James E. Tomlinson

Using gene expression profiling to identify the molecular basis of the synergistic actions of hepatocyte growth factor and vascular endothelial growth factor in human endothelial cells

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are two potent endothelial mitogens with demonstrated angiogenic activities in animal models of therapeutic angiogenesis. Several recent studies suggest that these growth factors may act synergistically, although the mechanism of this interaction is not understood. Changes in the gene expression profile of human umbilical vein endothelial cells treated with HGF, VEGF or the combination of the two were analyzed with high‐density oligonucleotide arrays, representing approximately 22,000 genes. Notably, the genes significantly up‐ and downregulated by VEGF versus HGF exhibited very little overlap, indicating distinct signal transduction pathways. The combination of HGF and VEGF markedly increased the number of significantly up‐ and downregulated genes. At 4 h, the combination of the two growth factors induced a number of chemokine and cytokines and their receptors (IL‐8, IL‐6, IL‐11, CCR6, CXCR1,CXC1 and IL17RC), numerous genes involved in growth factor signal transduction (egr‐1, fosB, grb10, grb14,MAP2K3,MAP3K8, MAPKAP2,MPK3, DUSP4 and DUSP6), as well as a number of other growth factors (PDGFA, BMP2, Hb‐EGF, FGF16, heuregulin beta 1, c‐kit ligand, angiopoietin 2 and angiopoietin 4 and VEGFC). In addition, the VEGF receptors neuropilin‐1 and flt‐1 were also upregulated. At 24 h, a clear ‘cell cycle’ signature is noted, with the upregulated expression of various cell cycle control proteins and gene involved in the regulation of mitosis and mitotic spindle assembly. The receptor for HGF, c‐met, is also upregulated. These data are consistent with the hypothesis that the combination of HGF and VEGF results in the cooperative upregulation of a number of different molecular pathways leading to a more robust proliferative response, that is, growth factor(s), receptors, molecules involved in growth factor signal transduction, as well as, at later time points, upregulation of the necessary cellular proteins required for cells to escape cell cycle arrest and enter the cell cycle.

Gene program for cardiac cell survival induced by transient ischemia in conscious pigs

Therapy for ischemic heart disease has been directed traditionally at limiting cell necrosis. We determined by genome profiling whether ischemic myocardium can trigger a genetic program promoting cardiac cell survival, which would be a novel and potentially equally important mechanism of salvage. Although cardiac genomics is usually performed in rodents, we used a swine model of ischemia/reperfusion followed by ventricular dysfunction (stunning), which more closely resembles clinical conditions. Gene expression profiles were compared by subtractive hybridization between ischemic and normal tissue of the same hearts. About one-third (23/74) of the nuclear-encoded genes that were up-regulated in ischemic myocardium participate in survival mechanisms (inhibition of apoptosis, cytoprotection, cell growth, and stimulation of translation). The specificity of this response was confirmed by Northern blot and quantitative PCR. Unexpectedly, this program also included genes not previously described in cardiomyocytes. Up-regulation of survival genes was more profound in subendocardium over subepicardium, reflecting that this response in stunned myocardium was proportional to the severity of the ischemic insult. Thus, in a swine model that recapitulates human heart disease, nonlethal ischemia activates a genomic program of cell survival that relates to the time course of myocardial stunning and differs transmurally in relation to ischemic stress, which induced the stunning. Understanding the genes up-regulated during myocardial stunning, including those not previously described in the heart, and developing strategies that activate this program may open new avenues for therapy in ischemic heart disease.

Characterization of pDJA1, a cardiac-specific chaperone found by genomic profiling of the post-ischemic swine heart

BACKGROUND Previously, we showed by subtractive hybridization in a swine model of ischemia/reperfusion that an upregulation of genes participating in mechanisms of cell survival is a potential genomic mechanism to tilt the balance from necrosis to functional reversibility. METHODS AND RESULTS We present here the full-length sequencing and characterization of a novel gene that was found in this subtraction, encoding a cardiac-specific DnaJ-like co-chaperone that we call Pig DnaJ-like protein A1 (pDJA1). The expression of pDJA1 was found to be restricted to the heart, as opposed to skeletal muscle, liver, lung, kidney, aorta, stomach and spleen. Expression of pDJA1 is restricted to cardiac myocytes, as determined by in situ hybridization. The transcript is expressed more in the left ventricle than in the other cardiac chambers. Remarkably, expression of pDJA1 follows a transmural gradient in the left ventricle, with the highest level of expression in the subendocardium. Expression of pDJA1 slightly increased during an episode of ischemia, but increased by 4-fold during the following period of reperfusion. Adenovirus-mediated transduction of pDJA1 in isolated rat neonatal cardiac myocytes decreased by 65% the rate of apoptosis induced by staurosporine. CONCLUSION Therefore, pDJA1 is a novel heart-specific, ventricle-enriched cardioprotective co-chaperone, which participates in the program of cell survival that limits irreversible damage in post-ischemic myocardium.

Hydroxamate based inhibitors of adenylyl cyclase. Part 2: The effect of cyclic linkers on P-site binding

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Previous work in our group identified novel inhibitors which possess an adenine ring joined to a metal-coordinating hydroxamic acid through flexible linkers. Considering the spatial positioning of the metals with respect to the adenine binding site coupled with potentially favorable entropic factors, conformational restriction of the tether through a stereochemistry based SAR employing a rigid cyclic scaffold was explored.

Application of industrial scale genomics to discovery of therapeutic targets in heart failure.

In recent years intense activity in both academic and industrial sectors has provided a wealth of information on the human genome with an associated impressive increase in the number of novel gene sequences deposited in sequence data repositories and patent applications. This genomic industrial revolution has transformed the way in which drug target discovery is now approached. In this article we discuss how various differential gene expression (DGE) technologies are being utilized for cardiovascular disease (CVD) drug target discovery. Other approaches such as sequencing cDNA from cardiovascular derived tissues and cells coupled with bioinformatic sequence analysis are used with the aim of identifying novel gene sequences that may be exploited towards target discovery. Additional leverage from gene sequence information is obtained through identification of polymorphisms that may confer disease susceptibility and/or affect drug responsiveness. Pharmacogenomic studies are described wherein gene expression‐based techniques are used to evaluate drug response and/or efficacy. Industrial‐scale genomics supports and addresses not only novel target gene discovery but also the burgeoning issues in pharmaceutical and clinical cardiovascular medicine relative to polymorphic gene responses.