Andrew Lam

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.

The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-beta (TGF-beta) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-beta-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-beta-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-beta and BNP for 24 and 48 hours. TGF-beta, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-beta effects; 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-beta-regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNFalpha-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-beta-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.

Inhibitors of angiogenic factors are known to be upregulated, and their levels increase in the maternal circulation before the onset of preeclampsia. We reproduced a previously characterized model of preeclampsia by adenoviral overexpression of the soluble vascular endothelial growth factor (VEGF) receptor sFlt-1 (also referred to as sVEGFR-1) in pregnant and nonpregnant Sprague-Dawley rats. Animals were treated with VEGF121 at 0, 100, 200, or 400 microg/kg once or twice daily (n=8 per group; 64 total) and compared with normal control animals (n=4 per group) by examination of systolic blood pressure, urinary albumin and creatinine, renal histopathology, and glomerular gene expression profiling. sFlt-1 expression induced hypertension with proteinuria and glomerular endotheliosis and significant changes in gene expression. VEGF121 treatment alleviated these symptoms and reversed 125 of 268 sFlt-1-induced changes in gene expression. VEGF121 had beneficial effects in this rat model of preeclampsia without apparent harm to the fetus. Further study of VEGF121 as a potential therapeutic agent for preeclampsia is warranted.

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.