Protective effect of a polysaccharide from Salvia miltiorrhiza on isoproterenol (ISO)-induced myocardial injury in rats.

In this study, we investigated the cardioprotective effect of one purified polysaccharide (SMP1) from Salvia miltiorrhiza on isoproterenol (ISO)-induced myocardial infarction (MI) in rats. ISO-treated rats showed severe myocardial damage and high lipid peroxidation level, as well as decreased endogenous myocardial antioxidant function. Pretreatment with SMP1 (100 and 400mg/kg) for 30 days significantly increased the body weight, decreased the heart weight, attenuated the serum levels of creatine kinase (CK), creatine phospokinase-MB (CK-MB), dehydrogenase (LDH), alkaline phosphate (ALP), aspartate transaminase (AST), alanine transaminase (ALT), total cholesterol, triglyceride, and LDL-cholesterol (LDL-C), along with the increased concentration of HDL-cholesterol (HDL-C). In addition, SMP1 also enhanced myocardial superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities and elevated myocardial reduced glutathione (GSH) level, along with a decrease in thiobarbituric acid reactive substances (TBARS) concentration. Collectively, our results indicated that long-term oral administration of SMP1 offered significant protection against the damage induced by ISO in rat heart through enhancement of endogenous antioxidants and antihyperlipidemic activity.

Cardiovascular effects in vitro of a polysaccharide from Salvia miltiorrhiza.

A polysaccharide (SMP1) was isolated from the roots of Salvia miltiorrhiza. This study is designed to investigate whether SMP1 prevents H9c2 cells from hydrogen peroxide (H2O2)-induced apoptosis. The present study showed that exposure of H9c2 cells to 100mM H2O2 for 24h caused a significant increase in cell death and apoptosis, but pretreatment with SMP1 eliminated H2O2-induced apoptotic cell death. Furthermore, pretreatment with SMP1 significantly prevented the mitochondria disruption, cytochrome c release, the rise of the ratio between proapoptotic Bax and antiapoptotic Bcl-2 protein expression, and caspase-3 activation in H9c2 cells upon H2O2 stimulation. Moreover, the decline of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities together with the elevation of malondialdehyde (MDA) in PC12 cells exposed to H2O2 were remarkably reversed to normal levels by pretreatment with SMP1. These results suggest that SMP1 protects H9c2 cells from H2O2-induced apoptosis through inhibition of mitochondrial dysfunction, inactivation of caspase-3 cascade and enhancement of antioxidant capacity.

Optimization of extraction and purification of arctiin from Fructus arctii and its protection against glucose-induced rat aortic endothelial cell injury.

To develop an efficient method for extracting and purifying the active ingredient, arctiin, from Fructus arctii and to investigate the protective effect of arctiin against glucose-induced rat aortic endothelial cell (RAEC) injury was investigated. Using a L9 (34) orthogonal array and two-step column chromatography (with AB-8 macroporous resin) arctiin extraction was optimized using a reflux method with 70% ethanol. The RAECs were then treated with different concentrations of arctiin (1, 10, or 100 µg/ml). The effects of arctiin on cell viability in a high glucose medium, malondialdehyde (MDA) levels, and lactate dehydrogenase were measured using commercially available assays. After extraction, the purity of arctiin reached 95.7%. In rats, arctiin was shown to stimulate the proliferation of RAECs in a high glucose medium in a dose-dependent manner. Exposure of RAECs to high glucose resulted in a significant increase in MDA and release of lactate dehydrogenase. This was accompanied by significant increase in nitric oxide release and expression of antiendothelial nitric oxide synthase. This technique resulted in relatively pure arctiin extraction. Furthermore, the results from this study suggest that arctiin could potentially function as a protector against vascular endothelial cell injury and further investigation is warranted.

Hydrogen-rich saline prevents neointima formation after carotid balloon injury by suppressing ROS and the TNF-α/NF-κB pathway.

BACKGROUND: Reactive oxygen species (ROS) play a pivotal role in neointima hyperplasia after balloon injury. Molecular hydrogen has emerged as a novel antioxidant and has been proven effective in treating many diseases. OBJECTIVES: We aimed to determine the mechanism by which hydrogen affects neointima formation. METHODS: We assessed the influence of a hydrogen-rich saline solution (HRSS) by daily injection in rats. Rats were euthanized to evaluate the neointima. ROS, malondialdehyde (MDA) and superoxide dismutase (SOD) and reduced glutathione (GSH), were detected in the injured artery. Macrophage infiltration and the production of inflammatory factors (i.e., IL-6, TNF-α and NF-κB) were also observed. The in vitro effects of hydrogen on vascular smooth muscle cell (VSMC) proliferation were also measured. RESULTS: HRSS decreased the neointima area significantly. The neointima/media ratio was also reduced by HRSS. There was a decline in the number of PCNA-positive cells in the intima treated with HRSS. Meanwhile, HRSS ameliorated the ROS and MDA levels and increased SOD, reduced GSH levels in the injured carotid. In addition, the levels of inflammatory factors, such as IL-6, TNF-α and NF-κB p65, were attenuated by HRSS. In vitro studies also confirmed the anti-proliferative capability of the hydrogen solution and ROS generation in VSMCs induced by PDGF-BB. CONCLUSION: HRSS may have a protective role in the prevention of neointima hyperplasia and restenosis after angioplasty. HRSS may partially exert its role by neutralizing the local ROS and suppressing the TNF-α/NF-κB pathway.

CCN1 promotes the differentiation of endothelial progenitor cells and reendothelialization in the early phase after vascular injury.

Endothelial progenitor cells (EPCs) contribute to the process of reendothelialization and prevent neointimal formation after vascular injury. The present study was designed to investigate whether the cysteine-rich 61 (CYR61, CCN1), an important matricellular component of local vascular microenvironment, has effect on EPCs differentiation and reendothelialization in response to vascular injury in rat. Following balloon injury, CCN1 was rapidly induced and dynamically changed at vascular lesions. Overexpression of CCN1 by adenovirus (Ad-CCN1) accelerated reendothelialization and inhibited neointimal formation in the early phase (day 14) after vascular injury (p < 0.05), while no effect was shown on day 21. Ad-CCN1 treatment increased the adhering EPCs on the surface of injured vessels on day 7, and the ratio of GFP- and vWF-positive area to the total luminal length on day 14 was 2.3-fold higher in the Ad-CCN1-EPC-transplanted group than in controls. Consistent with these findings, CCN1-stimulated EPC differentiation in vitro and 20 genes were found differentially expressed during CCN1-induced EPC differentiation, including Id1, Vegf-b, Vegf-c, Kdr, Igf-1, Ereg, Tgf, Mdk, Ptn, Timp2, etc. Among them, negative transcriptional regulator Id1 was associated with CCN1 effect on EPC differentiation. Our data suggest that CCN1, from the microenvironment of injured vessels, enhances reendothelialization via a direct action on EPC differentiation, revealing a possible new mechanism underlying the process of vascular repair.

Inhibitor of DNA binding-1 promotes the migration and proliferation of endothelial progenitor cells in vitro.

Migration and proliferation of endothelial progenitor cells (EPCs) are the key mechanisms in re-endothelialization after vascular injury. Inhibitor of DNA binding-1 (Id1) function has been linked to the proliferation, migration, and senescence of cells, and studies have shed light on the relationship between Id1 and the biological functions of EPCs. On the basis of the available data concerning Id1 and the behavior of EPCs, we hypothesized that Id1 was an important regulator in modulating the migration and proliferation of EPCs. Culture of spleen-derived EPCs was done as previously described. Id1 was presented at low levels in EPCs. Id1 was localized predominantly in the cytoplasm, and was rapidly upregulated by stimulation with serum and vascular endothelial growth factor. The migration and proliferation of EPCs were extensively improved by overexpression of adenovirus-mediated exogenous Id1 and inhibited by silencing of endogenous Id1 in EPCs. These results suggest that Id1 has a direct role in regulation of the migration and proliferation in EPCs.

[Autologous mesenchymal stem cell implantation promotes myocardial expressions of growth factors and improves cardiac function in failing rat hearts].

OBJECTIVE: To explore the underlying mechanism of mesenchymal stem cells (MSCs) transfer induced cardiac function improvement in failing hearts. METHODS: Congestive heart failure (CHF) was induced in rats by cauterization of the heart wall. MSCs were cultured from autologous bone marrow and injected into the border zone and the remote myocardium 5 days after cauterization. RESULTS: Ten weeks later, cardiomyocyte nucleus mitotic index, capillary density and expression of insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) were significantly increased in the border zone and significantly reduced in the remote myocardium in CHF rats (all P<0.05 vs. sham). Besides cardiac function improvement and left ventricular remodeling attenuation evidenced by hemodynamic and echocardiographic examinations, expressions of IGF-1, HGF and VEGF in the remote myocardium and in the border zone were also significantly upregulated (P<0.05 or P<0.01 vs. CHF), and cardiomyocyte nucleus mitotic index as well as capillary density were significantly increased in CHF rats with MSCs (P<0.05 or P<0.01 vs. CHF). Moreover, collagen area was significantly reduced and myocardial area was significantly increased in the border zone in these rats too. CONCLUSION: Autologous MSC implantation upregulated expressions of growth factors enhanced cardioangiogenesis which might be the underlying mechanisms for improved cardiac function and attenuated left ventricular remodeling induced by MSCs transplantation in failing rat myocardium.

Transfection of HGF gene enhances endothelial progenitor cell (EPC) function and improves EPC transplant efficiency for balloon-induced arterial injury in hypercholesterolemic rats.

Risk factors for coronary heart disease can reduce the number of endothelial progenitor cells (EPCs) and impair EPC function, thus hindering their utility in the treatment of cardiovascular diseases. In the present study, we began exploring the feasibility of genetic modification of EPCs with hepatocyte growth factor (HGF) to counter the effects of these risk factors and enhance the biological functions of EPCs. The effects of HGF transfection on proliferation, migration and angiogenesis of EPCs were investigated. Additionally, the role of ERK1/2 in this process was evaluated through the observation of ERK1/2 and ERK1/2 phosphorylation as well as by pharmacological analysis. Finally, we evaluated the effect of HGF-transfected EPCs (HGF-EPCs) on neointima formation after balloon-induced arterial injury in hypercholesterolemic rats. Our data showed that EPCs transfected with the HGF gene released high levels of soluble HGF protein, which were maintained for at least nine days. Transfection with HGF also enhanced the proliferative, migratory and angiogenic capabilities of EPCs, and promoted the activation of ERK1/2 without affecting its expression. ERK1/2 blockade by the chemical inhibitor PD98059 partially inhibited these effects. In hypercholesterolemic rats, HGF-EPCs homed to the site of vascular injury at a significantly higher rate than did EPCs without the exogenous HGF gene. Furthermore, systemically applied HGF-EPCs were more effective in decreasing neointima formation and increasing re-endothelialization. These data suggest that gene delivery combined with EPC transplant may be a practical and promising therapy for the prevention of neointimal formation after vascular injury.

Isolation and characterization of human coronary artery-derived endothelial cells in vivo from patients undergoing percutaneous coronary interventions.

BACKGROUND/AIMS: Human coronary artery-derived endothelial cells (ECs) seem to be the most appropriate cells for the pathogenesis study of coronary artery disease. But limited availability of endothelial tissue is a major constraint. In this study, we developed a method to isolate human coronary artery ECs in vivo from patients. METHODS: Coronary guidewires were used to obtain EC samples from coronary arteries in 76 patients. Cells were eluted from wire tips and purified by immunomagnetic beads. Von Willebrand factor and CD31 were used as immunocytochemical markers to identify cells as endothelium. Cell viability was evaluated in terms of cell membrane integrity, energy-dependent uptake of DiI-labeled acetylated low-density lipoprotein, and apoptosis. Nitric oxide synthase (eNOS) expression and nitric oxide (NO) production of cells were detected to evaluate cell function. RESULTS: About 96 coronary artery ECs were obtained per guidewire. Cells manifested endothelial morphology and immunoreactivity for von Willebrand factor and CD31 with good viability. But eNOS expression and NO production of cells were decreased. CONCLUSIONS: Viable coronary endothelium could be obtained during routine percutaneous coronary interventions combined with immunomagnetic beads. These cells may be used for advanced cellular functional analyses such as immunocytochemistry and molecular biology. Such information could aid in understanding mechanisms of coronary artery diseases.

An essential role for stromal interaction molecule 1 in neointima formation following arterial injury.

AIMS: There is evidence to suggest that stromal interaction molecule 1 (STIM1) functions as a Ca2+ sensor on the endoplasmic reticulum, leading to transduction of signals to the plasma membrane and opening of store-operated Ca2+ channels (SOC). SOC have been detected in vascular smooth muscle cells (VSMCs) and are thought to have an essential role in the regulation of contraction and cell proliferation. We hypothesized that knockdown of STIM1 inhibits VSMC proliferation and suppresses neointimal hyperplasia. METHODS AND RESULTS: We examined the effect of the knockdown of STIM1 using a rat balloon injury model and cultured rat aortic VSMCs. Interestingly, knockdown of rat STIM1 by adenovirus delivery of small interfering RNA (siRNA) significantly suppressed neointimal hyperplasia in a rat carotid artery balloon injury model at 14 days after injury. The re-expression of human STIM1 to smooth muscle reversed the effect of STIM1 knockdown on neointimal formation. Rat aortic VSMCs were used for the in vitro assays. Knockdown of endogenous STIM1 significantly inhibited proliferation and migration of VSMCs. Moreover, STIM1 knockdown induced cell-cycle arrest in G0/G1 and resulted in a marked decrease in SOC. Replenishment with recombinant human STIM1 reversed the effect of siRNA knockdown. These results suggest STIM1 has a critical role in neointimal formation in a rat model of vascular injury. CONCLUSION: STIM1 may represent a novel therapeutic target in the prevention of restenosis after vascular interventions.

[Effects of C-reactive protein on bone marrow-derived endothelial progenitor cell function].

OBJECTIVE: To observe the effects of C reactive protein (CRP) on endothelial progenitor cell (EPCs) function. METHODS: Mononuclear cells (MNCs), isolated from bone marrow by density gradient centrifugation combined with adherent cell filtration, were plated on fibronectin coated culture dishes. After 7 days, adherent cells were cultured with different concentrations of CRP (0, 5, 10, 15, 20 microg/ml) for 48 hours. EPCs proliferation and migration ability were observed and adhesion assay was performed. The eNOS mRNA expression of EPCs were measured by RT-PCR. RESULTS: The number of EPCs in CRP groups (10, 15, 20microg/ml) was obviously lower than that in control group (54 +/- 3, 47 +/- 3, 39 +/- 5 vs.60 +/- 3, P < 0.01). EPCs proliferation capacity was inhibited in CRP groups (10, 15, 20 microg/ml) compared with that in control group (0.297 +/- 0.036, 0.273 +/- 0.013, 0.259 +/- 0.035 vs. 0.345 +/- 0.014, P < 0.01). EPCs migration capacity was inhibited significantly in CRP groups (5, 10, 15, 20 microg/ml) than that in control group (28 +/- 2, 22 +/- 3, 19 +/- 3, 16 +/- 2 vs. 30 +/- 2, P < 0.05). EPCs adhensive number was lower in CRP groups than that in control group (11 +/- 2, 9 +/- 2, 6 +/- 2, 5 +/- 1 vs. 12 +/- 2, P < 0.05). The mRNA expressions of eNOS in CRP groups were significantly lower in control group. And compared with control group, NOS activity decreased significantly in CRP groups (10, 15, 20 microg/ml) (57.44 +/- 3.25, 48.37 +/- 3.86, 36.82 +/- 4.89 vs. 68.56 +/- 2.82, P < 0.01). CONCLUSION: CRP could both reduce EPCs number and inhibit EPCs functions.

[The role of cyclooxygenase on angiogenesis and endothelial progenitor cell mobilization in rat ischemic myocardium].

OBJECTIVE: To investigate the effect of COX1 and COX2 on angiogenesis and endothelial progenitor cell mobilization in rats with experimental myocardial infarction (MI). METHODS: The rats were randomly divided into 3 groups: MI group, MI plus rofecoxib group and MI plus valeryl salicylate group. At the 7th day after operation, circulating EPCs, plasma VEGF and HIF-1alpha mRNA of ischemic myocardium were measured. At the 28th day post operation, capillary densities were also measured in ischemic myocardium. RESULT: Compared with the MI group and the MI plus valeryl salicylate group, circulating EPCs, plasma VEGF, HIF-1alpha mRNA and capillary densities of ischemic myocardium were all decreased in MI plus rofecoxib group. CONCLUSION: The present study revealed that COX2 play an important role with angiogenesis and endothelial progenitor cell mobilization in rat with experimental MI by modulating expression of VEGF and HIF-1alpha.

[Sirolimus inhibits the differentiation, proliferation and migration of endothelial progenitor cells in vitro].

OBJECTIVE: To investigate the effect of sirolimus on differentiation, proliferation, adhesion and migration of endothelial progenitor cells (EPC) in vitro. METHODS: (1) Mononuclear cells (MNC) were isolated from rat bone marrow by Ficoll density gradient centrifugation and cultured on fibronectin-coated culture dishes with or without sirolimus (0.01 - 100 ng/ml) for 12 days. (2) After 8 days cultured, attached cells were treated with sirolimus (0.1 - 200 ng/ml) or vehicle for various time points (12 h, 24 h, 48 h and 96 h). EPC were identified as adherent cells double positive stained for FITC-UEA-I and DiI-acLDL under laser confocal immunofluence microscope. EPC proliferation, migration were assayed with MTT assay and modified Boyden chamber assay respectively. RESULTS: EPC number differentiated from MNC at 12 days was significantly lower in sirolimus treated cells in a dose-dependent manner than that of vehicle-treated cells. Sirolimus also significantly inhibited the proliferative, migratory and adhesive capacity of EPC in a time and dose dependent manner. CONCLUSION: Present results suggested that sirolimus could inhibit EPC differentiation from MNC and reduce the proliferation, migration and adhesion capacities of EPC.