Eleanor Hilliard

Cardiac muscle ring finger-1 increases susceptibility to heart failure in vivo

Muscle ring finger (MuRF)1 is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates and heterodimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal, whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy, indicating cooperative control of muscle mass by MuRF1 and MuRF2. To identify the unique role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg+) hearts exhibited a nonprogressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by transaortic constriction (TAC) induced rapid failure of MuRF1 Tg+ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg+ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg+ mice did not differ from wild-type mice despite the depressed contractility following TAC. In comparing the level and activity of creatine kinase (CK) between wild-type and MuRF1 Tg+ hearts, we found that mCK and CK-M/B protein levels were unaffected in MuRF1 Tg+ hearts; however, total CK activity was significantly inhibited. We conclude that increased expression of cardiac MuRF1 results in a broad disruption of primary metabolic functions, including alterations in CK activity that leads to increased susceptibility to heart failure following TAC. This study demonstrates for the first time a role for MuRF1 in the regulation of cardiac energetics in vivo.

Comparative Effects of Paclitaxel and Rapamycin on Smooth Muscle Migration and Survival Role of Akt-Dependent Signaling

Objective—Advances in stent technology have enabled the delivery of drugs to improve outcomes after stent deployment. However, the optimal payloads for stents are not clear, and the appropriate stent-based therapies for high-risk patients, such as diabetics, have not been clearly established. Methods and Results—We used smooth muscle cell culture models to compare the activities of rapamycin and paclitaxel. Smooth muscle cells were grown in normal or high glucose to induce insulin resistance. Both paclitaxel and rapamycin activate mitogen-activated protein kinase pathways similarly. However, rapamycin potently activates AKT-dependent signaling, an effect that overrides the downregulation of this pathway by insulin resistance and that causes phosphorylation of the AKT-dependent transcription factor FOXO1. This effect is associated with attenuation of the anti-migratory effects of rapamycin under high glucose conditions that are not observed with paclitaxel, as well as with increased protection against ceramide-induced cytotoxicity, both of which are dependent on FOXO1 phosphorylation. Conclusions—Differences between the ability of rapamycin and paclitaxel to activate AKT may account for their differential cell survival and antichemotactic activities. These observations may provide a basis for understanding clinical differences between rapamycin- and paclitaxel-coated stents. The approaches used in these studies can be expanded to other candidate stent payloads as a method for triage in preclinical studies.

Secreted Frizzle-Related Protein 2 Stimulates Angiogenesis via a Calcineurin/NFAT Signaling Pathway

Secreted frizzle-related protein 2 (SFRP2), a modulator of Wnt signaling, has recently been found to be overexpressed in the vasculature of 85% of human breast tumors; however, its role in angiogenesis is unknown. We found that SFRP2 induced angiogenesis in the mouse Matrigel plug assay and the chick chorioallantoic membrane assay. SFRP2 inhibited hypoxia induced endothelial cell apoptosis, increased endothelial cell migration, and induced endothelial tube formation. The canonical Wnt pathway was not affected by SFRP2 in endothelial cells; however, a component of the noncanonical Wnt/Ca2+ pathway was affected by SFRP2 as shown by an increase in NFATc3 in the nuclear fraction of SFRP2-treated endothelial cells. Tacrolimus, a calcineurin inhibitor that inhibits dephosphorylation of NFAT, inhibited SFRP2-induced endothelial tube formation. Tacrolimus 3 mg/kg/d inhibited the growth of SVR angiosarcoma xenografts in mice by 46% (P = 0.04). In conclusion, SFRP2 is a novel stimulator of angiogenesis that stimulates angiogenesis via a calcineurin/NFAT pathway and may be a favorable target for the inhibition of angiogenesis in solid tumors.

The Role of Calcineurin/NFAT in SFRP2 Induced Angiogenesis—A Rationale for Breast Cancer Treatment with the Calcineurin Inhibitor Tacrolimus

Tacrolimus (FK506) is an immunosuppressive drug that binds to the immunophilin FKBPB12. The FK506-FKBP12 complex associates with calcineurin and inhibits its phosphatase activity, resulting in inhibition of nuclear translocation of nuclear factor of activated T-cells (NFAT). There is increasing data supporting a critical role of NFAT in mediating angiogenic responses stimulated by both vascular endothelial growth factor (VEGF) and a novel angiogenesis factor, secreted frizzled-related protein 2 (SFRP2). Since both VEGF and SFRP2 are expressed in breast carcinomas, we hypothesized that tacrolimus would inhibit breast carcinoma growth. Using IHC (IHC) with antibodies to FKBP12 on breast carcinomas we found that FKBP12 localizes to breast tumor vasculature. Treatment of MMTV-neu transgenic mice with tacrolimus (3 mg/kg i.p. daily) (n = 19) resulted in a 73% reduction in the growth rate for tacrolimus treated mice compared to control (n = 15), p = 0.003; which was associated with an 82% reduction in tumor microvascular density (p<0.001) by IHC. Tacrolimus (1 µM) inhibited SFRP2 induced endothelial tube formation by 71% (p = 0.005) and inhibited VEGF induced endothelial tube formation by 67% (p = 0.004). To show that NFATc3 is required for SFRP2 stimulated angiogenesis, NFATc3 was silenced with shRNA in endothelial cells. Sham transfected cells responded to SFRP2 stimulation in a tube formation assay with an increase in the number of branch points (p<0.003), however, cells transfected with shRNA to NFATc3 showed no increase in tube formation in response to SFRP2. This demonstrates that NFATc3 is required for SFRP2 induced tube formation, and tacrolimus inhibits angiogenesis in vitro and breast carcinoma growth in vivo. This provides a rationale for examining the therapeutic potential of tacrolimus at inhibiting breast carcinoma growth in humans.

Muscle ring finger 1 and muscle ring finger 2 are necessary but functionally redundant during developmental cardiac growth and regulate E2F1-mediated gene expression in vivo

Muscle ring finger (MuRF) proteins have been implicated in the transmission of mechanical forces to nuclear cell signaling pathways through their association with the sarcomere. We recently reported that MuRF1, but not MuRF2, regulates pathologic cardiac hypertrophy in vivo. This was surprising given that MuRF1 and MuRF2 interact with each other and many of the same sarcomeric proteins experimentally.

A Novel Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits Tumor Growth

Secreted frizzled-related protein 2 (SFRP2) is overexpressed in human angiosarcoma and breast cancer and stimulates angiogenesis via activation of the calcineurin/NFATc3 pathway. There are conflicting reports in the literature as to whether SFRP2 is an antagonist or agonist of β-catenin. The aims of these studies were to assess the effects of SFRP2 antagonism on tumor growth and Wnt-signaling and to evaluate whether SFRP2 is a viable therapeutic target. The antiangiogenic and antitumor properties of SFRP2 monoclonal antibody (mAb) were assessed using in vitro proliferation, migration, tube formation assays, and in vivo angiosarcoma and triple-negative breast cancer models. Wnt-signaling was assessed in endothelial and tumor cells treated with SFRP2 mAb using Western blotting. Pharmacokinetic and biodistribution data were generated in tumor-bearing and nontumor-bearing mice. SFRP2 mAb was shown to induce antitumor and antiangiogenic effects in vitro and inhibit activation of β-catenin and nuclear factor of activated T-cells c3 (NFATc3) in endothelial and tumor cells. Treatment of SVR angiosarcoma allografts in nude mice with the SFRP2 mAb decreased tumor volume by 58% compared with control (P = 0.004). Treatment of MDA-MB-231 breast carcinoma xenografts with SFRP2 mAb decreased tumor volume by 52% (P = 0.03) compared with control, whereas bevacizumab did not significantly reduce tumor volume. Pharmacokinetic studies show the antibody is long circulating in the blood and preferentially accumulates in SFRP2-positive tumors. In conclusion, antagonizing SFRP2 inhibits activation of β-catenin and NFATc3 in endothelial and tumor cells and is a novel therapeutic approach for inhibiting angiosarcoma and triple-negative breast cancer. Mol Cancer Ther; 12(5); 685–95. ©2013 AACR.

Stress-dependent Daxx-CHIP interaction suppresses the p53 apoptotic program.

Our previous studies have implicated CHIP (carboxyl terminus of Hsp70-interacting protein) as a co-chaperone/ubiquitin ligase whose activities yield protection against stress-induced apoptotic events. In this report, we demonstrate a stress-dependent interaction between CHIP and Daxx (death domain-associated protein). This interaction interferes with the stress-dependent association of HIPK2 with Daxx, blocking phosphorylation of serine 46 in p53 and inhibiting the p53-dependent apoptotic program. Microarray analysis confirmed suppression of the p53-dependent transcriptional portrait in CHIP+/+ but not in CHIP−/− heat shocked mouse embryonic fibroblasts. The interaction between CHIP and Daxx results in ubiquitination of Daxx, which is then partitioned to an insoluble compartment of the cell. In vitro ubiquitination of Daxx by CHIP revealed that ubiquitin chain formation utilizes non-canonical lysine linkages associated with resistance to proteasomal degradation. The ubiquitination of Daxx by CHIP utilizes lysines 630 and 631 and competes with the sumoylation machinery of the cell at these residues. These studies implicate CHIP as a stress-dependent regulator of Daxx that counters the pro-apoptotic influence of Daxx in the cell. By abrogating p53-dependent apoptotic pathways and by ubiquitination competitive with Daxx sumoylation, CHIP integrates the proteotoxic stress response of the cell with cell cycle pathways that influence cell survival.

Stable patterns of gene expression regulating carbohydrate metabolism determined by geographic ancestry

Background Individuals of African descent in the United States suffer disproportionately from diseases with a metabolic etiology (obesity, metabolic syndrome, and diabetes), and from the pathological consequences of these disorders (hypertension and cardiovascular disease). Methodology/Principal Findings Using a combination of genetic/genomic and bioinformatics approaches, we identified a large number of genes that were both differentially expressed between American subjects self-identified to be of either African or European ancestry and that also contained single nucleotide polymorphisms that distinguish distantly related ancestral populations. Several of these genes control the metabolism of simple carbohydrates and are direct targets for the SREBP1, a metabolic transcription factor also differentially expressed between our study populations. Conclusions/Significance These data support the concept of stable patterns of gene transcription unique to a geographic ancestral lineage. Differences in expression of several carbohydrate metabolism genes suggest both genetic and transcriptional mechanisms contribute to these patterns and may play a role in exacerbating the disproportionate levels of obesity, diabetes, and cardiovascular disease observed in Americans with African ancestry.

Tobacco use induces anti-apoptotic, proliferative patterns of gene expression in circulating leukocytes of Caucasian males

BackgroundStrong epidemiologic evidence correlates tobacco use with a variety of serious adverse health effects, but the biological mechanisms that produce these effects remain elusive.ResultsWe analyzed gene transcription data to identify expression spectra related to tobacco use in circulating leukocytes of 67 Caucasian male subjects. Levels of cotinine, a nicotine metabolite, were used as a surrogate marker for tobacco exposure. Significance Analysis of Microarray and Gene Set Analysis identified 109 genes in 16 gene sets whose transcription levels were differentially regulated by nicotine exposure. We subsequently analyzed this gene set by hyperclustering, a technique that allows the data to be clustered by both expression ratio and gene annotation (e.g. Gene Ontologies).ConclusionOur results demonstrate that tobacco use affects transcription of groups of genes that are involved in proliferation and apoptosis in circulating leukocytes. These transcriptional effects include a repertoire of transcriptional changes likely to increase the incidence of neoplasia through an altered expression of genes associated with transcription and signaling, interferon responses and repression of apoptotic pathways.

Clinical Evidence Supports a Protective Role for CXCL5 in Coronary Artery Disease

Our goal was to measure the association of CXCL5 and molecular phenotypes associated with coronary atherosclerosis severity in patients at least 65 years old. CXCL5 is classically defined as a proinflammatory chemokine, but its role in chronic inflammatory diseases, such as coronary atherosclerosis, is not well defined. We enrolled individuals who were at least 65 years old and undergoing diagnostic cardiac catheterization. Coronary artery disease (CAD) severity was quantified in each subject via coronary angiography by calculating a CAD score. Circulating CXCL5 levels were measured from plasma, and both DNA genotyping and mRNA expression levels in peripheral blood mononuclear cells were quantified via microarray gene chips. We observed a negative association of CXCL5 levels with CAD at an odds ratio (OR) of 0.46 (95% CI, 0.27-0.75). Controlling for covariates, including sex, statin use, hypertension, hyperlipidemia, obesity, self-reported race, smoking, and diabetes, the OR was not significantly affected [OR, 0.54 (95% CI, 0.31-0.96)], consistent with a protective role for CXCL5 in coronary atherosclerosis. We also identified 18 genomic regions with expression quantitative trait loci of genes correlated with both CAD severity and circulating CXCL5 levels. Our clinical findings are consistent with the emerging link between chemokines and atherosclerosis and suggest new therapeutic targets for CAD.