Hong Yu

Age-related molecular genetic changes of murine bone marrow mesenchymal stem cells.

BackgroundMesenchymal stem cells (MSC) are pluripotent cells, present in the bone marrow and other tissues that can differentiate into cells of all germ layers and may be involved in tissue maintenance and repair in adult organisms. Because of their plasticity and accessibility these cells are also prime candidates for regenerative medicine. The contribution of stem cell aging to organismal aging is under debate and one theory is that reparative processes deteriorate as a consequence of stem cell aging and/or decrease in number. Age has been linked with changes in osteogenic and adipogenic potential of MSCs.ResultsHere we report on changes in global gene expression of cultured MSCs isolated from the bone marrow of mice at ages 2, 8, and 26-months. Microarray analyses revealed significant changes in the expression of more than 8000 genes with stage-specific changes of multiple differentiation, cell cycle and growth factor genes. Key markers of adipogenesis including lipoprotein lipase, FABP4, and Itm2a displayed age-dependent declines. Expression of the master cell cycle regulators p53 and p21 and growth factors HGF and VEGF also declined significantly at 26 months. These changes were evident despite multiple cell divisions in vitro after bone marrow isolation.ConclusionsThe results suggest that MSCs are subject to molecular genetic changes during aging that are conserved during passage in culture. These changes may affect the physiological functions and the potential of autologous MSCs for stem cell therapy.

Human macrovascular endothelial cells: optimization of culture conditions.

SummaryThe purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.

Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

Cell surface receptors play major roles in the mobilization and homing of progenitor cells from the bone marrow to peripheral tissues. CXCR4 is an important receptor that regulates homing of leucocytes and endothelial progenitors in response to the chemokine stromal cell‐derived factor‐1 (SDF‐1). Ionic calcium is also known to regulate chemotaxis of selective bone marrow cells (BMCs) through the calcium‐sensing receptor, CaR. Here we show that calcium regulates CXCR4 expression and BMC responses to SDF‐1. CaCl2 treatment of BMC induced a time‐ and dose‐dependent increase in both the transcription and cell surface expression of CXCR4. BMC subpopulations expressing VEGFR2+, CD34+ and cKit+/Sca‐1+ were especially sensitive to calcium. The effects were blocked by calcium influx inhibitors, anti‐CaR antibody and the protein synthesis inhibitor cycloheximide, but not by the CXCR4 antagonist AMD3100. Calcium treatment also enhanced SDF‐1‐mediated CXCR4 internalization. These changes were reflected in significantly improved chemotaxis by SDF‐1, which was abolished by AMD3100 and by antibody against CXCR4. Calcium pre‐treatment improved homing of CD34+ BMCs to ischemic muscle in vivo, and enhanced revascularization in ischemic mouse hindlimbs. Our results identify calcium as a positive regulator of CXCR4 expression that promotes stem cell mobilization, homing and therapy.

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.

A novel CXCR4 antagonist derived from human SDF-1β enhances angiogenesis in ischaemic mice

AIMSnThe effects on angiogenesis of a novel CXC chemokine receptor 4 (CXCR4) antagonist, SDF-1betaP2G, derived from human stromal cell-derived factor-1beta (SDF-1beta), were examined in a model of hind limb ischaemia in mice.nnnMETHODS AND RESULTSnThe antagonistic activities of SDF-1betaP2G against CXCR4 were evaluated in vitro and in vivo and compared with phosphate-buffered saline and AMD3100 (a small bicyclam antagonist of SDF-1). Angiogenesis, muscle regeneration and the expression of pro-angiogenic factors were evaluated in ischaemic gastrocnemius muscles. Distant toxic effects of SDF-1betaP2G were evaluated by inflammatory and apoptotic markers. SDF-1betaP2G induced CXCR4 internalization and competitively inhibited the chemotaxis of SDF-1beta but did not mediate migration, calcium influx, or the phosphorylation of Akt and extracellular signal-regulated kinase in cultured T-lymphoblastic leukaemia cells or H9C2 cells. SDF-1betaP2G enhanced blood flow, angiogenesis, and muscle regeneration in ischaemic hind limbs, and the enhancement was significantly better than that of AMD3100. Markers of angiogenesis and progenitor cell migration, including phosphorylated Akt, vascular endothelial growth factor (VEGF), SDF-1 and CXCR4, were up-regulated by SDF-1betaP2G and co-localized with CD31-positive cells. Neutralization of VEGF with its specific antibody abolished SDF-1betaP2G-induced blood reperfusion and angiogenesis. No apparent inflammatory and apoptotic effects were found in heart, liver, kidneys, and testes after SDF-1betaP2G administration.nnnCONCLUSIONnOur findings indicate that the novel CXCR4 antagonist, SDF-1betaP2G, can efficiently enhance ischaemic angiogenesis, blood flow restoration, and muscle regeneration without apparent adverse effects, most likely through a VEGF-dependent pathway.

High efficiency in vitro gene transfer into vascular tissues using a pseudotyped retroviral vector without pseudotransduction

Murine leukemia virus (MuLV)-derived retroviral vectors have had limited application in vascular gene therapy because of low transduction efficiency of vascular tissues, both in vitro and in vivo. In this study, we compared the gene transfer efficiency of two retroviral vectors: amphotropic MuLV and a MuLV vector pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. Target vascular tissues included human endothelial cells (EC), smooth muscle cells (SMC) and saphenous veins (SV). Transduction efficiency of human EC and SMC was significantly higher for VSV-G pseudotyped MuLV vector (90%) than for Amphotropic MuLV (20%). Luminal surface en face analysis of transduced cultured SV showed a six- to 10-fold greater transduction efficiency with VSV-G pseudotyped MuLV. The tissue plasminogen activator (tPA) gene was transduced into EC using each vector. Four days following transduction, a 12-fold higher tPA antigen concentration and a 38-fold higher tPA enzymatic activity was measured from cells transduced with the VSV-G pseudotyped vectors as compared with the amphotropic MuLV. There was no detectable pseudotransduction (protein transfer) associated with the VSV-G MuLV vector. Both AZT inhibition of reverse transcriptase and cell division arrest by gamma irradiation inhibited transduction, indicating that viral transduction correlated with RNA reverse transcription and cell proliferation. MuLV pseudotyped with the VSV-G envelope glycoprotein is an effective retroviral vector for vascular gene therapy.

Smooth muscle cells improve endothelial cell retention on polytetrafluoroethylene grafts in vivo

OBJECTIVEnWe investigated the influence of smooth muscle cells (SMC) on endothelial cell (EC) retention on polytetrafluoroethylene (PTFE) grafts and the effect of SMC seeding on intimal hyperplasia in vivo in a rabbit model.nnnMETHODSnFibronectin-coated PTFE grafts (4 mm diameter) were seeded with either EC alone, SMC alone, or SMC followed 24 hours later by EC. The grafts were connected to an extracorporal aortic shunt for 1 hour or were individually implanted for 1, 30, and 100 days into the infrarenal aorta as an end-to-side bypass graft. The number of retained cells was compared at 1 hour and at 1 day after implantation. Neointimal thickness was measured 30 and 100 days after implantation.nnnRESULTSnAfter 1-hour exposure to blood flow, EC retention rate was greater (P <.005) if seeded on top of SMC (98% +/- 2%; n = 8) versus being seeded alone (65 +/- 11%; n = 8). SMC retention rate was 95 +/- 5% (n = 8) when seeded alone. Similar cell retention was obtained 1 day after implantation. After 30-day implantation the neointima was thicker in grafts seeded with EC and SMC (282 +/- 136 microm; n = 3) than with EC only (52 +/- 45 microm; n = 3; P <.001). However, the neointimal thickness for dual-cell-seeded grafts (126 +/- 60 microm; n = 3) was not significantly different (P =.09) from EC-seeded grafts (79 +/- 48 microm; n = 3) after 100-day implantation.nnnCONCLUSIONnEC retention on PTFE grafts in vivo is improved if seeded over a layer of SMC. Further studies are needed to determine whether overlying EC modulate proliferation of underlying SMC.

The glomerulosclerosis of aging in females: contribution of the proinflammatory mesangial cell phenotype to macrophage infiltration.

Age-associated renal changes may be an important cause of renal failure. We recently found that aged female B6 mice developed progressive glomerular lesions. This was associated with macrophage infiltration, a frequent finding in glomerulosclerosis. We used these mice as a model for studying the mechanisms of glomerular aging. We compared the gene expression profile of intact glomeruli from late postmenopausal (28-month-old) mice to that of intact glomeruli from premenopausal (5-month-old) mice. We found that inflammation-related genes, especially those expressed by activated macrophages, were up-regulated in the glomeruli of 28-month-old mice, a result correlating with the histological observation of glomerular macrophage infiltration. The mechanism for macrophage recruitment could have been stable phenotypic changes in mesangial cells because we found that mesangial cells isolated from 28-month-old mice expressed higher levels of RANTES and VCAM-1 than cells from 5-month-old mice. The elevated serum tumor necrosis factor (TNF)-alpha levels present in aged mice may contribute to increased RANTES and VCAM-1 expression in mesangial cells. Furthermore, cells from 28-month-old mice were more sensitive to TNF-alpha-induced RANTES and VCAM-1 up-regulation. The effect of TNF-alpha on RANTES expression was mediated by TNF receptor 1. Interestingly, mesangial cells isolated from 28-month-old mice had increased nuclear factor-kappaB transcriptional activity. Inhibition of nuclear factor-kappaB activity decreased baseline as well as TNF-alpha-induced RANTES and VCAM-1 expression in mesangial cells isolated from 28-month-old mice. Thus, phenotypic changes in mesangial cells may predispose them to inflammatory stimuli, such as TNF-alpha, which would contribute to glomerular macrophage infiltration and inflammatory lesions in aging.

Genetic engineering of stent grafts with a highly efficient pseudotyped retroviral vector

PURPOSEnThe purpose of this study was first to compare the gene transfer efficiency of amphotrophic murine leukemia viral vector (ampho-MuLV) with the efficiency of MuLV pseudotyped with the vesicular stomatitis virus G glycoprotein (VSVG-MuLV) in tissue of vascular origin. The second purpose of this study was to determine cell retention after the implantation of genetically engineered stent grafts.nnnMETHODSnGene transfer efficiency was ascertained with the b-galactosidase assay. The target tissues included endothelial cells (ECs), smooth muscle cells (SMCs), and human saphenous veins (HSVs). Polyurethane stent grafts were suffused with lac Z-transduced ECs and SMCs that were harvested from porcine jugular vein. The grafts were implanted into the iliac artery of each pig whose jugular vein had been harvested. Cell retention was analyzed at 1 and 4 weeks with X-Gal staining.nnnRESULTSnVSVG-MuLV transduction efficiency exceeded that of ampho-MuLV in human ECs (VSVG-MuLV, n = 24, 89% +/- 6%; ampho-MuLV, n = 18, 14% +/- 6%; P <. 001), human SMCs (VSVG-MuLV, n = 5, 92% +/- 3%; ampho-MuLV, n = 4, 17% +/- 2%; P <.001), pig ECs (VSVG-MuLV, n = 4, 81% +/- 2%; ampho-MuLV, n = 4, 13% +/- 3%; P <.001), and pig SMCs (VSVG-MuLV, n = 5, 89% +/- 3%; ampho-MuLV, n = 4, 16% +/- 1%; P <.001). As much as a 10-fold higher transduction efficiency was observed with VSVG-MuLV in HSVs. After the stent graft implantation, the engineered cells were retained and proliferated on the stent membrane, with ingrowth into the underlying intima.nnnCONCLUSIONnVSVG-MuLV significantly increased the gene transfer efficiency in vascular SMCs and ECs and in organ-cultured HSVs. The cells were retained and proliferated on stent grafts for the short term in the pig.

Defective CXCR4 expression in aged bone marrow cells impairs vascular regeneration.

The chemokine stromal cell‐derived factor‐1 (SDF‐1) plays a critical role in mobilizing precursor cells in the bone marrow and is essential for efficient vascular regeneration and repair. We recently reported that calcium augments the expression of chemokine receptor CXCR4 and enhances the angiogenic potential of bone marrow derived cells (BMCs). Neovascularization is impaired by aging therefore we suggested that aging may cause defects of CXCR4 expression and cellular responses to calcium. Indeed we found that both the basal and calcium‐induced surface expression of CXCR4 on BMCs was significantly reduced in 25‐month‐old mice compared with 2‐month‐old mice. Reduced Ca‐induced CXCR4 expression in BMC from aged mice was associated with defective calcium influx. Diminished CXCR4 surface expression in BMC from aged mice correlated with diminished neovascularization in an ischemic hindlimb model with less accumulation of CD34+ progenitor cells in the ischemic muscle with or without local overexpression of SDF‐1. Intravenous injection of BMCs from old mice homed less efficiently to ischemic muscle and stimulated significantly less neovascularization compared with the BMCs from young mice. Transplantation of old BMCs into young mice did not reconstitute CXCR4 functions suggesting that the defects were not reversible by changing the environment. We conclude that defects of basal and calcium‐regulated functions of the CXCR4/SDF‐1 axis in BMCs contribute significantly to the age‐related loss of vasculogenic responses.