Scott P. Kennedy

Overexpression of Eotaxin and the CCR3 Receptor in Human Atherosclerosis Using Genomic Technology to Identify a Potential Novel Pathway of Vascular Inflammation

BackgroundUnstable atherosclerotic lesions typically have an abundant inflammatory cell infiltrate, including activated T cells, macrophages, and mast cells, which may decrease plaque stability. The pathophysiology of inflammatory cell recruitment and activation in the human atheroma is incompletely described. Methods and ResultsWe hypothesized that differential gene expression with DNA microarray technology would identify new genes that may participate in vascular inflammation. RNA isolated from cultured human aortic smooth muscle cells treated with tumor necrosis factor-&agr; (TNF-&agr;) was examined with a DNA microarray with 8600 genes. This experiment and subsequent Northern analyses demonstrated marked increases in steady-state eotaxin mRNA (>20 fold), a chemokine initially described as a chemotactic factor for eosinophils. Because eosinophils are rarely present in human atherosclerosis, we then studied tissue samples from 7 normal and 14 atherosclerotic arteries. Immunohistochemical analysis demonstrated overexpression of eotaxin protein and its receptor, CCR3, in the human atheroma, with negligible expression in normal vessels. Eotaxin was predominantly located in smooth muscle cells. The CCR3 receptor was localized primarily to macrophage-rich regions as defined by immunopositivity for CD 68; a minority of mast cells also demonstrated immunopositivity for the CCR3 receptor. ConclusionsEotaxin and its receptor, CCR3, are overexpressed in human atherosclerosis, suggesting that eotaxin participates in vascular inflammation. These data demonstrate how genomic differential expression technology can identify novel genes that may participate in the stability of atherosclerotic lesions.

Transcriptional Profile of Mechanically Induced Genes in Human Vascular Smooth Muscle Cells

Vascular smooth muscle cells must monitor and respond to their mechanical environment; however, the molecular response of these cells to mechanical stimuli remains incompletely defined. By applying a highly uniform biaxial cyclic strain to cultured cells, we used DNA microarray technology to describe the transcriptional profile of mechanically induced genes in human aortic smooth muscle cells. We first identified vascular endothelial growth factor (VEGF) as a mechanically induced gene in these cells; VEGF served as a positive control for these experiments. We then used a DNA microarray with 5000 genes with putative functions to identify additional mechanically induced genes. Surprisingly, relatively few genes are mechanically induced in human aortic smooth muscle cells. Only 3 transcripts of 5000 were induced >2.5-fold: cyclooxygenase-1, tenascin-C, and plasminogen activator inhibitor-1. Downregulated transcripts included matrix metalloproteinase-1 and thrombomodulin. The transcriptional profile of mechanically induced genes in human aortic smooth muscle cells suggests a response of defense against excessive deformation. These data also demonstrate that in addition to identifying large clusters of genes that respond to a given stimulus, DNA microarray technology may be used to identify a small subset of genes that comprise a highly specific molecular response.

Selective adenosine A3 receptor stimulation reduces ischemic myocardial injury in the rabbit heart

OBJECTIVE The aim of this study was to determine whether selective activation of the adenosine A3 receptor reduces infarct size in a Langendorff model of myocardial ischemia-reperfusion injury. METHODS Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion. Infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). RESULTS Preconditioning by 5 min global ischemia and 10 min reperfusion reduced infarct size (IA/AAR) to 19 +/- 4% (controls: 67 +/- 5%). Replacing global ischemia with 5 min perfusion of the rabbit A3-selective agonist, IB-MECA (A3 Ki: 2 nM; A1 Ki: 30 nM) elicited a concentration-dependent reduction in infarct size; 50 nM IB-MECA reduced IA/AAR to 24 +/- 4%. The A1-selective agonist, R-PIA (25 nM) reduced IA/AAR to a similar extent (21 +/- 6%). However, while the cardioprotective effect of R-PIA was significantly inhibited (54 +/- 7% IA/AAR) by the rabbit A1-selective antagonist, BWA1433 (50 nM), the IB-MECA-dependent cardioprotection was unaffected (28 +/- 6% IA/AAR). A non-selective (A1 vs. A3) concentration of BWA1433 (5 microM) significantly attenuated the IB-MECA-dependent cardioprotection (61 +/- 7% IA/AAR). CONCLUSIONS These data clearly demonstrate that selective A3 receptor activation provides cardioprotection from ischemia-reperfusion injury in the rabbit heart. Furthermore, the degree of A3-dependent cardioprotection is similar to that provided by A1 receptor stimulation or ischemic preconditioning.

Induction of Tenascin-C in Cardiac Myocytes by Mechanical Deformation ROLE OF REACTIVE OXYGEN SPECIES

Mechanical overload may change cardiac structure through angiotensin II-dependent and angiotensin II-independent mechanisms. We investigated the effects of mechanical strain on the gene expression of tenascin-C, a prominent extracellular molecule in actively remodeling tissues, in neonatal rat cardiac myocytes. Mechanical strain induced tenascin-C mRNA (3.9 ± 0.5-fold, p < 0.01, n = 13) and tenascin-C protein in an amplitude-dependent manner but did not induce secreted protein acidic and rich in cysteine nor fibronectin. RNase protection assay demonstrated that mechanical strain induced all three alternatively spliced isoforms of tenascin-C. An angiotensin II receptor type 1 antagonist inhibited mechanical induction of brain natriuretic peptide but not tenascin-C. Antioxidants such as N-acetyl-l-cysteine, catalase, and 1,2-dihydroxy-benzene-3,5-disulfonate significantly inhibited induction of tenascin-C. Truncated tenascin-C promoter-reporter assays using dominant negative mutants of IκBα and IκB kinase β and electrophoretic mobility shift assays indicated that mechanical strain increases tenascin-C gene transcription by activating nuclear factor-κB through reactive oxygen species. Our findings demonstrate that mechanical strain induces tenascin-C in cardiac myocytes through a nuclear factor-κB-dependent and angiotensin II-independent mechanism. These data also suggest that reactive oxygen species may participate in mechanically induced left ventricular remodeling.

Discovery of zoniporide: A potent and selective sodium–hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility

Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule.

Extended half-life and elevated steady-state level of a single-chain Fv intrabody are critical for specific intracellular retargeting of its antigen, caspase-7

8 h) and high steady-state levels of protein accumulation, while the H2 intrabodies had a half-life of 2 h and less protein at steady state. These results suggest that the choice of sFv as an intrabody depends critically on the intracellular sFv protein having an extended half-life and elevated steady-state level. Thus, extended half-life must be considered together with sFv antibody specificity and affinity when choosing an optimal sFv intrabody for functional studies of cellular proteins.

Zoniporide: a potent and highly selective inhibitor of human Na(+)/H(+) exchanger-1.

We evaluated the in vitro pharmacological profile of a novel, potent and highly selective Na(+)/H(+) exchanger-1 (NHE-1) inhibitor, [1-(Quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine hydrochloride monohydrate (zoniporide or CP-597,396). The potency and selectivity of zoniporide were determined via inhibition of 22Na(+) uptake by PS-120 fibroblast cell lines overexpressing human NHE-1, -2 or rat NHE-3. Additionally, potency for endogenous NHE-1 was confirmed via ex vivo human platelet swelling assay (PSA), in which platelet swelling was induced by exposure to sodium propionate. The pharmacological profile of zoniporide was compared with that of eniporide and cariporide. Zoniporide inhibited 22Na(+) uptake in fibroblasts expressing human NHE-1 in a concentration-dependent manner (IC(50) = 14 nM) and was highly selective (157-fold and 15,700-fold vs. human NHE-2 and rat NHE-3, respectively). Zoniporide was 1.64- to 2.6-fold more potent at human NHE-1 than either eniporide or cariporide (IC(50) = 23 and 36 nM, respectively). Zoniporide was also more selective at inhibiting human NHE-1 vs. human NHE-2 than either eniporide or cariporide (157-fold selective compared with 27- and 49-fold, respectively). All three compounds inhibited human platelet swelling with IC(50) values in low nanomolar range. From these results, we conclude that zoniporide represents a novel, potent and highly selective NHE-1 inhibitor.

Delivery of tumor suppressor genes to reverse the malignant phenotype.

Despite early enthusiasm and excitement, the treatment of cancer via gene therapy is a long way from reaching fruition. The objective of this review is to describe the rationale as to why the delivery of genes encoding functional proteins whose activity has been lost during the initiation and development of cancer may be a feasible therapeutic option. In addition we will evaluate the limitations of the current delivery systems and discuss how these limitations have impacted upon the progress of gene therapy. Finally, we will describe and discuss the most recent attempts to deliver tumor suppressor genes to rodent models of human cancer and perhaps more importantly to human patients. As will become apparent during this review the excitement and enthusiasm for gene therapy remains high, however, this should not diminish the challenges that must be overcome before gene therapy becomes routine.