Secreted phosphoglucose isomerase is a novel biomarker of nonalcoholic fatty liver in mice and humans.

The current gold standard for diagnosis of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is through a liver biopsy, and there is an urgent need to develop non-invasive methods for early detection. We previously demonstrated metabolic remodeling in the mouse fatty liver, which is marked by increased hepatic expression and activities of phosphoglucose isomerase (PGI) and several other glycolytic enzymes. Since PGI is actively transported out of the cell, acting as a multifunctional cytokine referred to as autocrine motility factor (AMF), we explored the possibility that PGI secreted from the fatty liver may be targeted for early detection of the silent disease. We report here that mice with NASH exhibited significantly elevated serum PGI enzyme activities compared to normal control (P < 0.005). We further confirmed the finding using serum/plasma samples (n = 73) collected from a cohort of NASH patients who were diagnosed according to Kleiner's criteria, showing a normal mean PGI of 19.5 ± 8.8 IU/L and patient mean PGI of 105.6 ± 79.9 IU/L (P < 0.005). In addition, elevated blood PGI in NASH patients coincided with increased blood L-lactate. Cell culture experiments were then conducted to delineate the PGI-lactate axis, which revealed that treatment of HepG2 cells with recombinant PGI protein stimulated glycolysis and lactate output, suggesting that the disease-induced PGI likely contributed to the increased lactate in NASH patients. Taken together, the preclinical and clinical data validate secreted PGI as a useful biomarker of the fatty liver that can be easily screened at the point of care.

sFRP2 activates Wnt/ß-catenin signaling in cardiac fibroblasts: differential roles in cell growth, energy metabolism, and extracellular matrix remodeling.

Secreted Frizzled-related protein 2 (sFRP2) plays a key role in chronic fibrosis after myocardial infarction and in heart failure. The aim of this study was to elucidate the mechanisms through which sFRP2 may regulate the growth and extracellular matrix (ECM) remodeling of adult mouse cardiac fibroblasts (CFs). We found that sFRP2 activates CFs in part through canonical Wnt/ß-catenin signaling, as evidenced by increased expression of Axin2 and Wnt3a, but not Wnt5a, as well as accumulation of nuclear ß-catenin. In response to sFRP2, CFs exhibited robust cell proliferation associated with increased glucose consumption and lactate production, a phenomenon termed "the Warburg effect" in oncology. The coupling between CF expansion and anaerobic glycolysis is marked by upregulation of glyceraldehyde-3-phosphate dehydrogenase and tissue-nonspecific alkaline phosphatase. In conjunction with these phenotypic changes, CFs accelerated ECM remodeling through upregulation of expression of the matrix metalloproteinase (MMP) 1 and MMP13 genes, two members of the collagenase subfamily, and enzyme activities of MMP2 and MMP9, two members of the gelatinase subfamily. Consistent with the induction of multiple MMPs possessing collagenolytic activities, the steady-state level of collagen type 1 in CF-spent medium was reduced by sFRP2. Analysis of non-CF cell types revealed that the multifaceted effects of sFRP2 on growth control, glucose metabolism, and ECM regulation are largely restricted to CFs and highly sensitive to Wnt signaling perturbation. The study provides a molecular framework on which the functional versatility and signaling complexity of sFRP2 in cardiac fibrosis may be better defined.

Tissue-nonspecific alkaline phosphatase as a target of sFRP2 in cardiac fibroblasts.

Recent studies of myocardial infarction in secreted Frizzled-related protein 2 (sFRP2) knockout mice and our hamster heart failure therapy based on sFRP2 blockade have established sFRP2 as a key profibrotic cytokine in the heart. The failing hamster heart is marked by prominent fibrosis and calcification with elevated expression of sFRP2. Noting the involvement of tissue-nonspecific alkaline phosphatase (TNAP) in bone mineralization and vascular calcification, we determined whether sFRP2 might be an upstream regulator of TNAP. Biochemical assays revealed an approximately twofold increase in the activity of TNAP and elevated levels of inorganic phosphate (Pi) in the failing heart compared with the normal heart. Neither was this change detected in the liver or hamstring muscle nor was it associated with systemic hyperphosphatemia. TNAP was readily cloned from the hamster heart and upon overexpression increased the level of extracellular but not intracellular Pi, which is consistent with the cell surface location of the ectoenzyme. In line with the previous demonstration that sFRP2 blockade attenuated fibrosis, we show here that the therapy downregulated TNAP. This in vivo finding is corroborated by the in vitro study showing that cultured cardiac fibroblasts treated with recombinant sFRP2 protein exhibited progressive increase in the expression and activity of TNAP, which was completely abrogated by cycloheximide or tunicamycin. Induction of TNAP by sFRP2 is restricted to cardiac fibroblasts among the multiple cell types examined, and was not observed with sFRP4. The current work indicates that sFRP2 may promote cardiac fibrocalcification through coordinate activation of tolloid-like metalloproteinases and TNAP.

Secreted Frizzled-related protein 2 as a target in antifibrotic therapeutic intervention.

Progressive fibrosis is a pathological hallmark of many chronic diseases responsible for organ failure. Although there is currently no therapy on the market that specifically targets fibrosis, the dynamic fibrogenic process is known to be regulated by multiple soluble mediators that may be therapeutically intervened. The failing hamster heart exhibits marked fibrosis and increased expression of secreted Frizzled-related protein 2 (sFRP2) amenable to reversal by mesenchymal stem cell (MSC) therapy. Given the previous demonstration that sFRP2-null mice subjected to myocardial infarction exhibited reduced fibrosis and improved function, we tested whether antibody-based sFRP2 blockade might counteract the fibrogenic pathway and repair cardiac injury. Cardiomyopathic hamsters were injected intraperitoneally twice a week each with 20 µg of sFRP2 antibody. Echocardiography, histology, and biochemical analyses were performed after 1 mo. sFRP2 antibody increased left ventricular ejection fraction from 40 ± 1.2 to 49 ± 6.5%, whereas saline and IgG control exhibited a further decline to 37 ± 0.9 and 31 ± 3.2%, respectively. Functional improvement is associated with a ∼ 50% reduction in myocardial fibrosis, ∼ 65% decrease in apoptosis, and ∼ 75% increase in wall thickness. Consistent with attenuated fibrosis, both MSC therapy and sFRP2 antibody administration significantly increased the activity of myocardial matrix metalloproteinase-2. Gene expression analysis of the hamster heart and cultured fibroblasts identified Axin2 as a downstream target, the expression of which was activated by sFRP2 but inhibited by therapeutic intervention. sFRP2 blockade also increased myocardial levels of VEGF and hepatocyte growth factor (HGF) along with increased angiogenesis. These findings highlight the pathogenic effect of dysregulated sFRP2, which may be specifically targeted for antifibrotic therapy.

Autologous mesenchymal stem cells mobilize cKit+ and CD133+ bone marrow progenitor cells and improve regional function in hibernating myocardium.

RATIONALE: Mesenchymal stem cells (MSCs) improve function after infarction, but their mechanism of action remains unclear, and the importance of reduced scar volume, cardiomyocyte proliferation, and perfusion is uncertain. OBJECTIVE: The present study was conducted to test the hypothesis that MSCs mobilize bone marrow progenitor cells and improve function by stimulating myocyte proliferation in collateral-dependent hibe rnating myocardium. METHODS AND RESULTS: Swine with chronic hibernating myocardium received autologous intracoronary MSCs (icMSCs; ≈44 ×10(6) cells, n = 10) 4 months after instrumentation and were studied up to 6 weeks later. Physiological and immunohistochemical findings were compared with untreated hibernating animals (n = 7), sham-normal animals (n = 5), and icMSC-treated sham-normal animals (n = 6). In hibernating myocardium, icMSCs increased function (percent wall thickening of the left anterior descending coronary artery 24 ± 4% to 43 ± 5%, P < 0.05), although left anterior descending coronary artery flow reserve (adenosine/rest) remained critically impaired (1.2 ± 0.1 versus 1.2 ± 0.1). Circulating cKit+ and CD133+ bone marrow progenitor cells increased transiently after icMSC administration, with a corresponding increase in myocardial cKit+/CD133+ and cKit+/CD133- bone marrow progenitor cells (total cKit+ from 223 ± 49 to 4415 ± 866/10(6) cardiomyocytes, P < 0.05). In hibernating hearts, icMSCs increased Ki67+ cardiomyocytes (from 410 ± 83 to 2460 ± 610/10(6) nuclei, P < 0.05) and phospho-histone H3-positive cardiomyocytes (from 9 ± 5 to 116 ± 12/10(6) nuclei, P < 0.05). Myocyte nuclear number (from 75 336 ± 5037 to 114 424 ± 9564 nuclei/mm3, P < 0.01) and left ventricular mass (from 2.5 ± 0.1 to 2.8 ± 0.1 g/kg, P < 0.05) increased, yet myocytes were smaller (14.5 ± 0.4 versus 16.5 ± 0.4 µm, P < 0.05), which supports endogenous cardiomyocyte proliferation. In sham-normal animals, icMSCs increased myocardial bone marrow progenitor cells with no effect on myocyte proliferation or regional function. CONCLUSIONS: Our results indicate that icMSCs improve function in hibernating myocardium independent of coronary flow or reduced scar volume. This arises from stimulation of myocyte proliferation with increases in cKit+/CD133+ bone marrow progenitor cells and cKit+/CD133- resident stem cells, which increase myocyte number and reduce cellular hypertrophy.

Activation of Toll-like receptor 3 amplifies mesenchymal stem cell trophic factors and enhances therapeutic potency.

Clinical trials of bone marrow mesenchymal stem cell (MSC) therapy have thus far demonstrated moderate and inconsistent benefits, indicating an urgent need to improve therapeutic efficacy. Although administration of sufficient cells is necessary to achieve maximal therapeutic benefits, documented MSC clinical trials have largely relied on injections of ∼1 × 10(6) cells/kg, which appears too low to elicit a robust therapeutic response according to published preclinical studies. However, repeated cell passaging necessary for large-scale expansion of MSC causes cellular senescence and reduces stem cell potency. Using the RNA mimetic polyinosinic-polycytidylic acid [poly(I:C)] to engage MSC Toll-like receptor 3 (TLR3), we found that poly(I:C), signaling through multiple mitogen-activated protein kinase pathways, induced therapeutically relevant trophic factors such as interleukin-6-type cytokines, stromal-derived factor 1, hepatocyte growth factor, and vascular endothelial growth factor while slightly inhibiting the proliferation and migration potentials of MSC. At the suboptimal injection dose of 1 × 10(6) cells/kg, poly(I:C)-treated MSC, but not untreated MSC, effectively stimulated regeneration of the failing hamster heart 1 mo after cell administration. The regenerating heart exhibited increased CD34(+)/Ki67(+) and CD34(+)/GATA4(+) progenitor cells in the presence of decreased inflammatory cells and cytokines. Cardiac functional improvement was associated with a ∼50% reduction in fibrosis, a ∼40% reduction in apoptosis, and a ∼55% increase in angiogenesis, culminating in prominent cardiomyogenesis evidenced by abundant distribution of small myocytes and a ∼90% increase in wall thickening. These functional, histological, and molecular characterizations thus establish the utility of TLR3 engagement for enabling the low-dose MSC therapy that may be translated to more efficacious clinical applications.

Dental Pulp Cell Conditioning through Polyinosinic-Polycytidylic Acid Activation of Toll-like Receptor 3 for Amplification of Trophic Factors.

INTRODUCTION: Regeneration of the pulp-dentin complex hinges on functionally diverse growth factors, cytokines, chemokines, signaling molecules, and other secreted factors collectively referred to as trophic factors. The delivery of exogenous factors and the induced release of endogenous dentin-bound factors by conditioning agents have been explored toward these goals. The aim of this study was to investigate a promising regeneration strategy based on the conditioning of dental pulp cells (DPCs) with polyinosinic-polycytidylic acid (poly[I:C]) for the amplification of endogenous trophic factors. METHODS: DPCs were isolated from human dental pulps, propagated in culture, and treated with an optimized dose of poly(I:C). The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay and metabolite analysis were conducted to monitor the cytotoxicity of poly(I:C). Enzyme-linked immunosorbent assays and quantitative polymerase chain reaction assays were performed to quantify the induction of trophic factors in response to DPC conditioning. Statistical significance was P < .05. RESULTS: The analysis of 32 trophic factors involved in Wnt signaling, cell migration and chemotaxis, cell proliferation and differentiation, extracellular matrix remodeling and angiogenesis, and immunoregulation revealed that DPCs abundantly express many trophic factors including AMF, BDNF, BMP2, FGF1, FGF2, FGF5, HGF, MCP1, NGF, SDF1, TGFß1, TIMP1, TIMP2, TIMP3, and VEGFA, many of which were further induced by DPC conditioning; induction was significant for BDNF, EGF, HGF, LIF, MCP1, SDF1, IL6, IL11, MMP9, and TIMP1. Both DPC proliferation and lactate production (P < .05) were inhibited by 8 µg/mL poly(I:C) relative to the control. CONCLUSIONS: In vitro DPC conditioning through poly(I:C) activation of toll-like receptor 3 led to the amplification of trophic factors involved in tissue repair. The strategy offers promise for endodontic regeneration and tooth repair and warrants further investigation.

Intramuscular VEGF activates an SDF1-dependent progenitor cell cascade and an SDF1-independent muscle paracrine cascade for cardiac repair.

The skeletal muscle is endowed with an impressive ability to regenerate after injury, and this ability is coupled to paracrine production of many trophic factors possessing cardiovascular benefits. Taking advantage of this humoral capacity of the muscle, we recently demonstrated an extracardiac therapeutic regimen based on intramuscular delivery of VEGF-A(165) for repair of the failing hamster heart. This distal organ repair mechanism activates production from the injected hamstring of many trophic factors, among which stromal-derived factor-1 (SDF1) prominently mobilized multi-lineage progenitor cells expressing CXCR4 and their recruitment to the heart. The mobilized bone marrow progenitor cells express the cardiac transcription factors myocyte enhancer factor 2c and GATA4 and several major trophic factors, most notably IGF1 and VEGF. SDF1 blockade abrogated myocardial recruitment of CXCR4(+) and c-kit(+) progenitor cells with an insignificant effect on the hematopoietic progenitor lineage. The knockdown of cardiac progenitor cells led to deprivation of myocardial trophic factors, resulting in compromised cardiomyogenesis and angiogenesis. However, the VEGF-injected hamstring continued to synthesize cardioprotective factors, contributing to moderate myocardial tissue viability and function even in the presence of SDF1 blockade. These findings thus uncover two distinct but synergistic cardiac therapeutic mechanisms activated by intramuscular VEGF. Whereas the SDF1/CXCR4 axis activates the progenitor cell cascade and its trophic support of cardiomyogenesis intramuscularly, VEGF amplifies the skeletal muscle paracrine cascade capable of directly promoting myocardial survival independent of SDF1. Given that recent clinical trials of cardiac repair based on the use of marrow-mobilizing agents have been disappointing, the proposed dual therapeutic modality warrants further investigation.

Activation of host tissue trophic factors through JAK-STAT3 signaling: a mechanism of mesenchymal stem cell-mediated cardiac repair.

We recently demonstrated a cardiac therapeutic regimen based on injection of bone marrow mesenchymal stem cells (MSCs) into the skeletal muscle. Although the injected MSCs were trapped in the local musculature, the extracardiac cell delivery approach repaired the failing hamster heart. This finding uncovers a tissue repair mechanism mediated by trophic factors derived from the injected MSCs and local musculature that can be explored for minimally invasive stem cell therapy. However, the trophic factors involved in cardiac repair and their actions remain largely undefined. We demonstrate here a role of MSC-derived IL-6-type cytokines in cardiac repair through engagement of the skeletal muscle JAK-STAT3 axis. The MSC IL-6-type cytokines activated JAK-STAT3 signaling in cultured C2C12 skeletal myocytes and caused increased expression of the STAT3 target genes hepatocyte growth factor (HGF) and VEGF, which was inhibited by glycoprotein 130 (gp130) blockade. These in vitro findings were corroborated by in vivo studies, showing that the MSC-injected hamstrings exhibited activated JAK-STAT3 signaling and increased growth factor/cytokine production. Elevated host tissue growth factor levels were also detected in quadriceps, liver, and brain, suggesting a possible global trophic effect. Paracrine actions of these host tissue-derived factors activated the endogenous cardiac repair mechanisms in the diseased heart mediated by Akt, ERK, and JAK-STAT3. Administration of the cell-permeable JAK-STAT inhibitor WP1066 abrogated MSC-mediated host tissue growth factor expression and functional improvement. The study illustrates that the host tissue trophic factor network can be activated by MSC-mediated JAK-STAT3 signaling for tissue repair.

Vascular endothelial growth factor (VEGF) as a key therapeutic trophic factor in bone marrow mesenchymal stem cell-mediated cardiac repair.

We recently demonstrated a novel effective therapeutic regimen for treating hamster heart failure based on injection of bone marrow mesenchymal stem cells (MSCs) or MSC-conditioned medium into the skeletal muscle. The work highlights an important cardiac repair mechanism mediated by the myriad of trophic factors derived from the injected MSCs and local musculature that can be explored for non-invasive stem cell therapy. While this therapeutic regimen provides the ultimate proof that MSC-based cardiac repair is mediated by the trophic actions independent of MSC differentiation or stemness, the trophic factors responsible for cardiac regeneration after MSC therapy remain largely undefined. Toward this aim, we took advantage of the finding that human and porcine MSCs exhibit species-related differences in expression of trophic factors. We demonstrate that human MSCs when compared to porcine MSCs express and secrete 5-fold less vascular endothelial growth factor (VEGF) in conditioned medium (40+/-5 and 225+/-17 pg/ml VEGF, respectively). This deficit in VEGF output was associated with compromised cardiac therapeutic efficacy of human MSC-conditioned medium. Over-expression of VEGF in human MSCs however completely restored the therapeutic potency of the conditioned medium. This finding indicates VEGF as a key therapeutic trophic factor in MSC-mediated myocardial regeneration, and demonstrates the feasibility of human MSC therapy using trophic factor-based cell-free strategies, which can eliminate the concern of potential stem cell transformation.

Intramuscular VEGF repairs the failing heart: role of host-derived growth factors and mobilization of progenitor cells.

Skeletal muscle produces a myriad of mitogenic factors possessing cardiovascular regulatory effects that can be explored for cardiac repair. Given the reported findings that VEGF may modulate muscle regeneration, we investigated the therapeutic effects of chronic injections of low doses of human recombinant VEGF-A(165) (0.1-1 microg/kg) into the dystrophic hamstring muscle in a hereditary hamster model of heart failure and muscular dystrophy. In vitro, VEGF stimulated proliferation, migration, and growth factor production of cultured C2C12 skeletal myocytes. VEGF also induced production of HGF, IGF2, and VEGF by skeletal muscle. Analysis of skeletal muscle revealed an increase in myocyte nuclear [531 +/- 12 VEGF 1 microg/kg vs. 364 +/- 19 for saline (number/mm(2)) saline] and capillary [591 +/- 80 VEGF 1 microg/kg vs. 342 +/- 21 for saline (number/mm(2))] densities. Skeletal muscle analysis revealed an increase in Ki67(+) nuclei in the VEGF 1 microg/kg group compared with saline. In addition, VEGF mobilized c-kit(+), CD31(+), and CXCR4(+) progenitor cells. Mobilization of progenitor cells was consistent with higher SDF-1 concentrations found in hamstring, plasma, and heart in the VEGF group. Echocardiogram analysis demonstrated improvement in left ventricular ejection fraction (0.60 +/- 0.02 VEGF 1 microg/kg vs. 0.45 +/- 0.01 mm for saline) and an attenuation in ventricular dilation [5.59 +/- 0.12 VEGF 1 microg/kg vs. 6.03 +/- 0.09 for saline (mm)] 5 wk after initiating therapy. Hearts exhibited higher cardiomyocyte nuclear [845 +/- 22 VEGF 1 microg/kg vs. 519 +/- 40 for saline (number/mm(2))] and capillary [2,159 +/- 119 VEGF 1 microg/kg vs. 1,590 +/- 66 for saline (number/mm(2))] densities. Myocardial analysis revealed approximately 2.5 fold increase in Ki67+ cells and approximately 2.8-fold increase in c-kit(+) cells in the VEGF group, which provides evidence for cardiomyocyte regeneration and progenitor cell expansion. This study provides novel evidence of a salutary effect of VEGF in the cardiomyopathic hamster via induction of myogenic growth factor production by skeletal muscle and mobilization of progenitor cells, which resulted in attenuation of cardiomyopathy and repair of the heart.

Myocardial oxidative stress, osteogenic phenotype, and energy metabolism are differentially involved in the initiation and early progression of delta-sarcoglycan-null cardiomyopathy.

Dilated cardiomyopathy (DCM) is a common cause of heart failure, and identification of early pathogenic events occurring prior to the onset of cardiac dysfunction is of mechanistic, diagnostic, and therapeutic importance. The work characterized early biochemical pathogenesis in TO2 strain hamsters lacking delta-sarcoglycan. Although the TO2 hamster heart exhibits normal function at 1 month of age (presymptomatic stage), elevated levels of myeloperoxidase, monocyte chemotactic protein-1, malondialdehyde, osteopontin, and alkaline phosphatase were evident, indicating the presence of inflammation, oxidative stress, and osteogenic phenotype. These changes were localized primarily to the myocardium. Derangement in energy metabolism was identified at the symptomatic stage (4 month), and is marked by attenuated activity and expression of pyruvate dehydrogenase E1 subunit, which catalyzes the rate-limiting step in aerobic glucose metabolism. Thus, this study illustrates differential involvement of oxidative stress, osteogenic phenotype, and glucose metabolism in the initiation and early progression of delta-sarcoglycan-null DCM.

Adenoviral expression of vascular endothelial growth factor splice variants differentially regulate bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) are being explored for clinical applications, and genetic engineering represents a useful strategy for boosting the therapeutic potency of MSCs. Vascular endothelial growth factor (VEGF)-based gene therapy protocols have been used to treat tissue ischemia, and a combined VEGF/MSC therapeutics is appealing due to their synergistic paracrine actions. However, multiple VEGF splice variants exhibit differences in their mitogenicity, chemotactic efficacy, receptor interaction, and tissue distribution, and the differential regulatory effects of multiple VEGF isoforms on the function of MSCs have not been characterized. We expressed three rat VEGF-A splice variants VEGF120, 164, and 188 in MSCs using adenoviral vectors, and analyzed their effects on MSC proliferation, differentiation, survival, and trophic factor production. The three VEGF splice variants exert common and differential effects on MSCs. All three expressed VEGFs are potent in promoting MSC proliferation. VEGF120 and 188 are more effective in amplifying expression of multiple growth factor and cytokine genes. VEGF164 on the other hand is more potent in promoting expression of genes associated with MSC remodeling and endothelial differentiation. The longer isoform VEGF188, which is preferentially retained by proteoglycans, facilitates bone morphogenetic protein-7 (BMP7)-mediated MSC osteogenesis. Under serum starvation condition, virally expressed VEGF188 preferentially enhances serum withdrawal-mediated cell death involving nitric oxide production. This work indicates that seeking the best possible match of an optimal VEGF isoform to a given disease setting can generate maximum therapeutic benefits and minimize unwanted side effects in combined stem cell and gene therapy.

Intracoronary administration of AdvFGF-5 (fibroblast growth factor-5) ameliorates left ventricular dysfunction and prevents myocyte loss in swine with developing collaterals and ischemic cardiomyopathy.

BACKGROUND: Fibroblast growth factor (AdvFGF-5) improves regional function by stimulating myocyte hypertrophy without increasing myocardial perfusion in swine with hibernating myocardium. We performed the present study to determine whether AdvFGF-5 could prevent the progression of LV dysfunction in swine with ischemic cardiomyopathy. METHODS AND RESULTS: Swine were chronically instrumented with LAD and LCX stenoses to produce viable dysfunctional myocardium and studied 1 month after instrumentation in the closed-chest sedated state. Flow and regional function before and 30 days after intracoronary AdvFGF-5 (2x10(12) vp, n=9) were compared with animals receiving intracoronary AdvEGFP (2x10(12) vp, n=6). Histological analysis was performed to quantify myocyte size, myocyte nuclear density, apoptosis (TUNEL), and the frequency of myocytes in the proliferative phase of the cell cycle (Ki-67 staining). LAD wall-thickening (27+/-3 to 46+/-6%, P<0.05) and EF (39+/-4 to 56+/-3%, P<0.05) increased after AdvFGF-5. AdvFGF-5 increased maximal perfusion during adenosine vasodilation despite no differences in baseline flow or stenosis severity. After AdvFGF-5, TUNEL-positive myocytes decreased 6-fold and Ki-67 positive myocyte nuclei increased 2-fold. As a result, AdvFGF-5 produced a marked increase in myocyte nuclear density (957+/-54 to 1447+/-40 nuclei/mm2, P<0.05). CONCLUSION: These data indicate that AdvFGF-5 increases regional function and maximal perfusion distal to stenotic arteries when administered before the development of collaterals. This was associated with a reduction in myocyte apoptosis, an increase in Ki-67-positive myocytes, and an increase in myocyte number. Thus, AdvFGF-5 offers a potential therapeutic approach to prevent the progression of ischemic cardiomyopathy and heart failure.

Assessment of a nuclear affinity labeling method for tracking implanted mesenchymal stem cells.

Therapeutic implantation of mesenchymal stem cells (MSCs) is entering the realm of clinical trials for several human diseases, and yet much remains uncertain regarding their dynamic distribution and cell fate after in vivo application. Discrepancies in the literature can be attributed in part to the use of different cell labeling/tracking methods and cell administration protocols. To identify a stem cell detection method suitable for myocardial implantation in a large animal model, we experimented on three different MSC labeling methods: adenovirus-mediated expression of enhanced green fluorescence protein (EGFP) and beta-galactosidase (LacZ), and nuclear staining with DAPI. Intramuscular and intracoronary administrations of labeled porcine MSCs identified the nuclear affinity dye to be a reliable stem cell tracking marker. Stem cell identification is facilitated by an optimized live cell labeling condition generating bright blue fluorescence sharply confined to the nucleus. DAPI-labeled MSCs retained full viability, ceased proliferation, and exhibited an increased differentiation potential. The labeled MSCs remained fully active in expressing key growth factor and cytokine genes, and notably exhibited enhanced expression of the chemokine receptor CXCR4 and its ligand SDF1, indicating their competency in response to tissue injury. Histological analysis revealed that approximately half a million MSCs or approximately 2% of the administered MSCs remained localized in the normal pig heart 2 weeks after coronary infusion. That the vast majority of these identified MSCs were interstitial indicated the ability of MSCs to migrate across the coronary endothelium. No evidence was obtained indicating MSC differentiation to cardiomyocyte.

Upregulation of non-canonical Wnt ligands and oxidative glucose metabolism in NASH induced by methionine-choline deficient diet.

Wnt ligands regulate metabolic pathways, and dysregulation of Wnt signaling contributes to chronic inflammatory disease. A knowledge gap exists concerning the role of aberrant Wnt signaling in non-alcoholic steatohepatitis (NASH), which exhibits metabolic syndrome and inflammation. Using a mouse model of methionine-choline deficient diet (MCDD)-induced NASH, we investigated the Wnt signaling pathways in relation to hepatic glucose oxidation. Mice fed the MCD diet for 6 weeks developed prominent NASH marked by macrovesicular steatosis, inflammation and lipid peroxidation. qPCR analysis reveals differential hepatic expression of canonical and non-canonical Wnt ligands. While expression of Wnt3a was decreased in NASH vs chow diet control, expression of Wnt5a and Wnt11 were increased 3 fold and 15 fold, respectively. Consistent with activation of non-canonical Wnt signaling, expression of the alternative Wnt receptor ROR2 was increased 5 fold with no change in LRP6 expression. Activities of the metabolic enzymes glucokinase, phosphoglucoisomerase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and pyruvate dehydrogenase were all elevated by MCDD. NASH-driven glucose oxidation was accompanied by a 6-fold increase in lactate dehydrogenase (LDH)-B with no change in LDH-A. In addition, glucose-6-phosphate dehydrogenase, the regulatory and NADPH-producing enzyme of the pentose phosphate pathway, was elevated in NASH. These data support a role of accelerated glucose oxidation in the development of NASH, which may be driven by non-canonical Wnt signaling.

Cellular toxicity induced by SRF-mediated transcriptional squelching.

The transcriptional activator serum response factor (SRF) is a member of the immediate early gene family known to promote embryonic development, cell growth, and myogenesis through interaction with multiple nuclear protein factors. Previous studies have shown that SRF possesses potent transcriptional activation domains that can interfere with gene expression at artificially high expression levels through "transcriptional squelching." The current work sought to characterize toxicological aspects of SRF-mediated transcriptional squelching. An adenoviral expression system driven by the potent cytomegalovirus promoter was used to achieve up to a 50-fold increase in SRF protein levels. The overexpressed SRF is nuclear localized and interferes with gene expression independent of specific promoter interaction as expected for transcriptional squelching. SRF-mediated squelching elicits robust cell killing affecting multiple cell types including normal and abnormal proliferating cells as well as postmitotic cells such as cardiomyocytes in culture, and the cell killing is more pronounced than that mediated by the tumor suppressor protein p53. Although both the DNA-binding and transcriptional activation domains of SRF are normally required for the physiological roles of SRF, only the transcriptional activation domain is required for cell killing. Unlike c-myc-induced cell killing, squelching-induced cell death does not require serum withdrawal and cannot be effectively attenuated by blocking the caspase and calpain proteolytic pathways or by overexpression of the antiapoptotic gene bcl-xL. These findings suggest transcriptional squelching may be engineered for killing cancer cells, and the SRF gene may represent a novel molecular target for cancer therapeutics.

Adenoviral gene transfer of FGF-5 to hibernating myocardium improves function and stimulates myocytes to hypertrophy and reenter the cell cycle.

Fibroblast growth factors (FGFs) have diverse actions on the myocardium but the importance of stimulating angiogenesis versus direct effects of FGFs on cardiac myocytes is unclear. We used intracoronary injection of a replication-deficient adenoviral construct overexpressing FGF-5 (AdvFGF-5) to improve flow and function in swine with hibernating myocardium. Two-weeks after AdvFGF-5 (n=8), wall-thickening increased from 2.4+/-0.04 to 4.7+/-0.7 mm in hibernating LAD regions (P<0.05) whereas remote wall-thickening was unchanged (6.7+/-0.4 to 5.8+/-0.5 mm). This was associated with small increases in resting flow to dysfunctional myocardium, but flow during adenosine was unchanged (LAD 1.45+/-0.27 versus 1.46+/-0.23 mL/min per g and remote 4.84+/-0.23 versus 4.71+/-0.47 mL/min per g, P=NS). Unexpectedly, animals receiving AdvFGF-5 demonstrated a 29% increase in LV mass over the 2-week period (P<0.05 versus untreated animals with hibernating myocardium and normal shams). Histological analysis confirmed profound myocyte cellular hypertrophy in AdvFGF-5 treated myocardium (19.9+/-0.32 versus 15.2+/-0.92 microm in untreated, P<0.001). Myocytes in the proliferative phase of the cell cycle (Ki-67 staining) increased 7-fold after AdvFGF-5 (2,904+/-405 versus 409+/-233 per 10(6) myocyte nuclei in untreated, P<0.05). Myocyte nuclei in the mitotic phase (phosphorylated histone H3 staining) also increased after AdvFGF-5 (127+/-24 versus 35+/-13 per 10(6) myocyte nuclei in untreated, P<0.05). Thus, rather than angiogenesis, stimulation of hypertrophy and reentry of a small number of myocytes into the mitotic phase of the cell cycle are responsible for the effects of AdvFGF-5 on function. Although additional mechanisms may contribute to the improvement in wall-thickening, overexpression of AdvFGF-5 may afford a way to restore function in hibernating myocardium and ameliorate heart failure in chronic ischemic cardiomyopathy.

Changes in NMDA receptor expression in auditory cortex after learning.

Extensive practice on auditory learning tasks dramatically alters the functional organization and response properties of neurons in the auditory cortex. The cellular mechanisms responsible for this auditory learning-induced cortical plasticity are unclear; however, changes in synaptic function involving NMDA receptors have been strongly implicated. To test this hypothesis, we measured the change in gene expression of NMDA receptors and associated proteins in the auditory cortex of adult rats trained to perform an auditory identification task. NMDA receptor 2A and 2B gene expression in auditory cortex decreased significantly as auditory discrimination improved whereas expression of Arc, an immediate early gene involved in memory stabilization, increased. These results suggest that changes in NMDA receptors 2A and 2B and Arc enhance synaptic plasticity, thereby facilitating experience-dependent cortical remodeling and auditory learning.