Lenti-green fluorescent protein (GFP)-CGRP] was constructed. And mesenchymal stem cells (MSCs) were transfected with Lenti-GFP-CGRP, Lenti-GFP and phosphate buffer saline (PBS). Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to determine the CGRP gene and protein expression level of smooth muscle cells in each group respectively. Then MSCs were co-cultured with VSMCs. Experimental groups were MSCs(CGRP+/+)+VSMCs, MSCs(CGRP-/-)+VSMCs and MSCs(PBS)+VSMCs groups. The method of methyl thiazolyl tetrazolium (MTT) was employed to detect the proliferation of smooth muscle cells. Sacculus damaged atherosclerotic carotid was prepared according to the previous study. MSCs were transfected with Lv-CGRP-EGFP and Lv-CGRP and then transplanted into rat model. At Day 7 post-transplantation, injured carotid artery was harvested to detect the C expression of GRP by Western blot and the expression of CD31 by immunofluorescence. At Day 28 post-transplantation, injured carotid artery was harvested to assess organization morphology by hematoxylin and eosin stain and detect the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemical stain. As compared with control and Lenti-GFP groups, the expressions of CGRP protein and CGRP mRNA increased at 72 h after transfecting with Lenti-GFP-CGRP (P < 0.05). After 72-hour co-culturing with VSMCs, the proliferation ability of VSMCs was the lowest in MSCs(CGRP+/+)+VSMCs group versus other three groups (P < 0.05). At Day 7 post-transplantation, as compared with control and Lenti-GFP groups, the expression of CGRP proteins increased significantly in MSCs-CGRP group (P < 0.05). A continuous expression of CD31 was found in damaged carotid intima in MSCs-CGRP group, but not in control group. At Day 28 post-transplantation, the area of intimal hyperplasia was smaller in MSCs-CGRP group than that in control and Lenti-GFP groups. Also the expression of PCNA decreased more in MSCs-CGRP group than control and Lenti-GFP groups (P < 0.05). CGRP protein and CGRP mRNA are expressed after CGRP transfection. And CGRP can suppress the proliferation of VSMCs and reduce intimal hyperplasia so as to facilitate a recovery of damaged endothelium.

Vascular Calcification - Pathological Mechanism and Clinical Application - . Role of vascular smooth muscle cells in vascular calcification

Vascular calcification is commonly seen with aging, chronic kidney disese (CKD), diabetes, and atherosclerosis, and is closely associated with cardiovascular morbidity and mortality. Vascular calcification has long been regarded as the final stage of degeneration and necrosis of arterial wall and a passive, unregulated process. However, it is now known to be an active and tightly regulated process involved with phenotypic transition of vascular smooth muscle cells (VSMC) that resembles bone mineralization. Briefly, calcium deposits of atherosclerotic plaque consist of hydroxyapatite and may appear identical to fully formed lamellar bone. By using a genetic fate mapping strategy, VSMC of the vascular media give rise to the majority of the osteochondrogenic precursor- and chondrocyte-like cells observed in the calcified arterial media of MGP (- / -) mice. Osteogenic differentiation of VSMC is characterized by the expression of bone-related molecules including bone morphogenetic protein (BMP) -2, Msx2 and osteopontin, which are produced by osteoblasts and chondrocytes. Our recent findings are that (i) Runx2 and Notch1 induce osteogenic differentiation, and (ii) advanced glycation end-product (AGE) /receptor for AGE (RAGE) and palmitic acid promote osteogenic differentiation of VSMC. To understand of the molecular mechanisms of vascular calcification is now under intensive research area.

Vascular Calcification - Pathological Mechanism and Clinical Application - . Vascular calcification in klotho deficient environment

Klotho deficient (kl/kl) mice exhibit Möncheberg's vascular calcification in the tunica media due to hyperphosphatemia and hypercalcemia by mediating the disrupted signaling of FGF23/klotho axis. Recent studies have hypothesized the mechanism of medial vascular calcification : Vascular smooth muscle cells acquired excessive intake of phosphate ions undergo a phenotypic differentiation into osteoblasts and induce biological calcification in the tunica media. It is useful to clarify the underlying cellular mechanism of vascular calcification for the development of the treatment and preventive medicine. This review will introduce the histological and ultrastructual findings on medial vascular calcification in kl/kl mice.

Effect of aging on transcription and protein expressions of procollagen α polypeptide gene of vascular smooth muscle cells in rat

Objective To investigate the effects of aging on procollagen α polypeptide gene transcription and protein expression in rat vascular smooth muscle cells. Methods Vascular smooth muscle cells from thoracoabdominal aorta in neonate and 9 months old healthy Wistar rats were cultured in vitro. Results Transcription polymerase chain reaction(RT-PCR) and Western blotting were used to detect type I and Ⅲ pro-collagen α polypeptide mRNA and protein. The RT-PCR displayed that type I procollagen α polypeptide mRNA expression had no significant difference between young group and adult group 〔(76.62±1.05) vs. (78.37±2.42), P>0.05〕. Type Ⅲ procollagen α polypeptide mRNA expression was (105.40±2.66) in young group and (123.10±3.81) in adult group(P>0.05). Type I procollagen α polypeptide mRNA expression was (3.13±0.54) in young group and (4.63±1.03) in adult group(P=0.05). Type Ⅲ procollagen α polypeptide mRNA expression had no significant difference between the adult and young groups〔(6.86±0.41) vs. (7.68±0.63), P>0.05〕. Type I and type Ⅲ procollagen α polypeptide protein expressions were increased significantly in adult group as compared with the young group 〔(0.10±0.03) vs. (0.06±0.03), (0.58±0.06) vs. (0.40±0.02), both P<0.05〕. Conclusions Aging increases the procollagen α polypeptide level in vascular smooth muscle cell, which may involve in the development of vascular remodeling and atherosclerosis. Key words: Muscle, smooth, vascular; Procollagen; Transcription, genetic

Changes in open probability and protein expression of large conductance Ca2+ -activated K+ channel in retinal vascular smooth muscle cells of diabetic rats

Objective To observe the changes in open probability and protein expression of large conductance Ca2+-activated K+ (BK) channel in retinal vascular smooth muscle cells (RVSMCs) of diabetic rats. Methods Streptozotocin (STZ)-induced rat diabetic animal model was established by STZ injection intraperitoneally.RVSMCs were isolated by enzyme digestion. The BK currents in control and diabetic groups were recorded by patch clamp technique in single channel configuration. BK channel protein expression in control and diabetic group were measured by Western blot. Results Compared with control group, the NP0 of BK channels in diabetic group were significantly increased (t=4.260, P 0.05); however, β1-subunit protein expression was remarkably increased in diabetic group (t=5.146, P<0.05). Conclusion The NP0 of BK channels and β1-subunit protein expression are increased in RVSMCs of diabetic rats. Key words: Diabetic retinopathy/etiology; Retinal artery; Potassium channels; Patch-clamp techniques; Animal experimentation

L’éveil de la matrix-gla-protéine sonnera le glas des calcifications vasculaires

Matrix-gla-protein (MGP) is mainly secreted by chondrocytes and smooth vascular muscle cells. This potent inhibitor of vascular calcification need to undergo 2 post-transcriptional steps to be fully active: one phosphorylation of 3 serine residues (on 5) and a carboxylation of 5 glutamate residues (on 9). Like other “Gla” proteins, this carboxylation is vitamin K dependant. Several forms of MGP thus circulate in the plasma, some of them being totally inactive (the unphosphorylated and uncarboxylated MGP), some others being partially or fully active, according to the number of phosphorylated or carboxylated sites. A theoretical link exists between MGP, vitamin K, vascular calcifications and cardiovascular diseases. This link is even more evident in patients suffering from chronic kidney diseases (CKD), and notably hemodialysis patients. If this link has been demonstrated in different experimental studies, clinical studies are mainly observational and their results must be interpreted accordingly. MGP concentrations are definitely not yet a surrogate to estimate the levels of vascular calcification, but could allow the monitoring of vitamin K treatment. Modulation of MGP concentrations may reduce vascular calcification in hemodialyzed patients, if the large ongoing trials show an efficiency of this treatment. In this review, we will summarize the role of MGP in the vascular calcifications process, describe the problems linked to the analytical determination of MGP in plasma and finally describe the different clinical studies on MGP and vascular calcifications in the general population and in CKD patients.

Role of NF-κB/survivin signal pathway on intima hyperplasia of rat carotid balloon injury restenosis model

To investigate the role of NF-κB/survivin signal pathway in the intima hyperplasia of rat carotid balloon injury restenosis model. NF-κB siRNA lentivirus vector (titer was 1 × 10⁸ TU/ml) was established. Carotid balloon injury restenosis model was made in 33 SD rats. The rats were divided into 4 groups according to different processing methods, including negative control (NC) group (n = 11), NF-κB siRNA group (n =11), NF-κB siRNA+YM155 (survivin inhibitor) (n = 11), the uninjured carotid artery served as the normal control group (n = 11). After 7 days, the carotid sample (n = 5 each group) were harvested to detect the NF-κB and survivin mRNA expression by RT-PCR.The carotid sample were harvested on 28 days (n = 6 each group) for HE staining and measuring intima hyperplasia. Immunohistochemical method was also used to detect the expression of intima proliferation cell nuclear antigen (PCNA) and media α-SM-actin. (1) After 7 days, NF-κB and survivin mRNA expression was significant higher in NC group than in normal control group (P<0.05), the NF-κB mRNA expression was significantly lower in NF-κB siRNA group than in NC group (P<0.05) and similar between NF-κB siRNA group and NF-κB siRNA+YM155 group. The survivin mRNA expression was significantly lower in NF-κB siRNA group compared to NC group (P<0.05) and significantly higher in NF-κB siRNA group than in NF-κB siRNA+YM155 group (P<0.05). (2) After 28 days, intima hyperplasia was observed in NC (0.13 ± 0.01), NF-κB siRNA (0.11 ± 0.01) and NF-κB siRNA+YM155 group (0.09 ± 0.01) mm² (P<0.05). Media area was similar among NC group, NF-κB siRNA group and NF-κB siRNA+YM155 group (P>0.05). I/M ratio was gradually reduced among NC group (1.55 ± 0.07), NF-κB siRNA group (0.92 ± 0.08), NF-κB siRNA+YM155 group (0.76 ± 0.06, all P<0.05). Similar results were found in the residual restenosis rate: NC group (58.71 ± 0.02) %, NF-κB siRNA group (32.13 ± 0.05) %, NF-κB siRNA+YM155 group (26.42 ± 0.03) % (all P<0.05) and expression of vascular smooth muscle cell PCNA: NC group (45.32 ± 7.21) %, NF-κB siRNA group (36.54 ± 6.42) %, NF-κB siRNA+YM155 group (28.57 ± 6.31) % (all P<0.05). On the contrary, the IOD of α-SM-actin in media increased gradually: NC group (0.055 ± 0.006), NF-κB siRNA group (0.072 ± 0.011), NF-κB siRNA+YM155 group (0.084 ± 0.008, all P<0.05). Inhibiting NF-κB expression can significant decrease intima hyperplasia in this model, and this effect may be mediated by inhibiting survivin and reducing the proliferation of vascular smooth muscle cells.

Cyclic strain modulates the phenotype of vascular smooth muscle cells from bone mesenchymal stem cells

Objective To investigate the mechanism of cyclic strain modulates the phenotype of vascular smooth muscle cells(VSMCs) from bone mesenchymal stem cells. Methods Sprague-Dawley rat bone mesenchymal stem cells(BMSCs)were isolated and then cultured, Flow cytometry was performed for identification.BMSCs was verified pluripotent stem cells by differrentiating into osteoblasts,adipocytes and smooth muscle cells.The cells were used at 3-5 passages,and the cells were devided into 4 groups in accordiance with different inducing methods, group A:the complete medium containing transforming growth factor-β1(TGF-β1, 10μg/L), group B: the cells were treated by cyclic strain(5% magnitude and 1 Hz frequency), group C: the combination of cyclic strain(5% magnitude and 1 Hz frequency) and TGF-β1(5μg/L), group D: the cells were cultured with complete medium as a control. After three days, the makers of smooth muscle cells:α-actin, smooth muscle myosin heavy chain(SM-MHC), calponin1,calponin3 were ditected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). Results The cultured cells were identificated as BMSCs by Flow cytometry.Treated by cyclic strain 3 days, at group A, B, C, the expression levels of VSMCs markers were much higher than group D, especialy the SM-MHC and calponin1.In group B(0. 823 6±0. 024 6) the levels of SMMHC was showing significant difference between group A(0. 919 2±0. 028 1)and group C(1. 043 1± 0. 090 7, P<0. 05).In group C, the mRNA of calponin3 increased more than the Western blotting, both mRNA and Western blotting of calponin1 had equal variations. Conclusion Cyclic strain can modulate the phenotype of VSMC differentiation from BMSCs, the biochemical factor can enhance this effects. Key words: Cyclic strain; Transforming growth factor-β1; Bone mesenchymal stem cells; Vascular smooth muscle cells; Phenotype

Different propensity of cultured aortic valve interstitial cells to uptake native or aggregated low density lipoprotein

Atherosclerosis is a progressive disease characterized by modified low density lipoprotein (LDL) accumulation in the large artery walls, with subsequent phagocytosis by macrophages, transforming into foam cells, and vascular smooth muscle cells (VSMCs). In particular, native LDL (nLDL) can modify joining to each other giving rise to aggregated LDL (agLDL), which were reported to be internalized more avidly by VSMCs in in vitro conditions (1). Here, primarily cultured aortic valve interstitial cells (AVICs) were treated for 3 up to 21 days with 50 mg/ml blood-derived nLDL or agLDL, alone or combined with pro-calcific culture media, to ascertain whether (i) agLDL are taken up more rapidly than nLDL also by AVICs and (ii) treatment with LDL at low, normolipidemic-like concentration can influence AVIC mineralization. Ultrastructurally, LDL uptake was observed exclusively for agLDL-treated AVICs, which were characterized by a lot of endocytic vesicles and intracytoplasmic vacuoles entrapping agLDL particles. Consistently, treatment with agLDL resulted in a significant increase in intracellular amounts of both esterified cholesterol and triglycerides, as chromatographically assayed. Ultrastructural analyses also revealed pro-calcific AVIC degenerative process to occur, consisting in intracellular release of lipidic material and its layering at cell edges, there acting as major hydroxyapatite nucleator (2,3). The extent of pro-calcific effects resulted to be LDL-dependent, being mitigated in the presence of nLDL and exacerbated in the presence of agLDL. These findings were consistent with spectrophotometrical analyses showing decreased calcium amounts in AVIC cultures superstimulated with nLDL and increased mineral content in the counterpart superstimulated with agLDL. Since aortic valve stenosis is considered as a valve atherosclerotic lesion, these preliminary data suggest that avid uptake of agLDL by AVICs might strongly contribute to lipid accumulation within aortic valves, with agLDL possibly affecting subsequent valve tissue mineralization even at concentrations comprised within the normolipidemic range.

Atherosclerosis and beneficial effects of parnaparin

In recent years atherosclerosis has become a serious health challenge given its causal association with cardiovascular disease and stroke, which predictably leads to most of the morbidity and mortality worldwide [1]. The mechanism by which in this pathology there is an extensive proliferation of vascular smooth muscle cells (VSMCs) and their migration to the intima remain poorly understood. Recently, low molecular weight heparin (LMWH) has been shown to inhibit the proliferation of VSMCs and, in particular, a kind of these heparins, parnaparin (PNP), acts on the development and clinical course of atherosclerosis in animal models [2]. We hypothesized that PNP might modulate VSMCs migration and thereby decrease vascular remodelling in atherosclerosis. Vascular remodelling was evalu- ated in aortas of an animal model of atherosclerosis, (ApoE)-deficient mice (ApoE -/-) with normal or western diet. We treated animals with PNP to evaluate its pro- tective effect. Thereafter, we evaluated morphological changes and the expression of proteins linked to atherogenesis and atheroprogression in particular an enzyme involved in oxidative stress, iNOS, some inflammatory mediators, such as TNF-α, IL-1 and IL-6, and markers of VSMCs alterations (PAI-1 and TSP1). Our results demonstrated that PNP downregulates VSMCs proliferation and migration, mediated by PAI-1 and TSP1, and reduces inflammation and oxidative stress in vessels. These data suggested that LMWH, in particular PNP, could be a theoretically practical tool in the prevention of atherosclerotic vascular modification.

Effect of Desmuslin gene over-expression on proliferation and migration of smooth muscle cells derived from primary varicose veins

Objective To observe functional alterations in smooth muscle cells (SMCs) derived from varicose veins after over-expression of Desmuslin (DMN) gene by pRetroQ-AcGFP1-C1-DMN plasmid and further explore the effect of DMN gene involved in the development of varicose veins.Methods SMCs from varicose veins were cultured by the explant culture technique.The purity of the SMCs was identified by immunofluresence with a monoclonal antibody against human smooth muscle α-actin.DMN in SMCs was overexpressed by pRetroQ-AcGFP1-C1-DMN plasmid infected.The transfection efficiency was tested by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.CCK-8 assay was used to determine the proliferation of SMCs.Transwell assay was used to determine the migration of SMCs.Results Purity of these cultured SMCs was over 99％ identified by positive immunostaining with monoclonal SM-α-actin antibody.Levels of DMN mRNA and DMN protein in SMCs infected by pRetroQ-AcG-FP1-C1-DMN were significantly overexpressed compared with the empty vector group,(8.3 ± 1.1) × 104 and (4.5 ± 1.2) times respectively (P ＜ 0.01).The proliferation of SMCs of DMN over-expression group was (48.2 ± 4.5) ％ of the empty vector group by using CCK-8 assay (P ＜ 0.01).The migration ability of SMCs of DMN over-expression group was (44.3 ±4.7)％ of the empty vector group by using transwell assay (P ＜ 0.01).Conclusion The SMCs derived from varicose veins presented a significantly inhibited state of proliferation and migration after DMN over-expression,suggesting that DMN may play a key role in the development of varicose veins. Key words: Desmuslin; Retroviral vector; Vascular smooth muscle cells; Varicose veins

Effect of chitosan on vascular smooth muscle cells inhibiting proliferation from rabbit arteriovenous fistula and its mechanisms

Objective To explore the effect of chitosan on vascular smooth muscle cells inhibited proliferation from rabbit arteriovenous fistula and its mechanisms.Methods Established rabbit fistula model on carotid arteryinternal jugular vein.After 1 month cultured VSMCs with primary culture by tissue-pieces inoculation.Cultured VSMCs were divided into three groups:①normal control group.②FBS-treated group:cell were treated with 5％,10％,20％ for 48 h,respectively; established the model of rabbit VSMCs proliferation.③chitosan-treated group:VSMCs cultured with 20％ FBS were exposed to different doses of chitosan(10,100,500,1000,2000μg/ml) for 48 h.And VSMCs were treated for different time (0,12,24,48 h) with Chitosan 1000 μg/ml.Expression levels of PCNA and TLR4/ NF-κB were detected by Western blotting.RT-PCR were applied to measure the mRNA expression of PCNA and TLR4.The protein levels of TLR4 and NF-κB were detected by immunofluorescence.Results Compared with low concentration serum group,FBS-treated VSMCs exhibited a increase in mRNA and protein expression of PCNA and TLR4.FBS-induced protein expression of PCNA and TLR4/NF-κB were reduced by chitosan.Also mRNA expression of PCNA and TLR4 were reduced.They were dependent on concentration and time.In rabbit VSMCs TLR4 was mainly expressed in the cytoplasm and NF-κB expressed mainly in the nucleus.Compared with normal control group,TLR4 and NF-κB protein expression were significantly decreased by chitosan.Conclusion High concentration serum induced VSMCs proliferation.Chitosan can inhibit the proliferation of rabbit VSMCs.It is speculated that the mechanism may be related to the expression of TLR4 receptor activation,reducing expression of downstream factor MyD88 and NF-κB.It is suggest that chitosan can become potential new drugs of arteriovenous fistula prevention of intimal hyperplasia. Key words: Chitosan ; TLR4 ; Vascular smooth muscle cells (VSMCs) ; Inhibiting proliferation ; Arteriovenous fistula

Cardiovascular Toxicity and Sorafenib

We report a case of a 55-year-old male with chronic hepatitis C virus infection and compensated liver disease treated with sorafenib for advanced hepatocarcinoma (Barcelona Clinic Liver Cancer stage C). At follow-up, the patient developed hypertension, which was well controlled with beta-blocker medication, and an aortic dilation detected by abdominal computerized tomography and echocardiography. There are some reports of the side effects of sorafenib on the cardiovascular system. The patient had no cardiac or aortic pathology before the start of this palliative chemotherapy. There is an article that describes the development of an aortic aneurysm in a patient with uncontrolled hypertension, who received treatment with sorafenib for renal carcinoma. However, our patient had a good control of blood pressure. The adverse vascular effects of Sorafenib may be due to the inhibition of the proliferation of vascular endothelial muscle cells. We believe that this case illustrates a probable relationship between sorafenib and aortic dilatation according to the Karch and Lasagna causality algorithm.

Bone metabolism and cardiovascular function update. Role of vitamin D in the bone and vascular intercommunication

Vascular calcification, hypertension and cardiac hypertrophy have been often complicated in osteoporotic patients with low bone mass. Since there are many similarities among the processes of bone formation and vascular calcification, vitamin D insufficiency has been thought to be deeply involved in the pathogenesis of these diseases. Indeed, in animal studies, vitamin D receptor gene knockout mice have been shown to display severe vascular calcification, high blood pressure, and left ventricular hypertrophy. On the other hand, in clinical studies, active vitamin D restores vascular calcification and improves heart function in dialysis patients. Whether 1,25 (OH) ₂D₃ acts directly on vascular smooth muscle cells and cardiomyocytes or acts indirectly on them via regulating calcium metabolism remains unclear. The elucidation of the role of vitamin D in the bone and vascular intercommunication and its application toward drug development could be an important step forward in the realization of health and longevity society.

MicroRNA-363 regulation in proliferation and migration of human umbilical vascular smooth muscle cells

Objective To investigate microRNA-363 (miR-363) regulation in proliferation and migration of human umbilical vascular smooth muscle cells (VSMCs) and explore the possible mechanisms of miR-363 affecting the biological behavior of VSMCs.Methods Primary cultured human umbilical VSMCs were treated with 30 μg/L of platelet derived growth factor BB (PDGF-BB) to induce phenotypic switch,and miR-363 expression was quantified by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).VSMCs were transfected with miR-363 mimics and miR-NC for negative control,VSMCs proliferation and migration in both groups were evaluated using cell counting kit-8 assays (CCK-8) assay and Transwell migration assay,respectively; phenotypic proteins of α-SMA and Calponin-1 were detected by Western blotting analysis.Comparisons were made between the two groups.Target gene of miR-363 was predicted using bioinformatics analysis,and confirmed by Western blotting analysis.Results VSMCs switched to contractile phenotype 72 hours after they were co-cultured with PDGF-BB.Contractile phenotype protein of α-SMA was down-regulated by 28％ (P ＜0.05) ; the expression of miR-363 was down-regulated by 70％ (P ＜ 0.01).Compared to miR-NC group,significant inhibition of proliferation and migration were observed in miR-363 over-expressed VSMCs,by 32％ and 30％,respectively at 72 hours (P ＜ 0.05).Kruppel-like factor 4 (Klf4),the target gene of miR-363,was down-regulated by 19％ (P ＜ 0.05),52％ (P ＜ 0.01) and 37％ (P ＜ 0.01),respectively 24,48 and 72 hours after VSMCs were transfected with miR-363 ; however,the expression of α-SMA and Calponin-1 were not significant.Conclusion Our results suggest that up-regulation of miR-363 may inhibit proliferation and migration of human VSMCs,and these effects may be achieved partly through targeting the KLF4 expression. Key words: MicroRNA-363 ; Vascular smooth muscle cells ; Proliferation ; Migration; Kruppel-like factor 4

The significance of interferon-γ on Fas ligand-induced apoptosis of vascular smooth muscle cells

Objective To investigate the effect of interferon-γ （IFN-γ on Fas ligand （FasL） -in duced apoptosis of rat vascular smooth muscle cells （VSMCs） and explore the underlying mechanism. Methods VSMCs isolated from rat aorta were cultured in vitro and treated with IFN-γor FasL alone, or both in combination, with or without the pretreatment of Tunicamycin. Hoechst 33342 staining and flow cyto metric analysis of Annexin V-fluoresceine isothiocyanate （FITC）/propidium iodide （ PI ） staining were em ployed to define the effect of IFN-γon FasL-induced VSMCs apoplosis. Protein levels of surface Fas, cyto plasmic Fas and total Fas were detected by Western blotting in order to observe the expression and distribu tion of Fas. Results As shown by both Hoeehst 33342 staining, apoptosis of VSMCs was only slightly in duced by treatment with IFN-γ or FasL alone [ apoptotie index of （7.4 ± 1.9 ） % vs. （6. 8 ± 2. 5 ） % ], but significantly induced by IFN-γ and FasL in combination [ （20. 1 ± 2. 2）%, both P 〈 O. 01 ]. Increased sur face Fas （ relative level of O. 266 ± 0. 042 vs. O. 042 ± O. 017, P 〈 O. O1 ） , decreased cytoplasmic Fas （ O. 193 ±0. 038 vs. O. 426 ±0. 074, P 〈0. O1 ） but unchanged total Fas expression （0. 469 ＋0. 129/0. 383 ±0. 045 for 36 h/72 h vs. 0. 402 ± 0. 031 for 0 h, both P 〉 O. 05 ） were detected by Western blotting after IFN-γ treatment. And pretreatment with membrane trafficking inhibitor Tunicamycin significantly diminished FasL-indueed VSMCs apoptosis promoted by IFN-γ. The apoptotic index [ （6. 1 ± 0. 8）% ] were less than that of control [ （23.0 ± 1.0）% , P 〈0. 011- Flow eytometric analysis also confirmed the results of Ho echst 33342 staining. Conclusion Instead of inducing VSMCs apoptosis directly, IFN-γchanges the Fas distribution by promoting translocation of Fas receptor to the cell membrane, which significantly enhances the sensitivity of VSMCs to FasL-indueed apoptosis. Key words: Interferon-γ; Vascular smooth muscle cells; Fas ligand; Fas; Cell apoptosis

The effects of TSH on human vascular endothelial cells and smooth muscle cells

Objective To study the effect of thyroid-stimulating hormone (TSH) on human vascular endothelial cells and smooth muscle cells and to explore the roles of TSH in the development of atherosclerosis.Methods Human vascular endothelial cells and smooth muscle cells were cultured in vitro.MTT method was used to assay the effect of TSH on cell viability.Real-time PCR was used to determine the levels of endothelial nitric oxide synthase (eNOS),prostacyclin(PGI2),endothelin-1 (ET-1),plasminagen activator inhibitor(PAI-1) (mRNA) in endothelial cells and the phenotype transition of smooth muscle cells.The effect of TSH to the cycle of smooth muscle cells was detected by using flow cytometry.Immunocytochemistry and Western blot were used to investigate the expression of the cell cyclin A,D1,and the expression of endothelial cell associated factors eNOS and ET-1.Results Compared with the control group,eNOS and PGI2 mRNA levels decreased while ET-1 and PAI-1 mRNA levels increased when different concentrations of TSH were applied to endothelial cells(P＜0.05).The level of eNOS protein was decreased gradually while the level of ET-1 protein was gradually increased(P＜0.05).Different concentrations of TSH applied to smooth muscle cells could promote the transition of cell cycle phase G2 to phase M and increase the expression of cell cyclin A and D1.Conclusion TSH may damage the function of vascular endothelial cells and promote the proliferation of smooth muscle cells. Key words: Thyroid stimulating hormone; Vascular endothelial cells; Vascular smooth muscle cells; Atherosclerosis

Effects of core-binding factor α1 gene silenced by siRNA on calcification of vascular smooth muscle cells induced by high phosphate

To explore the effects of core-binding factor α1 (Cbfα-1) gene silenced by siRNA on osteogenic differentiation and calcification of vascular smooth muscle cells (VSMC) induced by high phosphate in vitro. VSMC were cultured in vitro and passaged 3 to 8 times. Four pairs of Cbfα-1 siRNA were designed and synthesized. Transfection was performed with cationic lipid vectors (Lipofectamine 2000). Transfection conditions were optimized by the FAM fluorescent labeling-siRNA to screen effective siRNA sequences by reverse transcription-polymerase chain reaction (RT-PCR). After transfection with effective siRNA sequences, VSMCs were divided into 4 groups: (1) normal phosphate (Pi 1.3 mmol/L); (2) high phosphate (Pi 2.6 mmol/L); (3) siRNA transfection: high phosphate+Cbfα-1-siRNA; (4) negative transfection control: high phosphate+negative control siRNA. Cbfα-1 and osteopontin (OPN) mRNA and protein expression were detected by RT-PCR and Western blotting. Calcium deposition was visualized by Alizarin stain method. The transfection efficiency was around 55% with a concentration of Cbfα-1 siRNA 100 nmol/L and Lipo 8 µl/ well. Cbfα-1 siRNA 1952 was chosen as the effective sequence with a suppression ratio up to 81.8%. At 24 and 48 h post-transfection, the expression of Cbfα-1 mRNA was significantly lower in siRNA transfection group than that in high phosphate group (0.335 ± 0.059 vs 0.714 ± 0.106, 0.574 ± 0.036 vs 0.726 ± 0.086, all P < 0.01) . At 48 and 72 h post-transfection, the expression of Cbfα-1 protein in siRNA transfection group was significantly lower than that in high phosphate group (both P < 0.01) . While Cbfα-1 gene was silenced by siRNA in siRNA transfection group, the mRNA and protein expression of OPN significantly declined (all P < 0.05) and calcium deposition in cell layers decreased. Cbfα-1 siRNA can effectively inhibit the expression of Cbfα-1 mRNA and protein in VSMC and thus suppress the transformation of VSMC into osteoblast-like cells and calcification induced by high phosphate. Cbfα-1 may become a potential therapeutic target in vascular calcification of chronic kidney disease.

Gene expression profiles in intracranial aneurysms

In this study, we extracted total RNA from 15 intracranial aneurysms and 17 superficial temporal artery samples, then performed genome-wide expression profiling using the Affymetrix U133 Plus 2.0 GeneChip. Genes that were differentially expressed between intracranial aneurysms and arterial samples were identified using significance analysis for microarrays, and the expression patterns of three randomly-selected genes were verified by real-time polymerase chain reaction analysis. We identified 3 736 differentially-expressed genes out of the 47,000 assayed transcripts. A total of 179 genes showed a >10-fold change in expression between the aneurysms and the arterial samples. Genes involved in the proliferation, migration, and apoptosis of vascular muscle cells, atherosclerosis, extracellular matrix disruption, and inflammatory reactions were associated with the formation of intracranial aneurysms. There were no significant differences in gene expression profile between unruptured and ruptured aneurysms.

Sildenafil for Noncompaction Cardiomyopathy Treatment in a Child: Case Report

Congestive heart failure (CHF) occurs when the heart can no longer maintain the baseline metabolic demand triggered by physiological venous pressure. In the pediatric population, it corresponds to a complex entity with multiple etiologies, depending on the age group assessed, being mainly associated with congenital structural malformations, cardiomyopathies, or secondary to arrhythmogenic, infectious, ischemic, toxic or infiltrative events1. The incidence of CHF in children also varies according to the underlying condition, ranging from 15% to 25% in patients with congenital heart defects. In structurally normal hearts, cardiomyopathy is the major factor associated with CHF, with a 40% incidence of CHF reported in those patients1. Isolated noncompaction cardiomyopathy is a rare disease, whose incidence in the general population ranges from 0.014% to 1.3%. It might result from a failure in compaction of the ventricular myocardium between weeks 5 and 8 of the embryonic life, leading to the persistence of myocardial trabeculations and deep recesses, which communicate with the ventricular cavity, generating myocardial thickening in two distinct layers (compacted and noncompacted). Initially reported in the pediatric population or with a congenital cardiopathy, it also affects adults with no other heart disease2,3. This failure in the regression of embryonic sinusoids is postulated to be due to the extremely high pressures that the ventricles undergo in that developmental period. However, the literature has reported a genetic aspect emphasizing the relationship of the disease with different genes, such as the mutation of gene G4.5 in families with severe infantile noncompaction cardiomyopathy, mutations P121L, CYPHER/ZASP, E101K, and a locus containing the disease gene in chromosome 11p153. The major complications of patients with noncompaction cardiomyopathy are pulmonary thromboembolism, arrhythmias and CHF3. In addition to the usual treatment of CHF, new classes of drugs, such as the phosphodiesterase type 5 (PDE-5) inhibitors, have been studied4. They are currently used for the treatment of erectile dysfunction and pulmonary arterial hypertension, and began to be studied for the treatment of CHF after the demonstration of higher PDE-5 expression in myocytes and vascular muscle cells, as well as of markers of oxidative stress in patients with CHF5. We report, for the first time, the clinical and laboratory findings of a patient with noncompaction cardiomyopathy after the addition of sildenafil (PDE-5 inhibitor) to the usual treatment.