Mesenchymal stem cells ameliorate myocardial fibrosis in diabetic cardiomyopathy via the secretion of prostaglandin E2.

BACKGROUND: Diabetic cardiomyopathy (DCM) is a cardiac complication of long-term uncontrolled diabetes and is characterized by myocardial fibrosis and abnormal cardiac function. Mesenchymal stem cells (MSCs) are multipotent cells with immunoregulatory and secretory functions in diabetes and heart diseases. However, very few studies have focused on the effect and the underlying mechanism of MSCs on myocardial fibrosis in DCM. Therefore, we aimed to explore the therapeutic potential of MSCs in myocardial fibrosis and its underlying mechanism in vivo and in vitro. METHODS: A DCM rat model was induced using a high-fat diet (HFD) combined with a low-dose streptozotocin (STZ) injection. After four infusions of MSCs, rat serum and heart tissues were collected, and the levels of blood glucose and lipid, cardiac structure, and function, and the degree of myocardial fibrosis including the expression levels of pro-fibrotic factor and collagen were analyzed using biochemical methods, echocardiography, histopathology, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). We infused prostaglandin E2 (PGE2)-deficient MSCs to DCM rats in vivo and established a system mimicking diabetic myocardial fibrosis in vitro by inducing cardiac fibroblasts with high glucose (HG) and coculturing them with MSCs or PGE2-deficient MSCs to further explore the underlying mechanism of amelioration of myocardial fibrosis by MSCs. RESULTS: Metabolic abnormalities, myocardial fibrosis, and cardiac dysfunction in DCM rats were significantly ameliorated after treatment with MSCs. Moreover, the levels of TGF-ß, collagen I, collagen III, and collagen accumulation were markedly decreased after MSC infusion compared to those in DCM hearts. However, PGE2-deficient MSCs had decreased ability to alleviate cardiac fibrosis and dysfunction. In addition, in vitro study revealed that the concentration of PGE2 in the MSC group was enhanced, while the proliferation and collagen secretion of cardiac fibroblasts were reduced after MSC treatment. However, MSCs had little effect on alleviating fibrosis when the fibroblasts were pretreated with cyclooxygenase-2 (COX-2) inhibitors, which also inhibited PGE2 secretion. This phenomenon could be reversed by adding PGE2. CONCLUSIONS: Our results indicated that MSC infusion could ameliorate cardiac fibrosis and dysfunction in DCM rats. The underlying mechanisms might involve the function of PGE2 secreted by MSCs.

Mesenchymal stem cells promote type 2 macrophage polarization to ameliorate the myocardial injury caused by diabetic cardiomyopathy.

BACKGROUND: Diabetic cardiomyopathy (DCM) is a common complication of diabetes and is characterized by chronic myocardial inflammation. Mesenchymal stem cell (MSC) infusions have recently been suggested to alleviate myocardial injury and ameliorate cardiac function. However, few studies have focused on the effects of MSCs in DCM. Therefore, we explored the effects of MSC-regulated macrophage polarization on myocardial repair in DCM. METHODS: A DCM rat model was induced by a high-fat diet and streptozotocin (STZ) administration and infused 4 times with MSCs. Rat blood and heart tissue were analyzed for blood glucose levels, lipid levels, echocardiography, histopathology, macrophage phenotype ratios and inflammatory cytokines, respectively. We mimicked chronic inflammation in vitro by inducing peritoneal macrophages with high glucose and LPS, then cocultured these macrophages with MSCs to explore the specific mechanism of MSCs on macrophage polarization. RESULTS: DCM rats exhibited abnormal blood glucose levels and lipid metabolism, cardiac inflammation and dysfunction. MSC infusion ameliorated metabolic abnormalities and preserved cardiac structure and function in DCM rats. Moreover, MSC infusion significantly increased the M2 phenotype macrophages and alleviated cardiac inflammation. Interestingly, this in vitro study revealed that the MSCs pretreated with a COX-2 inhibitor had little effect on M2 macrophage polarization, but this phenomenon could be reversed by adding prostaglandin E2 (PGE2). CONCLUSIONS: Our results suggested that MSC infusions can protect against cardiac injury in DCM rats. The underlying mechanisms may include MSC-enhanced M2 macrophage polarization via the COX-2-PGE2 pathway.

Infusion of adipose­derived mesenchymal stem cells inhibits skeletal muscle mitsugumin 53 elevation and thereby alleviates insulin resistance in type 2 diabetic rats.

It is widely accepted that infusion of mesenchymal stem cells (MSCs) ameliorates hyperglycemia by alleviating insulin resistance in rats with type 2 diabetes mellitus (T2D). However, the detailed underlying mechanisms are not clearly defined. Mitsugumin 53 (MG53) is an E3 ligase that has recently been implicated in the aggravation of insulin resistance by promoting the ubiquitinoylation of insulin receptor substrate­1 (IRS­1) in skeletal muscles. It was therefore hypothesized that MG53 may be involved in MSC­mediated therapeutic effects on insulin resistance. To test this hypothesis, in the present study, T2D rat models were induced by a high­fat diet combined with streptozotocin administration and MSC infusion was performed four times (once every 2 weeks for 8 weeks). The therapeutic effects of MSC infusion on insulin resistance were evaluated and the effect on the expression of MG53 and insulin receptor signaling elements in skeletal muscle was also investigated by immunofluorescence staining and western blotting. The results demonstrated that MSC infusion ameliorated hyperglycemia and insulin resistance in T2D rats. Furthermore, MSC infusion inhibited MG53 elevation and reversed the decreases in glucose transporter type 4, insulin receptor, IRS­1 and phosphorylated­AKT levels in the skeletal muscle of T2D rats. These results indicated that MSC infusion has therapeutic effects in rats and that MG53 in skeletal muscle may be a promising novel therapeutic target protein for MSC­mediated amelioration of insulin resistance in T2D.

Spleen-Derived Anti-Inflammatory Cytokine IL-10 Stimulated by Adipose Tissue-Derived Stem Cells Protects Against Type 2 Diabetes.

Considering that the spleen plays an important role in the occurrence and development of diabetes, we aimed at investigating the role of the spleen in the treatment of type 2 diabetes (T2D) with adipose tissue-derived stem cells (ADSCs). We established a T2D/splenectomy (SPX) rat model by using high-fat diet/streptozotocin administration with SPX, assessed the therapeutic effects of ADSCs, and explored the possible mechanism. A single ADSC infusion was found to ameliorate hyperglycemia and insulin resistance in diabetic rats, accompanied by a considerable number of ADSCs homing to the spleens in T2D rats. Moreover, four times of infusion of ADSCs resulted in a more significant reduction of blood glucose and insulin resistance, whereas SPX exacerbated hyperglycemia and insulin resistance and attenuated the effects of ADSCs. In addition, ADSC infusion promoted anti-inflammatory cytokine interleukin (IL)-10 expression and inhibited pro-inflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor (TNF)-α expression in both the spleen and serum of T2D rats without SPX. ADSCs also inhibited serum IL-6, IL-1ß, and TNF-α expression, but cannot promote IL-10 expression in T2D rats with SPX. Therefore, these data indicate that the effect of ADSCs ameliorating hyperglycemia and insulin resistance may be partially through promoting spleen-derived anti-inflammatory cytokine IL-10 expression. These novel findings further confirmed the essential role of the spleen in the ADSC treatment of T2D and provide an important theoretical basis for the potential application of ADSCs in T2D therapy.

Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer.

CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer.

Human umbilical cord-derived mesenchymal stem cells elicit macrophages into an anti-inflammatory phenotype to alleviate insulin resistance in type 2 diabetic rats.

Insulin resistance, a major characteristic of type 2 diabetes (T2D), is closely associated with adipose tissue macrophages (ATMs) that induce chronic low-grade inflammation. Recently, mesenchymal stem cells (MSCs) have been identified in alleviation of insulin resistance. However, the underlying mechanism still remains elusive. Thus, we aimed to investigate whether the effect of MSCs on insulin resistance was related to macrophages phenotypes in adipose tissues of T2D rats. In this study, human umbilical cord-derived MSCs (UC-MSCs) infusion produced significantly anti-diabetic effects and promoted insulin sensitivity in T2D rats that were induced by a high-fat diet combined with streptozotocin and directed ATMs into an alternatively activated phenotype (M2, anti-inflammatory). In vitro, MSC-induced M2 macrophages alleviated insulin resistance caused by classically activated macrophages (M1, pro-inflammatory). Further analysis showed that M1 stimulated UC-MSCs to increase expression of interleukin (IL)-6, a molecule which upregulated IL4R expression, promoted phosphorylation of STAT6 in macrophages, and eventually polarized macrophages into M2 phenotype. Moreover, the UC-MSCs effect on macrophages was largely abrogated by small interfering RNA (siRNA) knockdown of IL-6. Together, our results indicate that UC-MSCs can alleviate insulin resistance in part via production of IL-6 that elicits M2 polarization. Additionally, human obesity and insulin resistance were associated with increased pro-inflammatory ATMs infiltration. Thus, MSCs may be a new treatment for obesity-related insulin resistance and T2D concerning macrophage polarized effects.

[The effects of leptin on neuron apoptosis in mice with cerebral ischemia/reperfusion injury].

OBJECTIVE: To study the effect of leptin on neuron apoptosis in mice with cerebral ischemia injury. METHODS: Seventy-five male Kuming mice were randomly divided into 3 groups:sham, model and leptin intervention group, respectively. Focal cerebral ischemia/reperfusion injury model in mice was established by middle cerebral artery occlusion. Leptin intervention group was injected with leptin (1µg/g weight, I. P.) at 0 min of ischemic injury. Neuron apoptosis was detected by TUNEL staining. The mRNA expression of apoptosis relative gene bcl-2 and caspase-3 were detected by RT-PCR. The protein expression of bcl-2 and caspase-3 were detected by immunohistochemistry. RESULTS: In model group, most of the neurons in the central area of cerebral ischemia had necrosis obviously, and the amount of neuron apop-tosis was much higher than that in sham group (P<0.01). Compared with sham group, both expression of pro-apoptosis gene caspase-3 and anti-apoptosis gene bcl-2 increased significantly in model group (P<0.01). Compared with model group, the amount of neuron apoptosis and expression level of caspase-3 were decreased significantly (P<0.01), whereas the mRNA and protein expression of bcl-2 were increased sig-nificantly in leptin intervention group (P<0.01). CONCLUSIONS: Leptin could reduce neuron apoptosis through down-regulation the expression of caspase-3 and up-regulation the expression of bcl-2. It suggests that leptin could play a neuroprotective role in cerebral ischemia injury.

Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Progastrin stimulates colonic cell proliferation via CCK2R- and ß-arrestin-dependent suppression of BMP2.

BACKGROUND & AIMS: Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R)-partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R. METHODS: We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of ß-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division. RESULTS: The BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by ß-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44(+), bromodeoxyuridine+, and NUMB(+) cells, indicating an increase in symmetric divisions of putative cancer stem cells. CONCLUSIONS: Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and ß-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division.

Bone marrow cells as precursors of the tumor stroma.

Cancer is a systemic disease. Local and distant factors conspire to promote or inhibit tumorigenesis. The bone marrow is one important source of tumor promoting cells. These include the important mature and immature hematopoietic cells as well as circulating mesenchymal progenitors. Recruited bone marrow cells influence carcinogenesis at the primary site, within the lymphoreticular system and even presage metastasis through their recruitment to distant organs. In this review we focus on the origins and contribution of cancer-associated fibroblasts in tumorigenesis. Mesenchymal cells present an important opportunity for targeted cancer prevention and therapy.

FHL2 inhibits the Id3-promoted proliferation and invasive growth of human MCF-7 breast cancer cells.

BACKGROUND: Id3 plays a key role in the progression of breast cancer. Previously, four and a half LIM protein (FHL2) was identified as a repressor of Id family proteins by interacting with them. This study aimed to investigate the effects of FHL2 on the transcriptional regulation and oncogenic activities of Id3 in human breast cancer cells. METHODS: Cell transfection was performed with SuperFect reagent. Stable transfectants that overexpressed Id3 were obtained by selection on G418. The level of Id3 protein was determined by Western blotting analysis. Dual luciferase assays were used to measure the effect of Id3 and FHL2 on E47-mediated transcriptional activity in MCF-7 human breast cancer cells. The MTT assay was used to measure cell proliferation. The transwell assay was used to measure the invasive capacity of MCF-7 cancer cells. RESULTS: Id3 markedly repressed transcription mediated by the basic helix-loop-helix (bHLH) factor E47 in MCF-7 cells. This Id3-mediated repression was effectively antagonized by FHL2. Overexpression of Id3 markedly promoted the proliferation and invasive capacity of MCF-7 cells; however, these effects were significantly suppressed by the overexpression of FHL2. CONCLUSIONS: FHL2 can inhibit the proliferation and invasive growth of human breast cancer cells by repressing the functional activity of Id3. The functional roles of FHL2-Id3 signaling in the development of human breast cancer need further research.

Infusion of mesenchymal stem cells ameliorates hyperglycemia in type 2 diabetic rats: identification of a novel role in improving insulin sensitivity.

Infusion of mesenchymal stem cells (MSCs) has been shown to effectively lower blood glucose in diabetic individuals, but the mechanism involved could not be adequately explained by their potential role in promoting islet regeneration. We therefore hypothesized that infused MSCs might also contribute to amelioration of the insulin resistance of peripheral insulin target tissues. To test the hypothesis, we induced a diabetic rat model by high-fat diet/streptozotocin (STZ) administration, performed MSC infusion during the early phase (7 days) or late phase (21 days) after STZ injection, and then evaluated the therapeutic effects of MSC infusion and explored the possible mechanisms involved. MSC infusion ameliorated hyperglycemia in rats with type 2 diabetes (T2D). Infusion of MSCs during the early phase not only promoted ß-cell function but also ameliorated insulin resistance, whereas infusion in the late phase merely ameliorated insulin resistance. Infusion of MSCs resulted in an increase of GLUT4 expression and an elevation of phosphorylated insulin receptor substrate 1 (IRS-1) and Akt (protein kinase B) in insulin target tissues. This is the first report of MSC treatment improving insulin sensitivity in T2D. These data indicate that multiple roles and mechanisms are involved in the efficacy of MSCs in ameliorating hyperglycemia in T2D.

Leptin administration alleviates ischemic brain injury in mice by reducing oxidative stress and subsequent neuronal apoptosis.

BACKGROUND: Recent research has indicates that leptin plays a protective role in traumatic brain injury. We studied the protective effect of leptin on cerebral ischemia/reperfusion injury by using mice transient focal cerebral ischemia/reperfusion injury model. METHODS: The distribution of 125I-leptin in the mouse brain was assessed by radioimmunoassay method. Mouse models of transient focal cerebral ischemia were established by occlusion of the right middle cerebral artery for two hours followed by 24 hours reperfusion. The neurologic deficits and infarct volume were determined using the Longa's score and 2,3,5-triphenyltetrazolium chloride staining, respectively. Regional cerebral blood flow was monitored by a laser-Doppler blood flowmeter. The levels of malondialdehyde, nitric oxide, nitric oxide synthase, and superoxide dismutase were detected according to respective assay kit. The histologic changes and neuronal apoptosis were observed with hematoxylin and eosin and transferase-mediated dUTP-biotin nick end labeling staining, respectively. The expression of B-cell lymphoma/leukemia-2 (Bcl-2) and cysteineasparateprotease-3 (caspase-3) were investigated by Western blot and real-time polymerase chain reaction assay. RESULTS: Leptin decreased infarct volume and neurologic defects and improved regional cerebral blood flow and microvascular branch blood flow after injury. The malondialdehyde and nitric oxide levels were reduced, and superoxide dismutase level was increased after leptin treatment, which also minimized histologic changes and neuronal apoptosis, led to the upregulation of Bcl-2 and downregulation of caspase-3 expression after injury. CONCLUSIONS: Peripherally administered leptin crossed the blood-brain barrier and was distributed into multiple regions of the brain; in the brain, leptin directly alleviated the injury-evoked damages by reducing oxidative stress and neuronal apoptosis.

[The role of Leptin on neuron apoptosis in mice with cerebral ischemia/reperfusion injury].

OBJECTIVE: To study the effect of Leptin on neuron apoptosis in mice with cerebral ischemia injury and its mechanism. METHODS: Seventy-five mice were randomly divided into three groups. Focal cerebral ischemia/reperfusion injury model in mice was reproduced by middle cerebral artery occlusion for 2 hours followed by reperfusion. In Leptin intervention group mice were given Leptin 1 µg/g during cerebral ischemia by intraperitoneal injection. Mice in the model group were given equal amount of phosphate buffer saline. After reperfusion for 24 hours, the neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The mRNA and protein expression of apoptosis relative gene caspase-3 and bcl-2 were determined by reverse transcription-polymerase chain reaction (RT-PCR) and immuno histochemistry. RESULTS: Most of neuron necrosis was observed in cerebral ischemia center in model group. Compared with sham-operation group, neuron apoptosis rate, mRNA and protein expression of caspase-3 and bcl-2 in model group increased significantly [apoptosis rate: (68.65 ± 0.79)% vs. (4.40 ± 0.00)%, caspase-3 mRNA: 2.563 ± 0.250 vs. 0.153 ± 0.020, bcl-2 mRNA: 0.337 ± 0.100 vs. 0.125 ± 0.030, caspase-3 protein (absorbance value, A value): 0.57 ± 0.05 vs. 0.37 ± 0.03, bcl-2 protein (A value): 0.51 ± 0.04 vs. 0.35 ± 0.01, all P<0.01]. The apoptosis rate of penumbra neurons was reduced in Leptin intervention group significantly compared with model group [(42.30 ± 8.45)% vs. (68.65 ± 0.79)%, P<0.01]. Compared with model group, the mRNA and protein expression of caspase-3 in Leptin intervention group were reduced significantly [caspase-3 mRNA: 2.267 ± 0.040 vs. 2.563 ± 0.250, caspase-3 protein (A value): 0.45 ± 0.04 vs. 0.57 ± 0.05, P>0.05 and P<0.01], and the mRNA and protein expression of bcl-2 in Leptin intervention group upregulated significantly [bcl-2 mRNA: 0.662 ± 0.040 vs. 0.337 ± 0.100, bcl-2 protein (A value): 0.76 ± 0.09 vs. 0.51 ± 0.04, both P<0.01]. CONCLUSION: Leptin could reduce apoptosis of neurons through down-regulation of the expression of caspase-3 and up-regulation of the expression of bcl-2. The results suggest that Leptin plays a neuroprotective role in cerebral ischemia injury.

LRP16 integrates into NF-κB transcriptional complex and is required for its functional activation.

BACKGROUND: Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation. METHODOLOGY: GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples. RESULTS: We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens. CONCLUSIONS: Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.

MSCs: Biological characteristics, clinical applications and their outstanding concerns.

Mesenchymal stem cells (MSCs) are multi-potent adult stem cells harboring multi-lineage differentiation potential and immunosuppressive properties that make MSCs an ideal candidate cell type for immunomodulation and regenerative medicine. Currently, MSC-related researches and clinical trials have evoked exciting promise in a variety of disorders and tissue regeneration. However, it must be recognized that several critical potential problems have also emerged from current clinical trials, for example: (1) the indefinite association between the phenotypic characteristics and the biological functions of MSCs; (2) the lack of clinical data to support the long-term safety of MSCs; (3) the need for further clarification of multiple mechanisms of MSC transplant actions in vivo; and (4) the lack of comparability of MSC transplant efficacy. Therefore, MSC-based therapies could not yet be considered a routine treatment in the clinic. Based on these, we proposed that large-scale and multi-center clinical trials of MSC-based therapies should be initiated under strict supervision. These interventions might help to establish a new clinical paradigm to turn MSC transplantation into a routine therapy for at least some diseases in the near future.

The evaluation of catechins that contain a galloyl moiety as potential HIV-1 integrase inhibitors.

Four catechins with the galloyl moiety, including catechin gallate (CG), epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), and epicatechin gallate (ECG), were found to inhibit HIV-1 integrase effectively as determined by our ELISA method. In our docking study, it is proposed that when the HIV-1 integrase does not combine with virus DNA, the four catechins may bind to Tyr143 and Gln148, thus altering the flexibility of the loop (Gly140-Gly149), which could lead to an inhibition of HIV-1 integrase activity. In addition, after combining HIV-1 integrase with virus DNA, the four catechins may bind between the integrase and virus DNA, consequently, disrupt this interaction. Thus, the four catechins may reduce the activity of HIV-1 integrase by disrupting its interaction with virus DNA. The four catechins have a highly cooperative inhibitory effect (IC50=0.1 µmol/L). Our study suggests that catechins with the galloyl moiety could be a novel and effective class of HIV-1 integrase inhibitors.

[Effects of leptin on apoptosis of rat cerebral astrocytes with ischemia/hypoxia injury].

OBJECTIVE: To investigate the effect of leptin on apoptosis of rat cerebral astrocytes with ischemia/ hypoxia injury and its mechanism. METHODS: The cerebral astrocytes with ischemic/hypoxia injury were induced in neonatal SD rats. The cellular viability of injury of astrocytes was detected by MTT assay. The apoptosis of astrocyte were detected with Annexin V-FITC kit. The effect of leptin on the expression of apoptosis factor bcl-2, bax, caspase-3 was detected by RT-PCR and Western blot. RESULTS: Compared with the ischemia group, the cellular viability of leptin intervention group increased significantly (P < 0.01), while the astrocytes apoptosis of leptin intervention group decreased significantly (P < 0.01). The mRNA and protein expression level of antiapoptosis factor bcl-2 in leptin intervention group was much higher than that of ischemia group (P < 0.01), whereas the mRNA and protein expression of bax and caspase-3 was much lower than that of ischemia group (P < 0.01). CONCLUSION: Leptin could significantly decrease the apoptosis of astrocytes with ischemia/hypoxia injury, and it i relevant to the increase of bcl-2 expression and the decrease of bax caspase-3 expression level.

Keratin 18 attenuates estrogen receptor alpha-mediated signaling by sequestering LRP16 in cytoplasm.

BACKGROUND: Oncogenesis in breast cancer is often associated with excess estrogen receptor alpha(ERalpha) activation and overexpression of its coactivators. LRP16 is both an ERalpha target gene and an ERalpha coactivator, and plays a crucial role in ERalpha activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear. RESULTS: Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERalpha, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERalpha, attenuated ERalpha-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERalpha recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERalpha-mediated signaling and promoted cell cycle progression. CONCLUSIONS: K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERalpha-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERalpha and promotes the proliferation of ERalpha-positive breast tumor cells. K18 plays an important functional role in regulating the ERalpha signaling pathway.

[Protective effect of leptin against cerebral ischemia/reperfusion injury in mice].

OBJECTIVE: To investigate the protective effect of leptin against cerebral ischemia/reperfusion injury in mice. METHODS: Mouse models of transient focal cerebral ischemia were established by occlusion of the right middle cerebral artery for 2 h followed by 24 h reperfusion. The infarct volume and neurological deficit scores following leptin treatment were determined using TTC staining and the Longa's score, respectively, to evaluate the protective effect of leptin against ischemic cerebral injury. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA) and nitric oxide (NO) in the brain tissue were measured by colorimetry. The histopathological changes in the brain were observed with HE staining, and the expression of glial fibrillary acidicprotein (GFAP) was detected by immunohistochemistry. RESULTS: Leptin treatment markedly reduced cerebral infarct volume and neurological deficits induced by transient ischemia. The LDH, MDA and NO levels in the brain tissues were significantly decreased after leptin treatment, which also alleviated the histopathological injury, maintained the normal morphology of the astrocytes and increased the expression of GFAP. CONCLUSION: Leptin produces obvious protective effect against cerebral ischemia/reperfusion injury by inhibiting lipid peroxidation, stabilizing the internal environment and adjusting the activity of the astrocytes.