Yu Wan

Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells

In this study, high-throughput microRNA (miRNA) expression analysis revealed that the expression of miR-140 was associated with chemosensitivity in osteosarcoma tumor xenografts. Tumor cells ectopically transfected with miR-140 were more resistant to methotrexate and 5-fluorouracil (5-FU). Overexpression of miR-140 inhibited cell proliferation in both osteosarcoma U-2 OS (wt-p53) and colon cancer HCT 116 (wt-p53) cell lines, but less so in osteosarcoma MG63 (mut-p53) and colon cancer HCT 116 (null-p53) cell lines. miR-140 induced p53 and p21 expression accompanied with G1 and G2 phase arrest only in cell lines containing wild type of p53. Histone deacetylase 4 (HDAC4) was confirmed to be one of the important targets of miR-140. The expression of endogenous miR-140 was significantly elevated in CD133+hiCD44+hi colon cancer stem-like cells that exhibit slow proliferating rate and chemoresistance. Blocking endogenous miR-140 by locked nucleic acid-modified anti-miR partially sensitized resistant colon cancer stem-like cells to 5-FU treatment. Taken together, our findings indicate that miR-140 is involved in the chemoresistance by reduced cell proliferation through G1 and G2 phase arrest mediated in part through the suppression of HDAC4. miR-140 may be a candidate target to develop novel therapeutic strategy to overcome drug resistance.

VEGF/SDF-1 Promotes Cardiac Stem Cell Mobilization and Myocardial Repair in the Infarcted Heart

AIMS The objective of this study was to investigate whether vascular endothelial growth factor (VEGF) secreted by mesenchymal stem cells (MSC) improves myocardial survival and the engraftment of implanted MSC in infarcted hearts and promotes recruitment of stem cells through paracrine release of myocardial stromal cell-derived factor-1α (SDF-1α). METHODS AND RESULTS VEGF-expressing MSC ((VEGF)MSC)-conditioned medium enhanced SDF-1α expression in heart slices and H9C2 cardiomyoblast cells via VEGF and the vascular endothelial growth factor receptor (VEGFR). The (VEGF)MSC-conditioned medium markedly promoted cardiac stem cell (CSC) migration at least in part via the SDF-1α/CXCR4 pathway and involved binding to VEGFR-1 and VEGFR-3. In vivo, (VEGF)MSC-stimulated SDF-1α expression in infarcted hearts resulted in massive mobilization and homing of bone marrow stem cells and CSC. Moreover, VEGF-induced SDF-1α guided the exogenously introduced CSC in the atrioventricular groove to migrate to the infarcted area, leading to a reduction in infarct size. Functional studies showed that (VEGF)MSC transplantation stimulated extensive angiomyogenesis in infarcted hearts as indicated by the expression of cardiac troponin T, CD31, and von Willebrand factor and improved the left ventricular performance, whereas blockade of SDF-1α or its receptor by RNAi or antagonist significantly diminished the beneficial effects of (VEGF)MSC. CONCLUSION Exogenously expressed VEGF promotes myocardial repair at least in part through SDF-1α/CXCR4-mediated recruitment of CSC.

Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation?

OBJECTIVE The potential use of bone marrow stromal cells (MSCs) as a cellular therapy for chronic cardiac diseases relies on the ability of the cell to replicate extensively in vitro and to give rise to myogenic cells that can replace the damaged cardiomyocytes. For this reason the present study investigated the replication lifespan and chemical-induced cardiomyogenic differentiation of rat MSCs in vitro. METHODS The primary and the successively passaged Wistar rat MSCs were exposed to different concentrations (3, 5 and 10 microM) of 5-azacytidine using different methods (single- or repeat-treatment). The growth properties and the fate of the cells were compared to their untreated counterparts by cell counting, immunocytochemistry and Western analysis. RESULTS When seeded at a density of 2845 cells/cm(2) and cultured under common conditions, rat MSCs could be expanded up to 21.94 cell doublings in 30 days of successive subcultures. This was accompanied by a gradual loss of their replication ability with passages. When treated with 5-azacytidine for 24 h at day 3 of primary culture and the first subculture, the growth properties of the MSCs were not obviously affected. Neither the spontaneously beating cells nor the formation of myotubes were found in the primary and first passaged MSCs after a single treatment with 5-azacytidine and in cultures which underwent repeated 5-azacytidine-treatments during continuous subculturing to passage 2. The expressions of cardiac troponin I, cardiac myosin heavy chain and connexin 43 by the 5-azacytidine-treated MSCs were also undetectable at both immunocytochemistry and Western blot levels. The specificity and reliability of the detection methods were technically confirmed with cultured rat cardiomyocytes. CONCLUSIONS Rat MSCs cannot be extensively expanded in vitro or be induced to differentiate in an expected cardiomyogenic way by 5-azacytidine-treatment, if the cells are not immortalized.

Vascular endothelial growth factor promotes cardiac stem cell migration via the PI3K/Akt pathway

VEGF is a major inducer of angiogenesis. However, the homing role of VEGF for cardiac stem cells (CSCs) is unclear. In in vitro experiments, CSCs were isolated from the rat hearts, and a cellular migration assay was performed using a 24-well transwell system. VEGF induced CSC migration in a concentration-dependent manner, and SU5416 blocked this. Western blot analysis showed that the phosphorylated Akt was markedly increased in the VEGF-treated CSCs and that inhibition of pAkt activity significantly attenuated the VEGF-induced the migration of CSCs. In in vivo experiments, rat heart myocardial infarction (MI) was induced by left coronary artery ligation. One week after MI, the adenoviral vector expressing hVEGF165 and LacZ genes were injected separately into the infarcted myocardium at four sites before endomyocardial transplantation of 2x10(5) PKH26 labeled CSCs (50 muL) at atrioventricular groove. One week after CSC transplantation, RT-PCR, immunohistochemical staining, Western blot, and ELISA analysis were performed to detect the hVEGF mRNA and protein. The expression of hVEGF mRNA and protein was significantly increased in the infarcted and hVEGF165 transfected rat hearts, accompanied by an enhanced PI3K/Ak activity, a greater accumulation of CSCs in the infarcted region, and an improvement in cardiac function. The CSC accumulation was inhibited by either the VEGF receptor blocker SU5416 or the PI3K/Ak inhibitor wortmannin. VEGF signaling may mediate the migration of CSCs via activation of PI3K/Akt.

Tumor Expression of Human Growth Hormone and Human Prolactin Predict a Worse Survival Outcome in Patients with Mammary or Endometrial Carcinoma

CONTEXT Evidence suggests that human GH (hGH) and human prolactin (hPRL) possess an autocrine or paracrine oncogenic role in mammary and endometrial carcinoma. However, especially for hGH, the prognostic relevance of tumor expression of these hormones is not well defined. OBJECTIVE We investigated the potential association of tumor mRNA and protein expression of hGH and hPRL with the clinicopathological features of mammary and endometrial carcinoma. The prognostic relevance of the individual or combined expression of hGH and hPRL in mammary and endometrial carcinoma was also determined. DESIGN The expression of hGH and hPRL was analyzed in histopathological samples of mammary and endometrial carcinoma, and the respective normal tissues, by in situ hybridization and immunohistochemistry. Kaplan-Meier and Cox regression analysis was performed to examine the association of tumor hGH and hPRL expression with relapse-free survival and overall survival of patients. RESULTS hGH expression was significantly associated with lymph node metastasis, tumor stage, human epidermal growth factor receptor-2 status, and proliferative index in mammary carcinoma and with International Federation of Gynecology and Obstetrics grade, myometrial invasion, and ovarian metastases in endometrial carcinoma. hPRL expression was associated with lymph node metastasis, tumor grade, and tumor stage in mammary carcinoma and with International Federation of Gynecology and Obstetrics stage and myometrial invasion in endometrial carcinoma. Both hGH and hPRL expression, individually and combined, are associated with worse relapse-free survival and overall survival in patients with mammary or endometrial carcinoma. CONCLUSION Tumor expression of both hGH or hPRL in mammary or endometrial carcinoma is associated with a large and significant difference in survival outcome for patients with these tumors.

Inhibitory GH Receptor Extracellular Domain Monoclonal Antibodies: Three-Dimensional Epitope Mapping

GH receptor (GHR) mediates the anabolic and metabolic effects of GH. We previously characterized a monoclonal antibody (anti-GHR(ext-mAb)) that reacts with subdomain 2 of the rabbit GHR extracellular domain (ECD) and is a conformation-specific inhibitor of GH signaling in cells bearing rabbit or human GHR. Notably, this antibody has little effect on GH binding and also inhibits inducible metalloproteolysis of the GHR that occurs in the perimembranous ECD stem region. In the current study, we demonstrate that anti-GHR(ext-mAb) inhibits GH-dependent cellular proliferation and also inhibits hepatic GH signaling in vivo in mice that adenovirally express rabbit GHR, as assessed with our noninvasive bioluminescence hepatic signaling assay. A separate monoclonal antibody (anti-GHR(mAb 18.24)) is a sister clone of anti-GHR(ext-mAb). Here, we demonstrate that anti-GHR(mAb 18.24) also inhibits rabbit and human GHR signaling and inducible receptor proteolysis. Further, we use a random PCR-generated mutagenic expression system to map the three-dimensional epitopes in the rabbit GHR ECD for both anti-GHR(ext-mAb) and anti-GHR(mAb 18.24). We find that each of the two antibodies has similar, but nonidentical, discontinuous epitopes that include regions of subdomain 2 encompassing the dimerization interface. These results have fundamental implications for understanding the role of the dimerization interface and subdomain 2 in GHR activation and regulated GHR metalloproteolysis and may inform development of therapeutics that target GHR.

VEGF-A promotes cardiac stem cell engraftment and myocardial repair in the infarcted heart.

BACKGROUND The objective of this study was to determine whether vascular endothelial growth factor (VEGF)-A subtypes improve cardiac stem cell (CSC) engraftment and promote CSC-mediated myocardial repair in the infarcted heart. METHODS CSCs were treated with VEGF receptor (VEGFR) inhibitors, VCAM-1 antibody (VCAM-1-Ab), or PKC-α inhibitor followed by the treatment with VEGF-A. CSC adhesion assays were performed in vitro. In vivo, the PKH26-labeled and VCAM-1-Ab or PKC-α inhibitor pre-treated CSCs were treated with VEGF-A followed by implantation into infarcted rat hearts. The hearts were then collected for measuring CSC engraftment and evaluating cardiac fibrosis and function 3 or 28days after the CSC transplantation. RESULTS All three VEGF-A subtypes promoted CSC adhesion to extracellular matrix and endothelial cells. VEGF-A-mediated CSC adhesion required VEGFR and PKCα signaling. Importantly, VEGF-A induced VCAM-1, but not ICAM-1 expression in CSCs through PKCα signaling. In vivo, VEGF-A promoted the engraftment of CSCs in infarcted hearts, which was attenuated by PKCα inhibitor or VCAM-1-Ab. Moreover, VEGF-A-mediated CSC engraftment resulted in a reduction in infarct size and fibrosis. Functional studies showed that the transplantation of the VEGF-A-treated CSCs stimulated extensive angiomyogenesis in infarcted hearts as indicated by the expression of cardiac troponin T and von Willebrand factor, leading to an improved performance of left ventricle. Blockade of PKCα signaling or VCAM-1 significantly diminished the beneficial effects of CSCs treated with VEGF-A. CONCLUSION VEGF-A promotes myocardial repair through, at least in part, enhancing the engraftment of CSCs mediated by PKCα/VCAM-1 pathway.

Effects of estrogen on growth and smooth muscle differentiation of vascular wall-resident CD34+ stem/progenitor cells

OBJECTIVES To investigate the effects of estrogen on growth and smooth muscle cell (SMC)-differentiation of vascular wall-resident CD34(+) stem/progenitor cells (VRS/Pcs). METHODS AND RESULTS The existence of CD34(+) VRS/Pcs was confirmed by immunohistochemistry in the adventitia of arteries of young (2-month-old) and old (24-month-old) female SD rats with less CD34(+) adventitial cells detected in the old. The VRS/Pcs isolated from young animals were grown in Stem cell growth medium or induced to differentiate into SMC with PDGF-BB in the presence or absence of 17β-estrodiol (E2). Flow cytometry, RT-qPCR and Western blot showed that E2 promoted Brdu incorporation of the CD34(+) VRS/Pcs growing in Stem cell growth medium; but when the cells were incubated in PDGF-BB, the hormone enhanced their expression of SMC marker SM22. ChIP and IP assays showed that E2 significantly promoted the binding of pELK1-SRF complex to the promoter of c-fos gene in CD34(+) VRS/Pcs growing in the Stem cell growth medium; but when the cells were stimulated with PDGF-BB, an E2-enhanced binding of myocardin-SRF to the promoter of SM22 gene was found with enhanced expression of SRC3 and its binding to myocardin. The effects of E2 above could be blocked by the estrogen receptor antagonist ICI 182,780 or inhibited by SRF-siRNA. CONCLUSION Estrogen has dual effects on CD34(+) VRS/Pcs. For the undifferentiated VRS/Pcs, it accelerates their proliferation by enhancing binding of pELK1-SRF complex to c-fos gene; while for the differentiating VRS/Pcs, it promotes their differentiation to SMC through a mechanism of SRC3-mediated interaction of myocardin-SRF complex with SM22 gene.

Dual effects of estrogen on vascular smooth muscle cells: Receptor-mediated proliferative vs. metabolite-induced pro-senescent actions

OBJECTIVE To investigate the mechanism for the dual effects of estrogen on vascular smooth muscle cells (VSMCs). METHODS Cultured rat VSMCs were exposed to gradient concentrations (10(-9)-10(-5)M) of 17β-estradiol (E(2)) with or without pre-administration of a broad-spectrum CYP450 inhibitor 1-aminobenzotriazole (ABT) (10×10(-6)M) and an estrogen receptor (ER) antagonist ICI 182,780 (10(-6)M), respectively. The growth, cell cycle progression, premature senescence, estrogen metabolites, reactive oxygen species (ROS) and DNA damage of the cells were analyzed with cell counting assay, flow cytometry, Western blot, liquid chromatography-mass spectrometry and comet assay, respectively. RESULTS E(2) in its physiological levels from 10(-9)M to 10(-8)M had a concentration-dependent promoting effect on growth of VSMCs. However, when the concentration increased over 10(-8)M, the growth-promoting effect gradually reversed to a growth-inhibiting action. When the activity of CYP450s was blocked by ABT, the growth-promoting effect of E(2) increased and did not reverse at high concentrations. Whereas when the ERs were blocked by ICI 182,780, E(2) showed a pure growth-inhibiting effect. The E(2) metabolites 2- and 4-hydroxyestradiols accumulated with the increase of E(2) over 10(-8)M, which accompanied by increased ROS, DNA damage and cellular senescence. All of these changes were eliminated by block of CYP450s, indicating that the VSMC growth inhibition by E(2) is due to an increased production of ROS from accumulated E(2) metabolites which induces DNA damage, leading to VSMC premature senescence. CONCLUSION The complex effect of E(2) is due to two opposite actions: one ER-mediated and proliferative, and the other estrogen metabolite-induced and pro-senescent.

Changes in growth hormone (GH), GH receptor, and GH signal transduction in hippocampus of congenital hypothyroid rats

Recent studies have shown that, like thyroid hormone (TH), growth hormone (GH) plays a critical role in development of the brain. However, it is still unclear whether the functions of the two hormones are locally orchestrated in the brain or whether TH has a permissive effect on GH in the central nervous system as it does in the periphery. To address this question, the present study investigated the changes in local expression of GH and GH receptor (GHR) and the activity of GH signaling molecules in the hippocampus of congenitally hypothyroid (CHT) rats. As demonstrated by morphometric measurements and the Y‐maze test, CHT rats had decreased neurons and weaker Nissl staining in the stratum pyramidal/granule in the hippocampus and a reduced acquisition of safe place recognition memory. Analyses of QPCR and Western blot revealed a substantially decreased hippocampal expression of GH and GHR, accompanied by a corresponding decrease in phosphorylation of JAK2 and STAT5 in the CHT rats. These changes were, at least in part, corrected by systemic supplement of T3. The findings provide the first direct evidence suggesting that the functional autocrine and paracrine regulation of GH in the CNS is orchestrated by TH.