Effects of low-intensity swimming on radiation-induced leg contracture in mice.

OBJECTIVE: To evaluate the effects of low-intensity swimming on radiation-induced leg contracture. METHODS: Forty mice were randomly and equally divided into four groups: 1) irradiation; 2) swimming before irradiation; 3) swimming after irradiation; 4) swimming after contracture, and their left hind legs were exposed to gamma irradiation of 60 Gy. The mice were allowed to swim freely for 10 minutes, three times per day. For group 2, the mice were allowed to swim for only 1 week before irradiation. For group 3, the mice were allowed to swim immediately after irradiation until day 130, post-irradiation. For group 4, the mice were allowed to swim after leg contracture happened (on day 30 post-irradiation) until day 130, post-irradiation. The leg lengths and knee joint angles were measured. Leg contracture was defined as the decrease in the hind leg lengths and the knee joint angles of each animal. The ultrastructural changes of gastrocnemius muscles were observed using transmission electron microscopy. RESULTS: The radiation could result in leg contracture and mitochondrial injury of the muscles. However, the group of swimming immediately after irradiation had less leg contracture and no vacuolar degeneration in the mitochondria, compared with the other groups. CONCLUSION: Low-intensity swimming that began immediately after the mice were irradiated could effectively prevent the irradiated legs from contracture. Patients with irradiated mastication muscles were recommended to begin mouth-opening exercises immediately after radiotherapy.

Requirement of the NF-κB pathway for induction of Wnt-5A by interleukin-1ß in condylar chondrocytes of the temporomandibular joint: functional crosstalk between the Wnt-5A and NF-κB signaling pathways.

OBJECTIVE: We have previously reported that interleukin-1ß (IL-1ß) up-regulates the expression of Wnt-5A and the activation of Wnt-5A signaling induces matrix metalloproteinase (MMP) through the c-Jun N-terminal kinase pathway in condylar chondrocytes (CCs) of the temporomandibular joint (TMJ). These results suggest that Wnt-5A could play an essential role in IL-1ß-mediated cartilage destruction. The objective of this study was to investigate the molecular mechanism underlying IL-1ß-induced up-regulation of Wnt-5A in TMJ CCs. METHODS: Primary CCs, limb chondrocytes (LCs) and SW1353 human chondrosarcoma cells were treated with IL-1ß in the presence or absent of BAY 11-7082 (an inhibitor of IκBα-phosphorylation). Then, expression of Wnt-5A was estimated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunocytofluorescence. Transient transfection of p65 expression vector and chromatin immunoprecipitation (ChIP) assay was performed to define the effect of p65 on Wnt-5A expression. RESULTS: IL-1ß up-regulated Wnt-5A expression at both the RNA and protein levels in articular chondrocytes. The inhibitor of IκBα-phosphorylation, BAY 11-7082, blocked the induction of Wnt-5A by IL-1ß in a dose-dependent manner. Moreover, experiments with overexpression of p65 and ChIP established that induction of Wnt-5A by IL-1ß is mediated through the NF-κB pathway, especially the p65 subunit. CONCLUSION: These results clarify the molecular mechanism underlying up-regulation of Wnt-5A by IL-1ß in chondrocytes, suggesting an important functional crosstalk between Wnt-5A and NF-κB signaling pathways. This finding provides new insights into the involvement of Wnt signaling in the cartilage destruction caused by arthritis.

beta-Catenin/TCF/LEF1 can directly regulate phenylephrine-induced cell hypertrophy and Anf transcription in cardiomyocytes.

beta-Catenin/TCF/LEF1 signaling is implicated in cardiac hypertrophy. We demonstrate that knockdown of beta-catenin attenuates phenylephrine (PE)-induced cardiomyocyte hypertrophy and the up-regulation of the fetal gene Anf. We explore the mechanism through which beta-catenin regulates Anf expression and find a consensus binding sequence on the Anf promoter for TCF/LEF1 family members. LEF1 binds directly to the Anf promoter via this sequence, which shows functional significance, and PE stimulation enhances recruitment of beta-catenin onto the Anf promoter. Thus, we document a direct positive role of beta-catenin on PE-induced cardiomyocyte hypertrophy and identify a new target gene for beta-catenin/TCF/LEF1.

[Redox factor 1 increases proliferation of neonatal rat cardiac fibroblasts].

OBJECTIVE: To investigate the function of REF1 in the proliferation and collagen synthesis of neonatal rat cardiac fibroblasts, and the underlying mechanisms. METHODS: Neonatal rat cardiac fibroblasts were transfected with the adenoviral vector containing rat wild type Ref1 (Ad-Ref1) or mutated Ref1 (Ad-mutRef1). The mutations resulted in Cys to Ala at amino acids 65 and 93, which eliminated the redox function of the REF1 protein. MTT was used to check the cell viability and flow cytometry was used to analyze the cell proliferation with the count of cell numbers and the percentage of cells in S phase of the cell cycle. The expressions of Ref1, collagen I (Col I) and collagen III (Col III) were determined by RT-PCR and Western blot. The translocation of REF1 was examined by fluorescence staining and revealed under fluorescence microscope. Electrophoretic mobility shift assay (EMSA) was used to check the effect of REF1 on AP1 DNA binding ability. The high glucose medium (25 mmol/L) was applied to culture cardiac fibroblasts. The effect of high glucose on AP1 DNA binding activity, the expression and translocation of REF1 were examined. RESULTS: MTT analysis showed that Ad-Ref1 promoted the relative viability of cardiac fibroblasts (0.671+/-0.044 vs control 0.364+/-0.007, n=6, P<0.01). The percentage of cells in S phase of the cell cycle was increased significantly in the Ad-Ref1 transfected cells (16.8%+/-0.62% vs control 9.04%+/-0.43%, n=3, P<0.05), as demonstrated by flow cytometry analysis. The expressions of Col I and Col III at mRNA level were increased when cells transfected with Ad-Ref1, while Ad-mutRef1 did not show such effects. Compared with the redox-deficient mutant Ad-mutRef1 (C65/93A), EMSA results demonstrated that Ad-Ref1 resulted in a marked increase in AP1 DNA binding. We also found that the cardiac fibroblasts cultured in high glucose (25 mmol/L) medium resulted in an increase in AP1 DNA binding activity, which was similar as seen in Ad-Ref1 transfected cells. There was also an increased accumulation of nuclear REF1 protein when cells were cultured in high glucose medium, although the expressions of REF1 at both mRNA and protein levels were not affected. CONCLUSION: REF1 can increase proliferation and collagen synthesis of cardiac fibroblasts, which may be related to its ability to up-regulate AP1 DNA binding.

The LIM-homeodomain protein ISL1 activates insulin gene promoter directly through synergy with BETA2.

The LIM-homeodomain transcription factor ISL1 (islet factor 1) is essential for pancreatic islet cell and dorsal mesenchyme development. Mutations in ISL1 are associated with maturity-onset diabetes of the young and type 2 diabetes. Whether ISL1 plays a role in the insulin gene expression has not been fully elucidated. In the present study, we show that ISL1 can synergistically activate insulin gene transcription with BETA2 in pancreatic beta cells. The protein-protein interactions of ISL1 and BETA2 are directly mediated by the LIM domains of ISL1 and the basic helix-loop-helix domain of BETA2. Deletion of the two LIM domains of ISL1 enhances the transcriptional activation of the insulin gene, indicating a key role for the homeodomain in activating the insulin promoter. Furthermore, ISL1 can bind with the A3/4 box in the rat insulin gene capital I, Ukrainian promoter through its homeodomain. ISL1 expression is up-regulated at the mRNA level in type 2 diabetes (db/db mouse model) but down-regulated by dexamethasone in rat insulinoma cells. These results suggest that ISL1 is a transcriptional activator for insulin gene expression, and the interactions of ISL1 with BETA2 are required for the transcriptional activity of the insulin gene. Reduction in Isl1 gene expression appears to be involved in the impairment of insulin expression mediated by dexamethasone.

Carboxyl terminus of Nkx2.5 impairs its interaction with p300.

The transcription factor Nkx2.5 plays critical roles in controlling cardiac-specific gene expression. Previous reports demonstrated that Nkx2.5 is only a modest transactivator due to the auto-inhibitory effect of its C-terminal domain. Deletion of the C-terminal domain, mimicking conformational change, evokes vigorous transactivation activity. Here, we show that a C-terminal defective mutant of Nkx2.5 improves the occupation of p300 at the ANF promoter compared with full-length Nkx2.5, leading to hyperacetylation of histone H4. We reveal that p300 is a cofactor of Nkx2.5, markedly potentiating Nkx2.5-dependent transactivation, whereas E1A antigen impairs Nkx2.5 activity. Furthermore, p300 can acetylate Nkx2.5 and display an acetyltransferase-independent mechanism to coactivate Nkx2.5. Physical interaction between the N-terminal activation domain of Nkx2.5 and the C/H3 domain of p300 are identified by GST pull-down assay. Point mutants of the N-terminal modify the transcriptional activity of Nkx2.5 and interaction with p300. Deletion of the C-terminal domain greatly facilitates p300 binding and improves the susceptibility of Nkx2.5 to histone deacetylase inhibitor. These results establish that p300 acts as an Nkx2.5 cofactor and facilitates increased Nkx2.5 activity by relieving the conformational impediment of its inhibitory C-terminal domain.

[HLA expression in human fetal bone marrow mesenchymal stem cells].

OBJECTIVE: To investigate the expression of human leukocyte antigens (HLA) in human fetal bone marrow mesenchymal stem cells (MSCs) after long time culture as well as the changes in response to interferon-gamma (IFN-gamma) treatment. METHODS: Human fetal MSCs were collected from 23 to 24-weeks-old fetues with the approval of Ethic Committee of Peking University Health Science Center. The cells of passage 5 and passage 12 were analyzed for HLA expression before and after IFN-gamma (50 microg/L) treatment by flow cytometry at different time points. The passage 5 cells were also treated with an additional dose of IFN-gamma (5 microg/L) and the HLA expression was analyzed 24, 48, 72, 96 and 120 h after treatment. RT-PCR was used to evaluate HLA-G and HLA-E expression at mRNA level in human fetal MSCs. RESULTS: Flow cytometry results showed that fetal MSCs expressed high level of HLA class I (HLA-I) antigen, but extremely low level of HLA class II (HLA-II) antigen. The percentage of HLA-I positive MSCs in total MSCs was approximately over 50%, while that of HLA-II positive MSCs in total MSCs was less than 10%. IFN-gamma (50 microg/L) enhanced the HLA-I and HLA-II expression in a time dependent manner and increased the percentage of HLA-I positive cells in both passage 5 and passage 12 cells but preferably in passage 5 cells. The enhanced HLA-II expression was seen 48 h after IFN-gamma treatment in passage 5 cells (59.9%) but 72 h in passage 12 cells (48.1%). The treatment of 5 microg/L IFN-gamma also increased percentage of HLA-I and HLA-II positive MSCs, but with a relatively less extent compared to the 50microg/L IFN-gamma treatment group. RT-PCR result indicated HLA-G and HLA-E were expressed at mRNA level in human fetal MSCs. CONCLUSION: Human fetal MSCs can be induced to express both HLA class I and class II antigens by IFN-gamma. The long time culture might reduce the IFN-gamma effects. Human fetal MSCs express HLA-G and HLA-E at mRNA level.

Rat adipose-derived stromal cells expressing BMP4 induce ectopic bone formation in vitro and in vivo.

AIM: Bone morphogenetic protein 4 (BMP4) is one of the main local contributing factors in callus formation in the early phase of fracture healing. Adipose-derived stromal cells (ADSC) are multipotent cells. The present study was conducted to investigate the osteogenic potential of ADSC when exposed to adenovirus containing BMP4 cDNA (Ad-BMP4). METHODS: ADSC were harvested from Sprague-Dawley rats. After exposure to Ad-BMP4, ADSC were assessed by alkaline phosphatase activity (ALP) assay, RT-PCR and von Kossa staining. BMP4 expression was assessed by RT-PCR, immunofluorescence and Western blot analysis. ADSC transduced with Ad-BMP4 were directly injected into the hind limb muscles of athymic mice. ADSC Ad-EGFP(enhanced green fluorescence protein) served as controls. All animals were examined by X-ray film and histological analysis. RESULTS: The expression of BMP4 was confirmed at both mRNA and protein levels. The expression of the osteoblastic gene, ALP activity and von Kossa staining confirmed that ADSC transduced with Ad-BMP4 underwent rapid and marked osteoblast differentiation, whereas ADSC transduced with Ad-EGFP and cells left alone displayed no osteogenic differentiation. X-ray and histological examination confirmed new bone formation in athymic mice transplanted with ADSC transduced with Ad-BMP4. CONCLUSION: Our data demonstrated successful osteogenic differentiation of ADSC transduced with Ad-BMP4 in vitro and in vivo. ADSC may be an ideal source of mesenchyme lineage stem cells for gene therapy and tissue engineering.

[Quantitative study on copy numbers of porcine endogenous retroviruses in genome of Banna miniature swine inbred line].

OBJECTIVE: To investigate the copy numbers of porcine endogenous retroviruses in genome of Banna miniature swines for screening of donors in xenotransplantation with porcine transplants. METHODS: The genomic DNA of peripheral blood mononuclear cells(PBMCs) of 50 Banna miniature swines in 4 inbred sublines and 5 European Large White Pigs was extracted for the detection to pol, envA, envB, envC and envA/C recombinant in PERVs with real-time quantitative/semi-quantitative PCR and normal PCR. RESULTS: PCR products of pol, envA and envB of PERV could be obtained from all the Banna samples, and the mean amount of these products was 24.2+/-9.4,13.1+/-8.4,16.4+/-9.8 respectively and was significantly different among the inbred herds (P<0.01). The envC and envA/C were not amplified. CONCLUSION: The copy numbers of pol, envA and envB genes of PERV in porcine genome were significantly different among the inbred herds of Banna miniature swines, and envC and envA/C were absent in genome of Banna Miniature Swines.

[Bone marrow stem cells-based SERCA2a gene therapy for heart failure after acute myocardium infarction].

OBJECTIVE: Explore the possibility of MSC to be used to target delivery of therapeutic gene and evaluate the therapeutic effects among gene therapy, MSC transplantation and MSC-based gene therapy. METHODS: MSC were infected with an adenoviral expression vector carrying SERCA2a. SD female rats were used to make animal model with heart failure after AMI and divided into 4 groups randomly. Group I (n = 7) received SERCA2a gene therapy, group II (n = 7) received MSC transplantation, group III (n = 8) received MSC infected with SERCA2a gene transplantation, and group IV (n = 7) received empty adenoviral vector. Cardiac function was evaluated by echocardiography and physiological recorder. SERCA2a gene and protein expression were evaluated by RT-PCR and Western blot respectively. RESULTS: Compared to group IV, EF and FS of group I, group II and group III were elevated significantly on 14 days after therapy (EF: 67.7 +/- 3.9, 62.6 +/- 4.0, 67.9 +/- 3.7 versus 45.0 +/- 2.2; FS: 33.9 +/- 1.9, 31.1 +/- 2.0, 33.9 +/- 1.9 versus 22.5 +/- 1.1, P < 0.05). While the elevation values of EF and FS began to reduce in group I 14 days after, it continued to increase in both group II and group III. Absolute value of LVEDP at 21 days after treatment was increased in group I, group II and group III compared to group IV (5.3 mm Hg +/- 1.2 mm Hg, 6.0 +/- 1.3 mm Hg, 6.2 mm Hg +/- 1.2 mm Hg versus 1.5 mm Hg +/- 0.2 mm Hg, P < 0.05), as well as absolute value of DP/dtmin (4756 mm Hg/s +/- 270 mm Hg/s, 5028 mm Hg/s +/- 253 mm Hg/s, 5283 mm Hg/s +/- 363 mm Hg/s versus 3201 mm Hg/s +/- 211 mm Hg/s, P < 0.05). DP/dtmax at 21 days after treatment increased in group I, group II and group III compared to group IV (6026 mm Hg/s +/- 281 mm Hg/s, 6278 mm Hg/s +/- 319 mm Hg/s, 7057 mm Hg/s +/- 389 mm Hg/s versus 5293 mm Hg/s +/- 360 mm Hg/s, P < 0.05). SERCA2a expressions and enzyme activity were significantly stronger in group I and group III than in group II and group IV. CONCLUSION: It showed that all MSC transplantation, SERCA2a gene therapy and MSC-based gene therapy could enhance cardiac function. The recovered heart function continued to improve in MSC transplantation group and MSC-based gene therapy group up to 21 days, however slowed down in single gene therapy group in 21 days. Such therapeutic tendency of MSC-based gene therapy was stronger than that of MSC transplantation. Thus, MSC proved an effective platform for the targeted delivery of therapeutic gene.

[Bone marrow mesenchymal cell transplantation reduces left ventricular remodeling in heart failure following acute myocardial infarction].

OBJECTIVE: Bone marrow mesenchymal cell (MSC) transplantation has been shown to improve heart failure but the mechanism and the subsequent effects are unclear. We tested the hypothesis that MSC transplantation reduces left ventricular remodeling through the MMP/TIMP system in heart failure following acute myocardial infarction. METHODS: Female SD rats underwent coronary artery ligation to induce myocardial infarction. Four weeks later, the rats were divided into the test group (n = 7) and the control group (n = 7), respectively. The donor MSCs were harvested and expanded from male SD rats (5 x 10(6) in 50 microl PBS) and injected into the ischemic myocardium, while the control group received the same volume of PBS. Left ventricular morphology was then evaluated in both groups through staining with H&E and Masson's trichrome. Immunohistochemical staining was used to examine the expressions of MMP2 and TIMP1, as well as type I and type III collagens, in the scar zones. The protein levels of MMP2 and TIMP1 were determined by Western blotting. RESULTS: MSC differentiated into fibroblast-like cells at 21 days after transplantation in the test group. In addition, many inflammatory cells infiltrated and aggregated in the scar area, but this effect was reduced at day 7 after transplantation. The following changes were noted in the test group compared to the control group: ejection fraction and shortening fraction were higher [(63.43 +/- 3.97)% vs. (36.20 +/- 3.99)%, (31.71 +/- 1.98)% vs. (18.00 +/- 2.07)%, P < 0.05]; dp/dt(min) was reduced [(-4756.24 +/- 270.00) mm Hg/s vs. -2789.53 +/- 624.13) mm Hg/s, P < 0.05]; the left ventricular thinning ratio was significantly higher [(76.34 +/- 2.66)% vs. (64.37 +/- 2.36)%, P < 0.05]; the infarct size was smaller [(36.19 +/- 0.83)% vs. (42.12 +/- 1.88)%, P < 0.05]; type I collagen expression in the scar area was much higher; type III collagen expression was much lower; MMP2 expression was reduced and TIMP1 expression was increased. CONCLUSION: MSC transplantation led to dynamic changes in the collagen network through regulation of MMP2/TIMP1 system and consequently interrupted the progress of adverse LV remodeling in heart failure following acute myocardial infarction.

ISL1 physically interacts with BETA2 to promote insulin gene transcriptional synergy in non-beta cells.

ISL1 is a LIM homeodomain protein that plays an important role in insulin gene transcriptional activation and islet cell formation. BETA2 is a transcription factor in the basic helix-loop-helix (bHLH) family, which activates expression of tissue-specific genes in several developmental systems. In this study, we investigated the functional and physical interactions of ISL1 and BETA2 in promoting insulin gene transcription in non-beta cells. Using the luciferase assay, we demonstrated that ISL1 and BETA2 could activate insulin gene transcription synergistically. Co-immunoprecipitation also supported that ISL1 and BETA2 appear in one complex and this physical interaction mediates the synergy between these two proteins.

Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells.

AIM: To investigate the effects of bone morphogenetic protein-7 (BMP7)-expressing recombinant adeno-associated virus (AAV) vector on the differentiation of C2C12 cells. METHODS: AAV-BMP7 was packaged by infecting the stable cell clone BHK-21 (integrated with recombinant AAV vector plasmid pSNAV-BMP7) with recombinant herpes simplex virus type 1, which expresses AAV-2 Rep and Cap and possesses AAV packaging functions. Following infection with AAV-BMP7 at multiplicities of infection of 1 x 10(5) vector genomes per cell and subsequent culture, C2C12 cells were assessed qualitatively for BMP7 production, alkaline phosphatase activity, osteocalcin production and Cbfal and MyoD expression. RESULTS: C2C12 cells transduced with AAV-BMP7 could produce BMP7 protein until d 28. Alkaline phosphatase in the cultured C2C12 cell lysate was elevated. Secreted osteocalcin in the culture medium was detectable at d 12 and Cbfal mRNA expression level was upregulated, coinciding with downregulation of MyoD in a temporal manner. CONCLUSION: The present in vitro study demonstrated that AAV-BMP7 could infect and efficiently convert C2C12 cells from myoblasts into osteoblast lineage cells.

Heterogeneity in predisposition of hepatic cells to be induced into pancreatic endocrine cells by PDX-1.

AIM: The role of Pancreatic and Duodenal Homeobox-1 (PDX-1) as a major regulator of pancreatic development determines the function and phenotype of beta cell. In this study, potential plasticity of liver cells into pancreatic endocrine cells induced by PDX-1 was evaluated. METHODS: Human hepatoma cell line HepG2 was stably transfected with mammalian expression plasmid pcDNA3-PDX encoding human PDX-1 gene. Ectopic expression of PDX-1 and insulin were detected by RT-PCR, Western blot and/or immunostaining. PDX-1(+) HepG2 cells were transplanted under renal capsule of STZ-induced diabetic nude mice (n = 16) to examine the inducing effect in vivo. RESULTS: Exogenous PDX-1 transgene was proved to express effectively in HepG2 cell at both mRNA and protein levels. The expression of endogenous insulin and some beta cell-specific differentiation markers and transcription factors were not induced in PDX-1(+) HepG2 cells. When transplanted under renal capsule of STZ-induced diabetic nude mice, PDX-1(+) HepG2 cells did not generate insulin-producing cells. These data indicated that stable transfected PDX-1 could not convert hepatoma cell line HepG2 to pancreatic cells in vitro or in vivo. Mature hepatocytes might need much more complicated or rigorous conditions to be shifted to insulin-producing cells. CONCLUSION: The expression of exogenous PDX-1 is not sufficient to induce relatively mature hepatocytes differentiating into insulin-producing cells.