Deborah Damm

Altered Patterns of Gene Expression in Response to Myocardial Infarction

The use of cDNA microarrays has made it possible to simultaneously analyze gene expression for thousands of genes. Microarray technology was used to evaluate the expression of >4000 genes in a rat model of myocardial infarction. More than 200 genes were identified that showed differential expression in response to myocardial infarction. Gene expression changes were monitored from 2 to 16 weeks after infarction in 2 regions of the heart, the left ventricle free wall and interventricular septum. A novel clustering program was used to identify patterns of expression within this large set of data. Unique patterns were revealed within the transcriptional responses that illuminate changes in biological processes associated with myocardial infarction.

B-Type Natriuretic Peptide Exerts Broad Functional Opposition to Transforming Growth Factor-β in Primary Human Cardiac Fibroblasts. Fibrosis, Myofibroblast Conversion, Proliferation, and Inflammation

Abstract— The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-&bgr; (TGF-&bgr;) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-&bgr;–induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-&bgr;–induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-&bgr; and BNP for 24 and 48 hours. TGF-&bgr;, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-&bgr; effects; 88% and 85% of all TGF-&bgr;–regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-&bgr;–regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (&agr;-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNF &agr;-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate–dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-&bgr;–induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.

Inhibition of Transforming Growth Factor β Signaling Reduces Pancreatic Adenocarcinoma Growth and Invasiveness

Transforming growth factor β (TGFβ) is a pleiotropic factor that regulates cell proliferation, angiogenesis, metastasis, and immune suppression. Dysregulation of the TGFβ pathway in tumor cells often leads to resistance to the antiproliferative effects of TGFβ while supporting other cellular processes that promote tumor invasiveness and growth. In the present study, SD-208, a 2,4-disubstituted pteridine, ATP-competitive inhibitor of the TGFβ receptor I kinase (TGFβRI), was used to inhibit cellular activities and tumor progression of PANC-1, a human pancreatic tumor line. SD-208 blocked TGFβ-dependent Smad2 phosphorylation and expression of TGFβ-inducible proteins in cell culture. cDNA microarray analysis and functional gene clustering identified groups of TGFβ-regulated genes involved in metastasis, angiogenesis, cell proliferation, survival, and apoptosis. These gene responses were inhibited by SD-208. Using a Boyden chamber motility assay, we demonstrated that SD-208 inhibited TGFβ-stimulated invasion in vitro. An orthotopic xenograft mouse model revealed that SD-208 reduced primary tumor growth and decreased the incidence of metastasis in vivo. Our findings suggest mechanisms through which TGFβ signaling may promote tumor progression in pancreatic adenocarcinoma. Moreover, they suggest that inhibition of TGFβRI with a small-molecule inhibitor may be effective as a therapeutic approach to treat human pancreatic cancer.

Insulin-Like Growth Factor-Binding Protein-3 Induces Fetalization in Neonatal Rat Cardiomyocytes

We employed cDNA microarrays representing 4000 distinct sequences to profile changes in gene expression in a rodent model of heart disease, namely, progression to heart failure after myocardial infarction. Differential gene expression in the left ventricle was examined at 4-week intervals over a 12-week period after coronary artery ligation in rats. Over this time course, insulin-like growth factor-binding protein-3 (IGFBP-3) was found to have a greater expression than in nondiseased tissues. We then employed quantitative real-time PCR to analyze gene expression in neonatal rat cardiac myocytes that had been treated with recombinantly expressed IGFBP-3 to examine a number of transcriptional responses designed to reflect the heart failure phenotype. The IGFBP-3 protein was shown to induce transcription of atrial natriuretic factor (ANF) and beta-myosin heavy chain (B-MHC). Analysis of conditioned media taken from IGFBP-3-treated cardiac myocyte cultures demonstrated an increase in ANF protein as well as in protein synthesis, as determined by metabolic incorporation of a radiolabeled amino acid. However, transcriptional changes of troponin-1, endothelin-1, or angiotensin-II by IGFBP-3 were not observed.