Nip21 gene expression reduces coxsackievirus B3 replication by promoting apoptotic cell death via a mitochondria-dependent pathway.

Our previous studies, using differential mRNA display, suggested that the mouse Nip21 gene may be involved in myocarditis development in the coxsackievirus B3 (CVB3)-infected mouse heart. Sequence comparison indicated that the mouse Nip21 gene shares high sequence homology to human Nip2. This human protein is known to interact with both the apoptosis inhibitor Bcl-2 and a homologous protein, the adenovirus E1B 19-kDa protein. Such interactions implicate Nip21 gene in cell death pathways. To study the function of this gene, we have cloned Nip21 from mouse hearts and established a Tet-On doxycycline-inducible HeLa cell line and a cardiomyocyte H9c2 cell line expressing Nip21 to characterize gene function in relation to apoptosis. We demonstrated that Nip21 expression could induce apoptosis via caspase-depended mitochondria activation. To further determine the function of Nip21 in CVB3-induced apoptosis, the Tet-On/Nip21 HeLa cell line was induced by doxycycline followed by CVB3 infection. We found that activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase occurred 2 hours earlier than in vector-transfected control cells, suggesting that Nip21 expression enhances CVB3-induced apoptosis. We also demonstrated a significant decrease in HeLa cell and H9c2 cell viability. Particularly, as illustrated by viral plaque assay, CVB3 replication was dramatically reduced in Tet-On HeLa cells, due at least in part to the earlier killing of the host cells by Nip21 overexpression.

Ultrastructural evidence of early endothelial damage in coronary arteries of rat cardiac allografts.

BACKGROUND: Events that occur early after transplantation, particularly immune recognition of allo-endothelium, initiate transplant vascular disease (TVD). Previous work suggests an important compromise of endothelial integrity as the allo-immune milieu evolves, although mechanisms by which integrity is altered remain unclear. Increased vascular permeability caused by endothelial damage may allow inflammatory cells, lipoproteins, other proteins, and plasma fluid to enter the sub-endothelial space, thereby contributing to the initiation of atherosclerosis. In this study, we examined endothelial integrity in coronary arteries and the proximal aorta after cardiac transplantation in rats. METHODS: We used Lewis-to-Lewis and Lewis-to-F344 rat heterotopic cardiac transplant models. We studied the effects of cyclosporine (5mg/kg/day) therapy compared with saline-treated controls. En face silver nitrate staining was performed to demonstrate endothelial cell borders and gaps. We used scanning electron microscopy to extend silver nitrate findings and to further define the presence and nature of endothelial disruptions. We used transmission electron microscopy to further characterize immune cell identity and interaction with endothelium. RESULTS: Syngrafts and cyclosporine-treated allografts showed normal-looking endothelium similar to that observed in arteries from native hearts. However, saline-treated allografts displayed progressive endothelial destruction, including large intercellular gaps, missing cells, and areas of bare extracellular matrix. Exfoliated surfaces were covered by platelets at various stages of adhesion, activation, and spreading. Similarly, we observed numerous leukocytes as either adherent to the endothelial lining or transmigrating into the sub-endothelial space. Cessation of cyclosporine therapy was associated with the development of similar abnormalities. CONCLUSIONS: Our findings indicate that, especially when immunosuppression is insufficient, early endothelial damage may promote vascular permeability and thereby initiate TVD.

Quantitative analysis of human heart valves: does anorexigen exposure produce a distinctive morphological lesion?

The need for more detail regarding the clinical and morphological features of human heart valves has become evident due to recent controversy regarding anorexigen-associated valvular dysfunction. In the present study, we used quantitative digital image analysis of geometric and compositional features to compare the histopathology of cardiac valves excised from patients treated with anorexigens as compared to normal, floppy, rheumatic and carcinoid valves. Anorexigen-exposed valves had the greatest number of onlays/valve (P<.0001), while rheumatic valves showed the greatest average onlay size and thickness of the comparison groups studied (P=.01). The valve onlays from anorexigen-exposed, carcinoid and floppy valves contained a greater percentage of glycosaminoglycans (GAGs) as compared to normal and rheumatic valves (P=.01). The anorexigen-exposed valve propers contained more GAGs than any other comparison group (P=.02). Vessels were prominent in both onlay and valve proper regions of carcinoid valves, in the anorexigen-exposed valve onlays and in rheumatic valve propers. Thus, the number of onlays, their size, the degree of GAG deposition, and the presence and location of vessels and leukocytes were important features distinguishing anorexigen-exposed valves from normal valves. Discriminant analyses, based on geometry, color composition or color composition, and vessel and leukocyte counts combined, were able to separate the valves into distinguishable groups. Our findings demonstrate that specific microscopic features can be used to separate anorexigen-associated heart valve lesions from normal valves and valve lesions associated with other pathologies, and suggest that a distinctive pathological process may exist in many anorexigen-exposed valves.

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.

Ubiquitin-dependent proteolysis of cyclin D1 is associated with coxsackievirus-induced cell growth arrest.

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 G(1) phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [(3)H]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 G(1) progression and G(1)/S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G(1) cyclin-dependent kinase activities, accompanied by decreased levels of G(1) 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 GSK3beta pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3beta independent. Taken together, our results demonstrate that CVB3 infection disrupts host cell homeostasis by blocking the cell cycle at the G(1)/S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1.

A shine-dalgarno-like sequence mediates in vitro ribosomal internal entry and subsequent scanning for translation initiation of coxsackievirus B3 RNA.

Translation initiation of coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. However, the details of ribosome-template recognition and subsequent translation initiation are still poorly understood. In this study, we have provided evidence to support the hypothesis that 40S ribosomal subunits bind to CVB3 RNA via basepairing with 18S rRNA in a manner analogous to that of the Shine-Dalgarno (S-D) sequence in prokaryotic systems. We also identified a new site within both the 18S rRNA and the polpyrimidine-tract sequence of the IRES that allows them to form stronger sequence complementation. All these data were obtained from in vitro translation experiments using mutant RNAs containing either an antisense IRES core sequence at the original position or site-directed mutations or deletions in the polypyrimidine tract of the IRES. The mutations significantly reduced translation efficiency but did not abolish protein synthesis, suggesting that the S-D-like sequence is essential, but not sufficient for ribosome binding. To determine how ribosomes reach the initiation codon after internal entry, we created additional mutants: when the authentic initiation codon at nucleotide (nt) 742 was mutated, a 180-nt downstream in-frame AUG codon at nt 922 is able to produce a truncated smaller protein. When this mutation was introduced into the full-length cDNA of CVB3, the derived viruses were still infectious. However, their infectivity was much weaker than that of the wild-type CVB3. In addition, when a stable stem-loop was inserted upstream of the initiation codon in the bicistronic RNA, translation was strongly inhibited. These data suggest that ribosomes reach the initiation codon from the IRES likely by scanning along the viral RNA.

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 alpha-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 alpha-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.

Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.

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.

Overexpression of interferon-gamma-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-gamma-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.

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.