Janet E. Wilson

ABCA1 mRNA and Protein Distribution Patterns Predict Multiple Different Roles and Levels of Regulation

Mutations in ABCA1 cause the allelic disorders familial hypolipoproteinemia and Tangier Disease. To identify where ABCA1 was likely to have a functional role, we determined the cellular and tissue-specific patterns of murine ABCA1 expression. RT-PCR and Western blot analysis on dissected murine tissues demonstrated broad expression of ABCA1 mRNA and protein in many tissues with prominent protein expression in liver, testis, and adrenal tissue. In situ hybridization and immunohistochemistry experiments demonstrated specific patterns of ABCA1 expression at the cellular level, with hepatocytes, the epithelial lining of the digestive system and bladder, the proximal convoluted tubule of the kidney, and Purkinje and cortical pyramidal neurons containing abundant ABCA1 protein. Significant discordance between relative mRNA and protein expression patterns suggests the possibility of post-transcriptional regulation of ABCA1 expression in selected cells or tissues. We also show that ABCA1 protein levels are up-regulated specifically in the liver after exposure to an atherogenic diet for 7 days, supporting a major role for the liver in dietary modulation of HDL-C levels. Our observations show that ABCA1 is expressed in a pattern consistent with its role in HDL-C metabolism. Additionally, ABCA1 may have important functional roles in other cell types independent of HDL-C regulation.

Coxsackievirus B3 Replication Is Reduced by Inhibition of the Extracellular Signal-Regulated Kinase (ERK) Signaling Pathway

ABSTRACT Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21 ras GTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.

Allograft Inflammatory Factor-1: A Cytokine-responsive Macrophage Molecule Expressed in Transplanted Human Hearts1,2

Allograft inflammatory factor-1 (AIF-1), a cytokine-responsive macrophage molecule, was originally identified and cloned from rat cardiac allografts with chronic cardiac rejection. We performed the present study to determine whether AIF-1 was also involved in the inflammatory response associated with human cardiac transplant rejection. AIF-1 gene transcripts were identified by the reverse-transcriptase polymerase chain reaction in endomyocardial biopsy specimens from human heart transplants and in macrophage cell lines. In the 441-base pair coding region of the human and rat cDNAs, the nucleotide sequences were 86% identical and the deduced amino acid sequences were 90% identical. Consistent with our studies in the rat. AIF-1 was selectively expressed in human macrophage-like cell lines, and immunostaining in human heart allografts localized the AIF-1 gene product to a subset of CD68+ macrophages in the interstitial and perivascular spaces. The parallels between rat and human AIF-1 expression suggest that AIF-1 may have a common effect on the function of activated macrophages in cardiac allografts.

Overexpression of Interferon-γ-inducible GTPase Inhibits Coxsackievirus B3-induced Apoptosis through the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway and Inhibition of Viral Replication

Our previous studies using differential mRNA display have shown that interferon-γ-inducible GTPase (IGTP), was up-regulated in coxsackievirus B3 (CVB3)-infected mouse hearts. In order to explore the effect of IGTP expression on CVB3-induced pathogenesis, we have established a doxycycline-inducible Tet-On HeLa cell line overexpressing IGTP and have analyzed activation of several signaling molecules that are involved in cell survival and death pathways. We found that following IGTP overexpression, protein kinase B/Akt was strongly activated through phosphorylation, which leads to phosphorylation of glycogen synthase kinase-3 (GSK-3). Furthermore, in the presence of CVB3 infection, the intensity of the phosphorylation of Akt was further enhanced and associated with a delayed activation of caspase-9 and caspase-3. These data indicate that IGTP expression appears to confer cell survival in CVB3-infected cells, which was confirmed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell viability assay. However, the ability of IGTP to induce phosphorylation of Akt and to promote cell survival was attenuated by the phosphotidylinositol-3 kinase (PI3-K) inhibitor LY294002. Transient transfection of the cells with a dominant negative Akt construct followed by doxycycline induction and CVB3 infection reversed Akt phosphorylation to basal levels and returned caspase-3 activity to levels similar to those when the PI3-K inhibitor LY294002 was added. Moreover, IGTP expression inhibited viral replication and delayed CVB3-induced cleavage of eukaryotic translation initiation factor 4G, indicating that IGTP-mediated cell survival relies on not only the activation of PI3-K/Akt, inactivation of GSK-3 and suppression of caspase-9 and caspase-3 but also the inhibition of viral replication.

Ubiquitin-Dependent Proteolysis of Cyclin D1 Is Associated with Coxsackievirus-Induced Cell Growth Arrest

ABSTRACT Coxsackievirus group B3 (CVB3) replication is influenced by host cell cycle status. However, the effect of CVB3 infection on cell cycle regulation and the mechanisms involved are not precisely defined. In this study, we examined cell cycle progression and regulation when the infection was initiated in late G1 phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [3H]thymidine uptake compared to that of sham-infected cells. To further clarify the effects of CVB3 on the host cell cycle, we examined the cell cycle regulatory proteins involved in G1 progression and G1/S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G1 cyclin-dependent kinase activities, accompanied by decreased levels of G1 cyclin protein expression (cyclin D1 and cyclin E). We further investigated the mechanisms by which CVB3 infection down-regulates cyclin D1 expression. Northern blotting showed that cyclin D1 mRNA levels were modestly increased following CVB3 infection, suggesting that cyclin D1 regulation occurs by a posttranscriptional mechanism. Viral infection resulted in only a 20 to 30% inhibition of cyclin D1 protein synthesis 3 h postinfection. However, the proteasome inhibitors MG132 and lactacystin prevent CVB3-induced cyclin D1 reduction, indicating that CVB3-induced down-regulation of cyclin D1 is facilitated by ubiquitin-proteasome proteolysis. Finally, using GSK3β pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3β independent. Taken together, our results demonstrate that CVB3 infection disrupts host cell homeostasis by blocking the cell cycle at the G1/S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1.

Correction of Hypertriglyceridemia and Impaired Fat Tolerance in Lipoprotein Lipase–Deficient Mice by Adenovirus-Mediated Expression of Human Lipoprotein Lipase

Humans homozygous or heterozygous for mutations in the lipoprotein lipase (LPL) gene demonstrate significant disturbances in plasma lipoproteins, including raised triglyceride (TG) and reduced HDL cholesterol levels. In this study we explored the feasibility of adenovirus-mediated gene replacement therapy for LPL deficiency. A total of 5 x 10(9) plaque-forming units (pfu) of an E1/E3-deleted adenovirus expressing either human LPL (Ad-LPL) or the bacterial beta-galactosidase gene (Ad-LacZ) as a control were administered to mice heterozygous for targeted disruption in the LPL gene (n = 57). Peak expression of total postheparin plasma LPL activity was observed at day 7 in Ad-LPL mice versus Ad-LacZ controls (834 +/- 133 vs 313 +/- 89 mU/mL, P < .01), and correlated with human-specific LPL activity (522 +/- 219 mU/mL) and mass (9214 +/- 782 ng/mL), a change that was significant to 14 and 42 days, respectively. At day 7, plasma TGs were significantly reduced relative to Ad-LacZ mice (0.17 +/- 0.07 vs 1.90 +/- 0.89 mmol/L, P < .01) but returned to endogenous levels by day 42. Ectopic liver expression of human LPL was confirmed by in situ hybridization analysis and from raised LPL activity and mass in liver homogenates. Analysis of plasma lipoprotein composition revealed a marked decrease in VLDL-derived TGs. Severely impaired oral and intravenous fat-load tolerance in LPL-deficient mice was subsequently corrected after Ad-LPL administration and closely paralleled that observed in wild-type mice. These findings suggest that liver-targeted adenovirus-mediated LPL gene transfer offers an effective means for transient correction of altered lipoprotein metabolism and impaired fat tolerance due to LPL deficiency.

Quantitative evaluation of inflammation in biopsy specimens from idiopathically failing or irritable hearts: Experience in 80 pediatric and adult patients

The histologic criteria for the diagnosis of myocarditis on an endomyocardial biopsy specimen are troubled by varying institutional criteria and interobserver differences. A comprehensive approach to tissue evaluation including quantitative assessment of mononuclear cell populations enhances the accuracy and the specificity of the morphologic diagnosis. Future efforts to characterize the infiltrating cell lines in inflammatory conditions of the heart will aid in the ultimate refinement of therapeutic efficacy.

Granzyme B in Atherosclerosis and Transplant Vascular Disease: Association with Cell Death and Atherosclerotic Disease Severity

Apoptosis of intimal cells is an important contributor to the pathogenesis of atherosclerosis and transplant vascular disease (TVD). Since the activated immune response may be a key regulator of apoptosis in these lesions, we used immunohistochemistry to characterize the presence and localization of granzyme B, a major mediator of the cytotoxic immune response, in advanced atherosclerosis and TVD. Formalin-fixed, paraffin-embedded transverse sections from human left anterior descending coronary arteries were cut serially and stained with antibodies specific for granzyme B, smooth muscle α-actin, CD68, and CD3. The amount of granzyme B staining was semi-quantitated on a 0–5+/5+ scale. Also, TUNEL staining and in situ hybridization was performed to visualize cells undergoing cellular damage suggestive of apoptosis, and to localize granzyme B mRNA, respectively. Granzyme B localization was similar in both diseases. This protease was absent in arteries with mild atherosclerosis, but was abundant in the intima and media of vessels with advanced atherosclerosis and TVD. Within the intima, granzyme B localized to TUNEL-positive foam cells surrounding lipid-rich atheromas. Staining of serial sections with granzyme B and either smooth muscle α-actin, anti-CD68, or anti-CD3 showed that granzyme B localized to smooth muscle cells, macrophages, and T-cells. Further, in situ hybridization for granzyme B mRNA in TVD cases localized its expression to infiltrating leukocytes and not foam cells. In conclusion, the presence of granzyme B in advanced atherosclerotic lesions and TVD is associated with increasing disease severity and cell death. These observations suggest that granzyme B-mediated apoptosis may contribute to the pathogenesis of these diseases.

The challenge of defining normality for human mitral and aortic valves: Geometrical and compositional analysis

Advances in digital imaging technology and in tools for obtaining detailed quantitation of morphological features have facilitated a new approach to pathological assessment of many tissues, including heart valves. In the present study, we quantitatively examined the tissue geometry and composition of structurally normal mitral and aortic valves removed at autopsy or surgery from patients aged 15-84 years. Through univariate analyses of quantitative variables, we have determined which features change distinctively with age. The anterior mitral valve leaflet (AMV) underwent a statistically significant decrease in area of the valve proper and an increase in the number of superficial tissue accumulations called onlays as the patients aged. For all geometric variables measured in the aortic valve, increases were seen with age, leading to a thicker valve, with enlargement of the valve proper and onlays, and with changes in the number of onlays. The mitral valve proper, composed largely of collagen in younger individuals, showed significant increases in glycosaminoglycans and elastin and a relative decrease in collagen with age. The compositional characteristics of the aortic valve proper were similar to those of the mitral valve, with a dramatic relative increase in elastin and a decrease in collagen with age. Valve onlays, when present, were similar in composition to the valve proper for both valves. Our findings regarding normal valve tissue composition, when taken in the context of geometrical features, and together with evidence of age-related changes in the relative amounts of specific constituents, provide a basis on which to analyze human heart valves affected by various known or putative diseases.

Viral Myocarditis: Identification of Five Differentially Expressed Genes in Coxsackievirus B3–Infected Mouse Heart

Differences in host susceptibility to viral myocarditis caused by a given strain of coxsackievirus B3 (CVB3) are known to be largely related to host genetic factors. Little is known, however, about the key genes that encode determinants (mediators) of myocarditis development or the nature of injury. To identify these genes and further understand the molecular mechanisms of the disease process, we have used a murine model and the differential display technique to fingerprint mRNAs from CVB3-infected mouse hearts. Total RNA was extracted from hearts of 4- and 10-week-old A/J(H-2(a)) mice at day 4 after CVB3 infection, and mRNAs were detected by reverse transcriptase-polymerase chain reaction and subsequently analyzed on polyacrylamide DNA sequencing gels. The differentially displayed bands were confirmed by Northern hybridization using the bands as cDNA probes. Twenty-eight upregulated or downregulated bands were selected from the sequencing gels; among these, 2 upregulated and 3 downregulated cDNA fragments were confirmed by Northern hybridization. DNA sequence analysis and GenBank searching have determined that 4 of the 5 candidate genes are homologous to genes encoding Mus musculus inducible GTPase, mouse mitochondrial hydrophobic peptide (a subunit of NADH dehydrogenase), mouse beta-globin, and Homo sapiens cAMP-regulated response element binding protein (CREB) binding protein (CBP), respectively. The remaining candidate gene matches an unpublished cDNA clone, M musculus Nip21 mRNA (GenBank accession number, AF035207), which is homologous to human Nip2, a Bcl-2 binding protein. Our data suggest preliminarily that both structural and nonstructural genes are involved in myocarditis development. For the structural gene, beta-globin, we further confirmed its downregulation at the protein level by measuring the mean cell volume of red blood cells and found it was marginally reduced in the CVB3-infected group (P<0.06), with no change in hemoglobin concentration. Cardiac myoglobin concentration was also measured and found to be decreased (P<0.005), with a parallel decrease in total soluble protein in the CVB3-infected mouse myocardium (P<0.01). We also noted that the ratio of myoglobin to total protein was not significantly changed; this may be due to the downregulation of additional genes in the host heart, a number being observed on the differential display gels. The significant downregulation of beta-globin major gene expression in the heart may be relevant to impaired cardiac function in both the early and late postinfection period. The other identified nonstructural genes are known to be involved in regulation of gene expression, signal transduction pathways, and apoptotic cell death. The altered expression of structural and nonstructural genes may play important roles in the mediation of myocarditis development and perhaps other pathological processes in the heart.