Malat1 promotes macrophage-associated inflammation by increasing PPAR-γ methylation through binding to EZH2 in acute myocardial infarction.

The inflammatory microenvironment of macrophage plays an important role in acute myocardial infarction (AMI), but the regulatory mechanism is unknown. Here, we aimed to investigate the role of Malat1 on inflammation microenvironment of macrophage in AMI. Our study found that Malat1 expression was increased in AMI, which mainly expressed in macrophages. Malat1 inhibition improved collagen deposition and inflammation in infarcted heart. In vitro, Malat1 inhibition evidently reduced macrophage-associated inflammation. The results from ribonucleic acid pull-down (RNA pull-down) and RNA Immunoprecipitation (RIP) assay demonstrated that Malat1 directly binds to EZH2. Malat1 and EZH2 complex could increase histone H3K27me3 expression and further inhibit the production of PPAR-γ. In vivo, inhibition of Malat1 also leaded to the down-regulation of both EZH2 and H3K27me3, as well as up-regulation of PPAR-γ in infarcted heart. Therefore, these findings demonstrate a novel mechanism of Malat1 on inflammation microenvironment of macrophage in AMI, which provide a new target for its treatment.

FURIN suppresses the progression of atherosclerosis by promoting macrophage autophagy.

FURIN, a member of the mammalian proprotein convertases (PCs) family, can promote the proteolytic maturation of proproteins. It has been shown that FURIN plays an important role in the progression of atherosclerosis (AS). Current evidence indicates that autophagy widely participates in atherogenesis. This study aimed to explore whether FURIN could affect atherogenesis via autophagy. The effect of FURIN on autophagy was studied using aortic tissues from aortic dissection patients who had BENTALL surgery, as well as macrophages and ApoE-/- mice. In atherosclerotic plaques of aortic tissues from patients, FURIN expression and autophagy were elevated. In macrophages, FURIN-shRNA and FURIN-overexpression lentivirus were used to intervene in FURIN expression. The results showed that FURIN overexpression accelerated LC3 formation in macrophages during the autophagosome formation phase. Furthermore, FURIN-induced autophagy resulted in lower lipid droplet concentrations in macrophages. The western blot revealed that FURIN regulated autophagy via the AMPK/mTOR/ULK1/PI3KIII signaling pathway. In vivo, FURIN overexpression resulted in increased macrophage LC3 formation in ApoE-/- mice atherosclerotic plaques, confirming that FURIN could inhibit the progression of AS by promoting macrophage autophagy. The present study demonstrated that FURIN suppressed the progression of AS by promoting macrophage autophagy via the AMPK/mTOR/ULK1/PI3KIII signaling pathway, which attenuated atherosclerotic lesion formation. Based on this data, current findings add to our understanding of the complexity of AS.

Ozone protects cardiomyocytes from myocardial ischemia-reperfusion injury through miR-200c/FOXO3 axis.

PURPOSE: Myocardial ischemia-reperfusion injury (I/R) is a detrimental process contributing to the pathological progression of coronary artery diseases. Studies indicate that miRNAs are implicated in ischemic heart disease, and ozone therapy could protect the heart from ischemic heart disease. In this study, we investigated the effect of ozone on miR-200c expression and the potential role of miR-200c in an I/R myocardial injury model. METHODS: A myocardial cellular model of I/R was established to detect the expression of miR-200c. Cardiomyocytes with I/R induction were treated with ozone as a cellular model to detect miR-200 expression and investigate its functional roles. The downstream target of miR-200c was predicted with Starbase online tools and validated by dual luciferase reporter assay. The function of miR-200c/FOXO3 axis in I/R was examined by CCK-8 proliferation and apoptotic assays. RESULTS: miR-200c was upregulated in primary cardiomyocytes of the I/R model. In cardiomyocyte cells, cell proliferation in the I/R group was significantly impaired, which could be partially rescued by miR-200c inhibitor or ozone treatment. Cell death detected by LDH release and apoptosis assay in the I/R model could also be inhibited by miR-200c inhibitor or ozone treatment. FOXO3 was identified as a downstream target of miR-200c, which was induced by ozone treatment and suppressed by miR-200c. Silencing FOXO3 abrogated the protective effect of ozone treatment on the I/R cell model. CONCLUSION: Overall, our results suggest that ozone plays a cardio-protective role in I/R through regulating miR-200/FOXO3 axis, and indicate that targeting miR-200/FOXO3 axis could potentially alleviate I/R.

miR-188-3p Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Targeting Fibroblast Growth Factor 1 (FGF1).

BACKGROUND As one of the crucial causes leading to cardiovascular disease, atherosclerosis (AS) develops in association with the dysfunction of vascular smooth muscle cells (VSMCs). However, the associated mechanism of the proliferation and migration in VSMCs requires further elucidation. MATERIAL AND METHODS Human VSMCs and ApoE-knockout (ApoE-/-) mice were used to establish AS cell and animal models, respectively. Expression levels of miR-188-3p and fibroblast growth factor 1 (FGF1) mRNA were detected using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blot was used to assess FGF1 protein expression. The proliferation, migration, and apoptosis of the cells were determined using MTT, BrdU, and Transwell assays, as well as flow cytometry analysis. The interaction between miR-188-3p and FGF1 was validated using dual-luciferase reporter gene assay, qRT-PCR, and Western blot analysis. RESULTS MiR-188-3p was found to be significantly decreased in the serum of AS patients and ApoE-/- mice as well as VSMCs of ApoE-/- mice and human VSMCs treated with oxidized low-density lipoprotein. MiR-188-3p repressed the proliferation and migration of VSMCs but promoted apoptosis of VSMCs. The binding site between miR-188-3p and 3' untranslated region (3'-UTR) of FGF1 was identified, and FGF1 was verified as a target gene of miR-188-3p. Restoration of FGF1 reversed the effects of miR-188-3p on VSMCs. CONCLUSIONS MiR-188-3p suppresses the proliferation and migration of VSMCs and induces their apoptosis through targeting FGF1.

Tanshinone IIA prevents platelet activation and down-regulates CD36 and MKK4/JNK2 signaling pathway.

BACKGROUND: Tanshinone IIA (TS IIA), a multi-pharmaceutical compound from traditional Chinese herb, is effective for treatment of atherothrombosis. However, the underlying mechanisms of TS IIA-mediated anti-platelet activation effect are still poorly understood. As shown in our previous study, platelet-derived microvesicles (PMVs) generated in response to oxidant insult could activate CD36/mitogen-activated protein kinase kinase 4/Jun N-terminal kinase 2 (CD36/MKK4/JNK2) signals and lead to platelet activation. The present study aims to investigate the effect of TS IIA on platelet activation and the possible mechanisms. METHODS: The production of PMVs induced by Interleukin 6 (IL-6) was detected by flow cytometry. We performed activating studies of platelets with PMVs derived from IL-6-treated platelets (IL-6-PMVs) in vitro. Sometimes, platelet suspensions were incubated with serial concentrations of TS IIA for 15 min before being stimulated with IL-6-PMVs. Expression of platelet integrin αIIbß3 and CD36 was detected by flow cytometry. Phosphorylation of MKK4 and JNK were detected by immunoblotting. RESULTS: Here we demonstrated firstly that TS IIA could prevent platelet activation induced by PMVs and down-regulates CD36 and MKK4/JNK2 signaling pathway. CD36 may be the target of atherosclerosis (AS)-related thrombosis. CONCLUSIONS: This study showed the possible mechanisms of TS IIA-mediated anti-platelet activation and may provide a new strategy for the treatment of AS-related thrombosis by targeting platelet CD36.

Evaluation of pulmonary vein anatomy using 256-slice computed tomography

Background/aim: This study aimed to evaluate pulmonary vein (PV) anatomy using 256-slice computed tomography (CT), which may be necessary for electrophysiologists to know before radiofrequency catheter ablation (RFCA) therapy.Materials and methods: A total of 102 patients with paroxysmal and persistent atrial fibrillation underwent 256-slice cardiac CT angiography prior to catheter ablation. PV morphology, ostial diameter, ostial orientation, and distance from ostium to first bifurcation were evaluated using three-dimensional volume-rendering and multiplanar-reformatting technology. Results: We found that 72.5% of patients had four conventional PVs. On the right, 22.5% of patients had one accessory PV, 4.9% had 2 accessory PVs, 1% had one common PV, and 1% had one top vein. On the left, 27.5% of patients had one common PV. Additionally, 9.8% of patients had bilateral PV variation. Ostial size was larger for superior PVs than inferior PVs and larger for right PVs than left PVs. PV ostia on the right tended to be more circular. There was a rather wide variation of projective angle and distance from ostium to first bifurcation. Early branching occurred more often in the right inferior PV. Conclusion: 256-Slice CT can depict PV anatomy and afford substantial data, which will help electrophysiologists conduct the RFCA procedure safely and efficiently.

Low shear stress induced HMGB1 translocation and release via PECAM-1/PARP-1 pathway to induce inflammation response.

Low shear stress (LSS) plays a critical role in the site predilection of atherosclerosis through activation of cellular mechanosensors, such as platelet endothelial cell adhesion molecule 1 (PECAM-1). Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that regulates the expression of various inflammatory cytokines. The nuclear enzyme high mobility group box 1 (HMGB1) can induce inflammation response by binding to toll-like receptor 4 (TLR4). In the present study, we aimed to investigate the role and mechanism of HMGB1 in LSS induced inflammation in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated by undisturbed shear stress (USS, 1 Pa) and LSS (0.4 Pa) in our experiments. Gene expression was inhibited by small interfering RNA (siRNA). ICAM-1 expression was regulated by LSS in a time dependent manner. LSS can induce HMGB1 translocation from nucleus to cytoplasm and release. Compared with the USS, LSS could increase the protein expression of PECAM-1 and PARP-1 as well as the secretion of TNF-α and IL-1ß. LSS induced the translocation of HMGB1 from nucleus to cytoplasm. Inhibition of HGMB1 reduced LSS-induced inflammatory response. Inhibition of PARP-1 suppressed inflammatory response through inhibiting TLR4 expression and HMGB1 translocation. PECAM-1 inhibition reduced LSS-induced ICAM-1 expression, TNF-α and IL-1ß secretion, and monocytes adhesion. LSS can induce inflammatory response via PECAM-1/PARP-1/HMGB1 pathway. PARP-1 plays a fundamental role in HMGB1 translocation and TLR4 expression. Inhibition of PARP-1 may shed light on the treatment of HMGB1 involved inflammation during atherosclerosis.

Mesh optimization of vessel surface model for computer-aided simulation of percutaneous coronary intervention.

Percutaneous coronary intervention is the gold standard to coronary diseases in the past decades due to much less trauma and quick recovery. However, due to the traits of minimal invasiveness, clinicians have to defeat the difficulties in eye-hand coordination during the procedure, which also makes it a non-trivial task in the catheterization lab. The computer-aided surgical simulation is designed to provide a reliable tool for the early stage of the training of the procedure. In this simulation system, the surface model of the vessels contribute the major part in the virtual anatomic environment. On the other hand, heavy interactions between the virtual surgical tools and the model surface occur during the training. In order to achieve acceptable performances, the patient-specific vessel surface model needs further process to adapt to this situation. We proposed in this paper an approach to optimize the meshes that consist the surface model with its application in consideration. The connectivity of the surface model is firstly checked. Next a smooth processing is applied without modifying the geometry of the largest-connected surface. Then the quantities of the polygons consisting the model surface are eliminated both dramatically and appropriately. The resultant surface model is applied in the validation test interacting with the virtual guidewire.

A 3D virtual reality simulator for training of minimally invasive surgery.

For the last decade, remarkable progress has been made in the field of cardiovascular disease treatment. However, these complex medical procedures require a combination of rich experience and technical skills. In this paper, a 3D virtual reality simulator for core skills training in minimally invasive surgery is presented. The system can generate realistic 3D vascular models segmented from patient datasets, including a beating heart, and provide a real-time computation of force and force feedback module for surgical simulation. Instruments, such as a catheter or guide wire, are represented by a multi-body mass-spring model. In addition, a realistic user interface with multiple windows and real-time 3D views are developed. Moreover, the simulator is also provided with a human-machine interaction module that gives doctors the sense of touch during the surgery training, enables them to control the motion of a virtual catheter/guide wire inside a complex vascular model. Experimental results show that the simulator is suitable for minimally invasive surgery training.

Tumor necrosis factor-alpha G-308 A polymorphism and risk of coronary heart disease and myocardial infarction: A case-control study and meta-analysis.

OBJECTIVES: The tumor necrosis factor-alpha (TNF-α) gene may play an important role in coronary heart disease (CHD) and myocardial infarction (MI) risk. Recently, controversial results regarding the association of the G-308 A (rs1800629)polymorphism of the TNF-α gene with CHD/MI have been reported. We herein examine a possible association between the G-308 A (rs1800629)polymorphism of the TNF-α gene and CHD/MI in a sample of the Chinese Han population. MATERIALS AND METHODS: We determined the genotypes of TNF-α G-308 A (rs1800629) in 535 unrelated Chinese patients with CHD, 420 patients with MI, and 1020 coronary artery disease-free controls. Additionally, a meta-analysis of all previous studies on the TNF-α G-308 A polymorphism and the risk of CHD and MI was performed. RESULTS: AA genotypes in the G-308 A (rs1800629)polymorphism of the TNF-α gene did not occur more frequently in CHD/MI patients than in controls; odds ratios (95% confidence intervals) were 1.743 (0.325 to 1.423) for CHD and 1.731 (0.442 to 1.526) for MI, after adjusting for conventional risk factors. Further stratification for age, gender, and other cardiovascular risk factors did not alter the prior negative findings. Pooled meta-analysis of 23 studies also found no statistically significant associations between the TNF-α polymorphism and CHD/MI risk in the genetic additive, dominant, and recessive models. Subgroup analyses showed no association between the TNF-α polymorphism and CHD/MI in Asian and Caucasian populations. CONCLUSION: Our study showed no association between the G-308 A (rs1800629) polymorphism of the TNF-α gene (presence of A allele) and CHD/MI in the Chinese Han population. There was no evidence of a difference in risk effects of rs1800629 between Caucasians and Asians.

Recombinant proteins secreted from tissue-engineered bioartificial muscle improve cardiac dysfunction and suppress cardiomyocyte apoptosis in rats with heart failure.

BACKGROUND: Tissue-engineered bioartificial muscle-based gene therapy represents a promising approach for the treatment of heart diseases. Experimental and clinical studies suggest that systemic administration of insulin-like growth factor-1 (IGF-1) protein or overexpression of IGF-1 in the heart exerts a favorable effect on cardiovascular function. This study aimed to investigate a chronic stage after myocardial infarction (MI) and the potential therapeutic effects of delivering a human IGF-1 gene by tissue-engineered bioartificial muscles (BAMs) following coronary artery ligation in Sprague-Dawley rats. METHODS: Ligation of the left coronary artery or sham operation was performed. Primary skeletal myoblasts were retrovirally transduced to synthesize and secrete recombinant human insulin-like growth factor-1 (rhIGF-1), and green fluorescent protein (GFP), and tissue-engineered into implantable BAMs. The rats that underwent ligation were randomly assigned to 2 groups: MI-IGF group (n = 6) and MI-GFP group (n = 6). The MI-IGF group received rhIGF-secreting BAM (IGF-BAMs) transplantation, and the MI-GFP group received GFP-secreting BAM (GFP-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: S-IGF group (n = 6) and S-GFP group (n = 6). The S-IGF group underwent IGF-1-BAM transplantation, and S-GFP group underwent GFP-BAM transplantation. IGF-1-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats after two weeks of operation was performed. Four weeks after the treatment, hemodynamics was performed. IGF-1 was measured by radioimmunoassay, and then the rats were sacrificed and ventricular samples were subjected to immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of bax and Bcl-2. TNF-α and caspase 3 expression in myocardium was examined by Western blotting. RESULTS: Primary rat myoblasts were retrovirally transduced to secrete rhIGF-1 and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted consistent levels of hIGF (0.4 - 1.2 µg×BAM(-1)×d(-1)). When implanted into syngeneic rat, IGF-BAMs secreted and delivered rhIGF. Four weeks after therapy, the hemodynamics was improved significantly in MI rats treated with IGF-BAMs compared with those treated with GFP-BAMs. The levels of serum IGF-1 were increased significantly in both MI and sham rats treated with IGF-BAM. The mRNA expression of bax was lower and Bcl-2 expression was higher in MI-IGF group than MI-GFP group (P < 0.05). Western blotting assay showed TNF-α and caspase 3 expression was lower in MI-IGF group than MI-GFP group after therapy. CONCLUSIONS: rhIGF-1 significantly improves left ventricular function and suppresses cardiomyocyte apoptosis in rats with chronic heart failure. Genetically modified tissue-engineered BAMs provide a method delivering recombinant protein for the treatment of heart failure.

Recombinant human growth hormone secreted from tissue-engineered bioartificial muscle improves left ventricular function in rat with acute myocardial infarction.

BACKGROUND: Experimental studies and preliminary clinical studies have suggested that growth hormone (GH) treatment may improve cardiovascular parameters in chronic heart failure (CHF). Recombinant human GH (rhGH) has been delivered by a recombinant protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. The present study aimed to examine a new method for delivery of rhGH using genetically modified bioartificial muscles (BAMs), and investigate whether the rhGH delivered by this technique improves left ventricular (LV) function in rats with CHF. METHODS: Primary skeletal myoblasts were isolated from several Sprague-Dawley (SD) rats, cultured, purified, and retrovirally transduced to synthesize and secrete human rhGH, and tissue-engineered into implantable BAMs. Ligation of the left coronary artery or sham operation was performed. The rats that underwent ligation were randomly assigned to 2 groups: CHF control group (n = 6) and CHF treatment group (n = 6). The CHF control group received non-rhGH-secreting BAM (GFP-BAMs) transplantation, and the CHF treatment group received rhGH-secreting BAM (GH-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: sham control group (n = 6) and sham treatment group (n = 6). The sham control group underwent GFP-BAM transplantation, and the sham treatment group underwent GH-BAM transplantation. GH-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats with ligation of the left coronary artery or sham operation was performed. Eight weeks after the treatment, echocardiography was performed. hGH, insulin-like growth factor-1 (IGF-1) and TNF-alpha levels in rat serum were measured by radioimmunoassay and ELISA, and then the rats were killed and ventricular samples were subjected to immunohistochemistry. RESULTS: Primary rat myoblasts were retrovirally transduced to secrete rhGH and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 1 to 2 microg of bioactive rhGH per day. When implanted into syngeneic rat, GH-BAMs secreted and delivered rhGH. Eight weeks after therapy, LV ejection fraction (EF) and fractional shortening (FS) were significantly higher in CHF rats treated with GH-BAMs than in those treated with GFP-BAMs ((65.0 +/- 6.5)% vs (48.1 +/- 6.8)%, P < 0.05), ((41.3 +/- 7.4)% vs (26.5 +/- 7.1)%, P < 0.05). LV end-diastolic dimension (LVEDD) was significantly lower in CHF rats treated with GH-BAM than in CHF rats treated with GFP-BAM (P < 0.05). The levels of serum GH and IGF-1 were increased significantly in both CHF and sham rats treated with GH-BAM. The level of serum TNF-alpha decreased more significantly in the CHF treatment group than in the CHF control group. CONCLUSIONS: rhGH significantly improves LV function and prevents cardiac remodeling in rats with CHF. Genetically modified tissue-engineered bioartificial muscle provides a method delivering recombinant protein for the treatment of heart failure.

Change of p16(INK4a) and PCNA protein expression in myocardium after injection of hIGF-1 gene modified skeletal myoblasts into post-infarction rats.

This study examined the change of p16(INK4a) and PCNA protein expression in myocardium after injection of hIGF-1 gene modified skeletal myoblasts into post-infarction rats. HIGF-1 gene modified skeletal myoblasts (hIGF-1-myoblasts) were injected into hind limb muscles of 18 post-infraction rats (experimental group). Primary-myoblasts were injected into 18 post-infraction rats (control group) and 12 non-infarction rats (sham group). Expression of p16(INK4a) and PCNA protein in myocardiums were separately detected immunocytochemically 1, 2 and 4 weeks after the injection. The level of hIGF-1 and rIGF-1 protein in serum and myocardium were detected by enzyme-linked immunosorbent assay (ELISA). Compared with the sham group, the percentage of p16(INK4a) and PCNA positive cells reached a peak after 1 week in the control group and the experimental group (P<0.01). Moreover, the percentage of p16(INK4a)-positive cells in the experimental group was lower than in control group whereas the percentage of PCNA-positive cells was lower in the control group than in the experimental group (P<0.01). The percentage of p16(INK4a)-positive cells in the experimental group and the percentage of PCNA-positive cells in the control group were close to that in the sham group from the 2nd week (P>0.05). ELISA analysis disclosed that the myocardium level of rIGF-1 protein increased gradually in the controls and especially in the experimental group (P<0.01). The serum level of rIGF-1 decreased significantly in post-infraction rats, but these conditions were improved in the experimental group (P<0.01). The hIGF-1 protein in serum and myocardium were detected from the 1st week to the 4th week in the experimental group. Statistical analysis revealed significant associations of myocardium level of hIGF-1 protein with expression of p16(INK4a) and PCNA protein (r=-0.323, P<0.05; r=0.647, P<0.01). It is concluded that genetically hIGF-1-myoblast provides a means for constant synthesis and release of hIGF-1. It could not only improve the expression of rIGF-1 and PCNA protein in myocardium, but also suppress the expression of p16(INK4a) protein for 30 days in post-infraction rats. Myoblasts-mediated IGF-1 gene therapy may provide a new alternative for the clinical treatment of heart failure.

Effects of transplanted myoblasts transfected with human growth hormone gene on improvement of ventricular function of rats.

BACKGROUND: Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human growth hormone (hGH) has been shown to promote angiogenesis and attenuate apoptosis in the experimental animal. This study was conducted to explore the effects of myoblast-based hGH gene therapy on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences between myoblast-based hGH gene therapy and myoblast therapy. METHODS: Myoblasts were isolated from several SD rats, cultured, purified, and transfected with plasmid pLghGHSN and pLgGFPSN. Radioimmunoassay (RIA) was used to detect the expression of hGH in these myoblasts. SD rats underwent the ligation of the left anterior descending coronary artery so as to establish a heart ischemia model. Thirty surviving rats that underwent ligation were randomly divided into 3 equal groups 2 weeks after left coronary artery occlusion: pLghGHSN group received myoblast infected with hGH gene transplantation; pLgGFPSN group received myoblast infected with GFP gene transplantation; control group: received cultured medium only. Four weeks after the injection the surviving rat underwent evaluation of cardiac function by echocardiography. The rats were killed and ventricular samples were undergone immunohistochemistry with hematoxylin-eosin and factor VIII. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of vascular endothelial growth factor (VEGF), bax and Bcl-2. hGH expression in myocardium was examined by Western blot. RESULTS: Myoblast can be successfully isolated, cultured and transfected. The expression of hGH in transfected myoblast was demonstrated with RIA. Four weeks after therapy, the cardiac function was improved significantly in pLghGHSN group and pLgGFPSN group. Fractional shortening (FS) and ejection fraction (EF) in pLghGHSN group were elevated significantly compared with pLgGFPSN group and control group after therapy (FS: 36.9+/-5.3 vs 29.5+/-3.5, 21.8+/-2.9; EF: 56.9+/-4.3 vs 47.1+/-3.6, 38.4+/-4.8, P<0.05). Left ventricular end-diastolic dimension (LVEDD) and heart infracted size in pLghGHSN group were decreased significantly compared with pLgGFPSN group and control group after therapy (LVEDD: 5.9+/-0.3 vs 6.8+/-0.2, 8.6+/-0.3; heart infracted size: (34.5+/-4.2)% vs (40.0+/-3.9)%, (46.1+/-3.8)%, P<0.05); Green fluorescence was detected in cryosection of pLgGFPSN group. The capillary density of the pLgGFPSN group was significantly greater than those of the pLghGHSN group and control group (P<0.05). The mRNA expression of VEGF and Bcl-2/bax in pLghGHSN group was higher than in pLgGFPSN group or control group (P<0.05). The expression of hGH gene in myocardium tissue can be detected by Western blot assay in pLghGHSN group. CONCLUSIONS: Transplantation of heart cells transfected with hGH induced greater angiogenesis and effect of antiapoptosis than transplantation of cells transfected with GFP. Combined GH gene transfer and cell transplantation provided an effective strategy for improving postinfarction ventricular function.

Change of p16INK4a and PNCA Protein Expression in Myocardium after Injection of hIGF-1 Gene Modified Skeletal Myoblasts into Post-infarction Rats / 华中科技大学学报（医学）（英德文版）

This study examined the change of p 16INK4a and PNCA protein expression in myocardium after injection of hIGF-1 gene modified skeletal myoblasts into post-infarction rats. HIGF-1 gene modified skeletal myoblasts (hIGF-1-myoblasts) were injected into hind limb muscles of 18post-infraction rats (experimental group). Primary-myoblasts were injected into 18 post-infraction rats (control group) and 12 non-infarction rats (sham group). Expression of p16INK4a and PCNA protein in myocardiums were separately detected immunocytochemically 1, 2 and 4 weeks after the inuection. The level of hIGF-1 and rIGF-1 protein in serum and myocardium were detected by enzyme-linked immunosorbent assay (ELISA). Compared with the sham group, the percentage of p16INK4a and PCNA positive cells reached a peak after 1 week in the control group and the experimental group (P＜0.01). Moreover, the percentage of p16INK4a-positive cells in the experimental group was lower than in control group whereas the percentage of PCNA-positive cells was lower in the control group than in the experimental group (P＜0.01). The percentage of p16INK4a-positive cells in the experimental group and the percentage of PCNA-positive cells in the control group were close to that in the sham group from the 2nd week (P＞0.05). ELISA analysis disclosed that the myocardium level of rIGF-1 protein increased gradually in the controls and especially in the experimental group (P＜0.01). The serum level of rIGF-1 decreased significantly in post-infraction rats, but these conditions were improved in the experimental group (P＜0.01). The hIGF-1 protein in serum and myocardium were detected from the 1st week to the 4th week in the experimental group. Statistical analysis revealed significant associations of myocardium level of hIGF-1 protein with expression of p16INK4a and PCNA protein (r=-0.323, P＜0.05; r=0.647, P＜0.01). It is concluded that genetically hIGF-1-myoblast provides a means for constant synthesis and release of hIGF-1. It could not only improve the expression of rIGF-1 and PCNA protein in myocardium, but also suppress the expression of p16INK4a protein for 30 days in post-infraction rats. Myoblasts-mediated IGF-1 gene therapy may provide a new alternative for the clinical treatment of heart failure.

Effects of recombinant retroviral vector mediated human insulin like growth factor-1 gene transfection on skeletal muscle growth in rat.

BACKGROUND: This study transferred a recombinant gene encoding human insulin like growth factor-1 (hIGF-1) into modified primary skeletal myoblasts with a retroviral vector (pLgXSN) and determined whether the hIGF-1 promoted growth of skeletal muscle in rat. METHODS: hIGF-1cDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochemistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghIGF-1SN were injected into hind limb muscles of 10 - 12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth. RESULTS: Recombinant retroviral plasmid vector pLghIGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 x 10(6) cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening. hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast (P < 0.05). The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast (P < 0.05). Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myoblast group (P < 0.05). CONCLUSIONS: The transfection of the human IGF-1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.