Essential role of HDL on endothelial progenitor cell proliferation with PI3K/Akt/cyclin D1 as the signal pathway.

High-density lipoprotein (HDL) is known as an important factor in vascular wall remodeling that also affects gene expression in cell proliferation and differentiation. In this article, the role of HDL on endothelial progenitor cell (EPC) proliferation, angiogenesis and the signal pathway involved was studied, particularly the influence of HDL in strengthening the promoting effect of EPCs on wound healing of the arterial wall in hypercholesterolemic rats. Mononuclear cells isolated from rat bone marrow displayed characteristics of EPCs after cultivation. The role of HDL on EPC function and the signal pathway involved were studied by Western blotting, in vitro migration and 'tube' formation. Re-endothelialization and the number of circulating EPCs were compared between normal rats, hypercholesterolemic rats and hypercholesterolemic rats with HDL treatment. Results showed that HDL participated in the healing process by promoting EPC proliferation, migration and 'tube' formation. HDL activates cyclin D1 via phosphatidylinositol 3-kinase (PI3K)/Akt stimulation. Inhibition of PI3K/Akt via pharmacological or small interfering RNA approaches significantly attenuated HDL-induced EPC migration, proliferation and 'tube' formation. Results of experiments in vivo showed that HDL increased the number of circulating EPCs and promoted re-endothelialization in wound healing. These findings demonstrate for the first time that PI3K/Akt-dependent cyclin D1 activation plays an essential role in HDL-induced EPC proliferation, migration and angiogenesis.

[Effects of rapamycin-loaded poly(lactic-co-glycolic) acid nanoparticles on distribution of cell cycle, expression of p27 protein, and proliferation of human umbilical arterial vascular smooth muscle cell in vitro].

OBJECTIVE: To evaluate the effects of rapamycin (RPM)-loaded poly (lactic-co- glycolic) acid (PLGA) nanoparticles (NPs) on the proliferation, distribution of cell cycle, and expression of p27 protein in human umbilical arterial vascular smooth muscle cell (HUASMC) in vitro. METHODS: The primarily culture model of HUASMC was successfully established by explant-attached method in vitro. The cells were administrated with different doses of RPM, and RPM-PLGA NPs were observed as treat groups compared with PLGA NPs and M231-SMGs medium cultured group. The effect of RPM-PLGA NPs on proliferation of HUASMC was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetry method. The influences of RPM-PLGA NPs on the cell cycle and cellular growth kinetics of HUASMCs were tested by flow cytometry. The effect of RPM-PLGA NPs on the expression of p27 protein of HUASMCs was assessed through an immunohistochemical method. RESULTS: Compared with the control group, the proliferation of HUASMCs was inhibited by 50 microg/L and higher concentration of RPM-PLGA NPs in a dose-dependent manner (P < 0.05). The numbers of cells entering cell cycle of S/G2/M phases were significantly lower in RPM-PLGA NPs and RPM treated groups. Histologically, the expression of p27 were up-regulated in 500 microg/L RPM-PLGA NPs and 100 microg/L RPM treated group (all P < 0.01 ) when compared with the control group. CONCLUSIONS: RPM-PLGA NPs has a similar effects as RPM in inhibiting the growth of in vitro cultured HUASMC. It can remarkably suppress the expression of in vitro cultured HUASMC p27 protein, arrest its cell cycle at G1/S phase, and inhibit its proliferation.

CHIP represses myocardin-induced smooth muscle cell differentiation via ubiquitin-mediated proteasomal degradation.

Myocardin, a coactivator of serum response factor (SRF), plays a critical role in the differentiation of vascular smooth muscle cells (SMCs). However, the molecular mechanisms regulating myocardin stability and activity are not well defined. Here we show that the E3 ligase C terminus of Hsc70-interacting protein (CHIP) represses myocardin-dependent SMC gene expression and transcriptional activity. CHIP interacts with and promotes myocardin ubiquitin-mediated degradation by the proteasome in vivo and in vitro. Furthermore, myocardin ubiquitination by CHIP requires its phosphorylation. Importantly, CHIP overexpression reduces the level of myocardin-dependent SMC contractile gene expression and diminishes arterial contractility ex vivo. These findings for the first time, to our knowledge, demonstrate that CHIP-promoted proteolysis of myocardin plays a key role in the physiological control of SMC phenotype and vessel tone, which may have an important implication for pathophysiological conditions such as atherosclerosis, hypertension, and Alzheimer's disease.

Regulation of the stability and transcriptional activity of NFATc4 by ubiquitination.

Nuclear factor of activated T cells (NFATc4) has been implicated as a critical regulator of the cardiac development and hypertrophy. However, the mechanisms for regulating NFATc4 stability and transactivation remain unclear. We showed that NFATc4 protein was predominantly ubiquitinated through the formation of Lysine 48-linked polyubiquitin chains, and this modification decreased NFATc4 protein levels and its transcriptional activity. Furthermore, activation of GSK3beta markedly enhanced NFATc4 ubiquitination and decreased its transactivation, whereas inhibition of GSK3beta had opposite effects. Importantly, ubiquitination and phosphorylation induced by GSK3beta repressed NFATc4-dependent cardiac-specific gene expression. These results demonstrate that the ubiquitin-proteasome system plays an important role in regulating NFATc4 stability and transactivation.

Proteasome-dependent inactivation of Akt is essential for 12-O-tetradecanoylphorbol 13-acetate-induced apoptosis in vascular smooth muscle cells.

Apoptosis of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling. Here we investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on SMC apoptosis. We found that TPA treatment induced SMC apoptosis through the rapid downregulation of Akt phosphorylation. The inhibition of Akt activation by TPA was markedly reduced by inhibitors of protein phosphatase 2A and proteasome. Moreover, TPA promoted the ubiquitination of p-Akt, whereas inhibition of TPA-induced PKC activation suppressed the downregulation and ubiquitination of p-Akt. Taken together, these results demonstrate that TPA triggers inactivation of Akt, at least in part, through PKC and Ubiquitin-proteasome degradation, thereby contributing to SMC apoptosis.

Biological behaviour and role of endothelial progenitor cells in vascular diseases.

OBJECTIVE: To review the biological behaviour of endothelial progenitor cells and their role in vascular diseases. Data sources The data used in this review were mainly from Medline and PubMed for relevant English language articles published from 1985 to March 2007. The search term was "endothelial progenitor cells". Study selection Articles about the biological behaviour of endothelial progenitor cells and their roles in the pathogenesis of vascular diseases such as atherogenesis were used. RESULTS: Progenitor cells in bone marrow, peripheral blood and adventitia can differentiate into mature endothelial cells (ECs). The progenitor cells, which express certain surface markers including AC133, CD34 and KDR, enable restoration of the microcirculation and ECs when injury or ischaemia occurs. Endothelial progenitor cells used in experimental models and clinical trials for ischaemic syndromes could restore endothelial integrity and inhibit neointima development. Moreover, their number and functional properties are influenced by certain cytokines and atherosclerotic risk factors. Impairment of the progenitor cells might limit the regenerative capacity, even lead to the development of atherosclerosis or other vascular diseases. CONCLUSIONS: Endothelial progenitor cells have a particular role in prevention and treatment of certain cardiovascular diseases. However, many challenges remain in understanding differentiation of endothelial progenitor cells, their mobilization and revascularization.

[The effect of serum high-density lipoprotein on the growth rate of human endothelial progenitor cells].

OBJECTIVE: To study the effect of high-density lipoprotein (HDL) on the proliferation of endothelial progenitor cells (EPC) isolated from human umbilical cord blood; to further explore its effect on prevention and development of atherogenesis. METHODS: EPC isolated by density gradient centrifugation were cultured in a M200 medium. Immunofluorescence staining for CD133, CD34, KDR and Factor VIII were adopted respectively as the specific markers for identification. The effect of HDL on EPC proliferation was estimated on the 7th day of cell cultivation using MTT assay, confocal microscopy and fluorescence activated cell sorting. RESULTS: HDL, when incubated with EPC, was able to promote remarkably the proliferation rate of EPC, dose- and time-dependent. HDL participated in the transcriptional regulation of cell cycle by affecting the regulatory proteins such as cyclin D1. CONCLUSIONS: A subtype of progenitor cells was isolated from human cord blood with a potential of differentiating into mature endothelial cells (known as endothelial progenitor cells). HDL plays an important role on EPC fluorescence activated cell sorting differentiation and proliferation. Further studies are required to identify the signal pathway and the molecular mechanism of HDL effect on EPC proliferation.

Effects of oxidized low density lipoprotein on the growth of human artery smooth muscle cells.

BACKGROUND: Studies have shown that oxidized low density lipoprotein (ox-LDL) promotes the pathogenesis and development of atherosclerosis (AS), and that the proliferation, migration and phenotype alteration of vascular smooth muscle cells (vSMCs) into foam cells are critical changes in AS. It is proposed that ox-LDL might play a novel role in the pathologic process of vSMCs. The present study was performed ex vivo to investigate the effects of ox-LDL on the growth of cultured human vSMCs. METHODS: Using NaBr density gradient centrifugation, LDL from human plasma was isolated and purified. ox-LDL was produced from LDL after being incubated with CuSO4. ox-LDL was then added to the culture medium at different concentrations (25 microg/ml, 50 microg/ml, 75 microg/ml, 100 microg/ml, 125 microg/ml, and 150 microg/ml) for 7 days. The influence of ox-LDL on vSMC growth was observed from several aspects as growth curve, mitosis index, lipid staining, and in situ determination of apoptosis. The digital results were analyzed with SPSS 10.0. RESULTS: The ox-LDL produced ex vivo had a good purity and optimal oxidative degree, which was similar to the intrinsic ox-LDL in atherosclerotic plaque. ox-LDL at a concentration of 25 microg/ml demonstrated the strongest proliferation. At the concentration of 125 microg/ml, ox-LDL suppressed the growth of vSMCs. At concentrations of 25 microg/ml and 50 microg/ml, ox-LDL presented powerful mitotic trigger. When the concentration of ox-LDL increased, the mitotic index of vSMCs decreased gradually. ox-LDL induced more foam cells from vSMCs with rich intracellular lipid accumulation at concentrations of 25 microg/ml and 50 microg/ml. ox-LDL at higher concentrations induced more apoptotic vSMCs. CONCLUSIONS: ox-LDL at lower concentrations may trigger proliferation and phenotype alteration into foam cells of vSMCs, and at higher concentrations it may induce apoptosis in vSMCs. ox-LDL plays an important role in the pathogenesis and development of atherosclerosis by its effect on vSMCs proliferation, phenotype alteration and apoptosis.

Effect of oxidized-LDL on NF-kappaB nuclear translocation in aortic smooth muscle cells originated from rats of different ages.

OBJECTIVE: To investigate the molecular mechanism of atherosclerosis that related to age. METHODS: Immunohistochemistry staining and Western blot were adopted to determine the nuclear translocation of nuclear factor-kappa B (NF-kappaB) and expression of platelet-derived growth factor B (PDGF-B) in smooth muscle cells (SMCs) co-cultured with low density lipoprotein (LDL), oxidized LDL (ox-LDL), and ox-LDL+high density lipoprotein (HDL) originated from rats of 2 and 10 months old respectively. Fat stain was used to identify the lipid intake in SMCs. RESULTS: The optimal stimulation time of ox-LDL to SMCs was 12 hours. NF-kappaB intensity increased in most nuclei of SMCs that originated from rats of either 2 or 10 months old co-cultured with ox-LDL. The intensity of NF-KB and the amount of intracellular lipid taken in SMCs were more obvious in cells from 10-month-old rats than from the younger ones. Change of PDGF-B expression in SMCs was not remarkable in each group of rats. CONCLUSIONS: The 10-month-old rats are more susceptive to ox-LDL than 2-month-old rats in activating nuclear translocation of NF-kappaB. Maybe this is one of the important reasons contributing to the difference between the older and younger rats on the initiation and development of atherosclerosis lesion. Expression of PDGF-B is not associated with the activity of nuclear translocation of NF-kappaB.

An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus.

A new SARS animal model was established by inoculating SARS coronavirus (SARS-CoV) into rhesus macaques (Macaca mulatta) through the nasal cavity. Pathological pulmonary changes were successively detected on days 5-60 after virus inoculation. All eight animals showed a transient fever 2-3 days after inoculation. Immunological, molecular biological, and pathological studies support the establishment of this SARS animal model. Firstly, SARS-CoV-specific IgGs were detected in the sera of macaques from 11 to 60 days after inoculation. Secondly, SARS-CoV RNA could be detected in pharyngeal swab samples using nested RT-PCR in all infected animals from 5 days after virus inoculation. Finally, histopathological changes of interstitial pneumonia were found in the lungs during the 60 days after viral inoculation: these changes were less marked at later time points, indicating that an active healing process together with resolution of an acute inflammatory response was taking place in these animals. This animal model should provide insight into the mechanisms of SARS-CoV-related pulmonary disease and greatly facilitate the development of vaccines and therapeutics against SARS.

Age-dependent increase of NF-kappaB translocation and PDGF-B expression in aortic endothelial cells of hypercholesterolemic rats.

The objective of this study was to determine whether there was an age-dependent difference in promoting nuclear translocation of NF-kappa B and platelet derived growth factor expression in aortic endothelial cells between two groups of Wistar rats of 2 and 10 months in age, respectively, accompanied by hypercholesterolemia. The serum cholesterol levels became significantly higher in the older rats after taking a cholesterol-enriched diet for 16 weeks (129.3+/-11.9 vs. 194.5+/-22.6 mg/dl, P<0.01). The increase in total cholesterol levels were due to an elevation of LDL cholesterol since serum HDL cholesterol concentrations were similar in rats on either a high cholesterol- or a standard diet. Immunohistologic staining revealed nuclear translocation of NF-kappaB in aortic endothelial cells of rats was responsible for a high cholesterol uptake by cells. PDGF-B production was also increased in the endothelial cells of these animals as identified by situ hybridization and immunohistologic staining. Interestingly, the intensity of PDGF-B expression in the 10 months old rats was markedly higher than those of 2 months old. Taken together, these findings provide evidence that there was an elevation of serum cholesterol levels coinciding with an increase of PDGF-B expression in older rats, in which NF-kappaB might be an important transcription factor in mediating a hypercholesterolemia-induced PDGF-B expression.

[Detection of NF-kappaB activation and platelet-derived growth factor-B expression in endothelial cells of hypercholesterolemic rats].

OBJECTIVE: To detect whether the activation of nuclear factor-kappa B (NF-kappaB) in endothelial cells induced by mm-LDL can promote platelet-derived growth factor-B (PDGF-B) expression in vitro, and whether it is also present in hypercholesterolemic rats in vivo, influence of age on NF-kappaB and PDGF-B signal transduction pathway. METHODS: Established hypercholesterolemic rat model by feeding with a high-cholesterol ration. The activation of NF-kappaB in aortic endothelial cells was identified by immunohistochemical staining, the expression of PDGF-B mRNA and PDGF-B protein were examined using in situ hybridization and immunohistochemistry respectively. RESULTS: In comparison with the control rats, a positive immunostaining of NF-kappaB in nuclei of aortic endothelial cells of the experimental rats was detected after a high cholesterol ration for 6 weeks. The number of endothelial cells expressing PDGF-B mRNA increased and the intensity was dependent upon the duration of high-cholesterol intake. NF-kappaB translocation (0.461 +/- 0.075 vs. 0.350 +/- 0.094, P < 0.05) and PDGF-B expression in 10-month old Wistar rats were more remarkable than that of 2-month old rats after having cholesterol for 16 weeks. Immunohistochemical staining for PDGF-B gave a similar result (0.230 +/- 0.040 vs. 0.185 +/- 0.037, P < 0.001). CONCLUSIONS: Hypercholesterolemia is capable of activating nuclear translocation of NF-kappaB and promoting expression of PDGF-B in rat aortic endothelial cells in vivo, this coincided with the results obtained in ox-LDL or mm-LDL experiments on endothelial cells in vitro. This phenomenon is much more evident in 10-month old rats which indicates that age might have a close relationship with NF-kappaB - PDGF-B signal transduction pathway.

[Molecular basis of preventive effect of human apolipoprotein-1 on murine vascular smooth muscle cell proliferation and lipid deposition induced by oxidized low density lipoprotein].

OBJECTIVE: To study the molecular basis of preventive effect of human apolipoprotein-1 (h-apoA-1) on vascular smooth muscle cell (vSMC) proliferation and lipid deposition induced by oxidized low density lipoprotein (ox-LDL). METHODS: Smooth muscle cells originated from the aortae of h-apo-A-1 transgenic mice were cultured and divided into 2 groups, one group was stimulated by ox-LDL (tester) and the other group was used as control (driver). Subtractive hybridization was used to enrich the genes differentially expressed in the vSMCs induced by ox-LDL. A subtractive library was thus established and confirmed by colony hybridization in situ and dot blot analysis. 15 clones out of the 57 differentially expressed clones were randomly chosen foe sequencing and homology analysis. The whole-length cDNA library of vSMC induced by ox-LDL was established using SMART technique. RESULTS: Three expression sequence tags (EST), all correlated with immune system, were confirmed: C1-inhibitor (C1-INH), lectin, and T cell receptor beta. The whole-length cDNA library contained 1.5 x 10(6) pfu/ml primary recombinants with insertions 0.5 - 3 kb in length. CONCLUSION: The 3 EST may be involved in the mechanism of atherogenesis by ox-LDL and the mechanism of the function of h-ApoA-1 in retarding the progression of atherogenesis induced by ox-LDL.

Cloning the full-length cDNA of a gene responsible for vascular smooth muscle cell proliferation in atherogenesis and study of function.

OBJECTIVE: To clone the full-length cDNA of a gene responsible for vascular smooth muscle cell (v-SMC) proliferation in atherogenesis, and study its function. METHODS: Oxidized low density lipoprotein (ox-LDL) at optimal concentration was used as the stimulant to induce v-SMC proliferation in culture medium. A cDNA subtractive library of v-SMC proliferation specific to ox-LDL stimulation was established using subtractive hybridization technique. Methods, including blotting, Northern hybridization and gene sequencing, were used to clone new gene fragments. By using full-length cDNA screening and protein expression techniques, one full-length cDNA was cloned and its function was studied. RESULTS: One full-length cDNA was cloned. The new gene (Genbank AF 174647) expressed a 44 kDa protein, which might be associated with the activity of ox-LDL. CONCLUSION: The new gene cloned may be associated with SMC proliferation in atherogenesis.

Expression of antisense thrombin receptor gene inhibits intimal hyperplasia of rat carotid artery after balloon injury.

OBJECTIVE: To study the mechanism of restenosis after angioplasty and to clarify the effect of thrombin and its receptor on restenosis development. METHODS: Balloon catheter-induced injury was adopted to induce intimal hyperplasia of the carotid arteries in rats. Antisense thrombin receptor (ATR) cDNA was transfected by perfusing recombinant LXSN ATR plasmid/nanoparticle complex into the segment of the injured carotid artery. RESULTS: PCR result showed integration of the recombined gene. Dot blot showed the expression of antisense TR mediated by recombinant LXSN ATR plasmid/nanoparticle complex in the wall of common carotid arteries of the experimental group rats, which enabled to inhibit TR gene expression and intimal hyperplasia of the injured arteries. CONCLUSIONS: Thrombin and its receptor play an important role in the formation of neointima after the injury, which provides a potential clue in developing a new approach for prevention and treatment of restenosis after angioplasty.

Effect of oxLDL on the uptake and clearance rate of cholesterol in v-SMC originated from human apoA1 transgenic mice.

OBJECTIVE: To study the inhibition effect of oxLDL on the uptake and clearance of intra-cellular (3)H-cholesterol in v-SMC from the human-apoA1 transgenic mice (C57BL/6) and the changes in human-apoA1 mRNA expression in v-SMC from human apoA1 transgenic mice after oxLDL stimulation. METHODS: v-SMC originally isolated from human apoA1 transgenic mice connected with a recombined mouse metallothionein-I (MT-I) promoter was used, and the effect of oxLDL on the uptake and clearance of intracellular (3)H-cholesterol was studied in v-SMC of the transgenic and control mice respectively, the study of h-apoA I mRNA expression from v-SMC of the transgenic mice were done by RT-PCR and Northern blot. RESULTS: oxLDL (30 microg/ml) strongly promoted the SMC proliferation. No difference was found in (3)H-cholesterol uptake between nSMC and trSMC, and the uptake rates of both kinds of SMC rose 100% after oxLDL stimulation. The efflux rates of (3)H-cholesterol in trSMC were much higher than those of nSMC (40% - 50%). After oxLDL stimulation, the clearance rates fell by 28% and 10%, respectively, for nSMC and trSMC. The result of RT-PCR and Northern blot showed that h-apoA1 gene expression was markedly increased by the stimulation of oxLDL. CONCLUSION: Expression of the h-apoA1 gene in C57BL/6 mice enables them to reduce the accumulation of cholesterol in v-SMC. The trSMC can alleviate the harmful effect of oxLDL due to the increase of h-apoA1 expression.

Effect of NF-kappaB on the induction of PDGF-B transcription by angiotensin II in the ECV304 cell line.

OBJECTIVE: To examine the effect of angiotensin II (Ang II) on nuclear factor-kappa B (NF-kappaB) activation in human endothelial cell line ECV304 and the molecular mechanism by which Ang II activates NF-kappaB. METHODS: ECV304 cells were transiently cotransfected with an NF-kappaB/luciferase reporter gene and inactive NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKK(alpha)), IkappaB kinase beta (IKK(beta)) mutants or vectors, respectively. The effect on NF-kappaB was detected by using an electrophoretic mobility shift assay (EMSA) and overexpression of the mutants enabled blocking of reporter gene activation induced by Ang II. With immunofluorescence and immuno-electronic microscope techniques, including confocal microscopy and gold particle labeled electronic microscopy, definite cytoplasmic-to-nuclear translocations of NF-kappaB activation were detected using subunits p50 and p65 induced by Ang II. RESULTS: The translocation of p50 in nuclei was highly remarkable 2 hours after Ang II stimulation, and the activity was somewhat reduced 6 hours after stimulation to the 18th hour. Northern blot also showed PDGF-B mRNA increased by stimulation of Ang II for 18 hours. CONCLUSION: Ang II is effective in stimulating NF-kappaB activation through a pathway dependent on NIK, IKK(alpha) and IKK(beta), and induces PDGF-B transcription in the endothelial cell line, ECV304.v

Nanoparticle as a new gene transferring vector in specific expression gene / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.</p><p><b>METHODS</b>Nanoparticle-DNA complex was prepared with Poly-dl-lactic-co-glycolic acid (PLGA) bearing anti-sense monocyte chemotactic protein-1 (A-MCP-1), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 microg), 6 with A-MCP-1 (200 microg) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.</p><p><b>RESULTS</b>The package efficiency of the nanoparticle-DNA complex was 0.9%, release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300 nm. SMC genomic DNA PCR showed that A-MCP-1 gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-1 mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.</p><p><b>CONCLUSION</b>Nanoparticle can act as a vector to transfect specific gene.</p>