First report of tamoxifen-induced baboon syndrome.

BACKGROUND: Baboon syndrome is a rare, type IV hypersensitivity reaction causing a maculopapular rash. Tamoxifen is an antineoplastic agent, working as an estrogen receptor antagonist, also called a selective estrogen receptor modulator. A variety of rashes were reported with Tamoxifen use to-date except baboon syndrome. The Tamoxifen-induced baboon syndrome seems to be reversible, as discontinuation of the drug improves clinical outcomes. AIM: Herein, we present the first case of Tamoxifen-induced baboon syndrome which occurred 8 years after initiation of Tamoxifen use. PATIENTS: A 44-year-old woman presented with papulovesicular eruption on her body and erythema on her face for a duration of 6 months. There was no evidence of ocular or mucosal involvement. She was diagnosed with breast cancer and treated with tamoxifen 10 mg twice daily over the past 8 years. She was not taking other medications or over-the-counter supplements at the time of presentation. The patient underwent urgent skin biopsies of two lesions on her buttock and thigh. No organisms were seen on Gram stain. The patient's skin biopsy revealed extensive hyperorthokeratosis, minimal parakeratosis, hypergranulosis, and lichenoid interface dermatitis in the irregularly acanthotic epidermis supporting diagnosis of fixed drug eruption. Following a multidisciplinary discussion, the patient was diagnosed with baboon syndrome or symmetrical drug-related intertriginous and flexural exanthema (SDRIFE) associated with Tamoxifen. RESULTS: Hence, Tamoxifen was immediately discontinued and treated with oral steroid along with topical agents. She showed improvement of clinical abnormalities within days after discontinuation of Tamoxifen. CONCLUSIONS: Given the widespread use of Tamoxifen in the management of patients with breast cancer, it is important that healthcare professionals monitor for rare, however clinically significant, and potentially life-threatening dermatological manifestations of Tamoxifen use, such as baboon syndrome.

First report of concomitant pheochromocytoma and duodenal neuroendocrine tumour in a sporadic multiple endocrine neoplasia type 1.

A 77-year-old woman was diagnosed with pheochromocytoma followed by adrenalectomy at age 57. Hyperparathyroidism without osteoprosis was diagnosed at age 58. At age 75, Dual Energy X-ray Absoptiometry (DEXA) revealed osteoporosis and sestamibi scan showed a left parathyroid adenoma. Criteria for parathyroidectomy were met, and she underwent parathyroidectomy. Furthermore, she presented with haematochezia at age 75. An abdominal CT demonstrated a mass in the second portion of the duodenum. Additionally, octreoscan revealed somatostatin receptor positive tissue in the duodenum and Gallium 68 dotatate scan also showed a well-differentiated duodenal neuroendocrine tumour (NET). Genetic testing for MEN1, MEN2 and MEN4 was negative. Diagnosis of sporadic MEN1 syndrome was made. The patient underwent resection of the duodenal NET at age 76. She is in good health 21 years after her first presentation of MEN1. In summary, we present the first sporadic case of MEN1 with concomitant pheochromocytoma and duodenal NET which occurred 20 years apart.

Acute Cholestatic Liver Injury From Hydralazine Intake.

Hydralazine is a commonly used oral antihypertensive agent. We report a rare case of hydralazine-induced hepatotoxicity in the form of subacute hepatic necrosis. A 75-year-old African American woman presented with jaundice of 7-day duration. She was started on hydralazine 100 mg 3 times a day 10 weeks before presentation. On physical examination, scleral icterus was noted. Workup revealed elevated liver transaminases, alkaline phosphatase, and conjugated bilirubin. She had no history of liver disease, and liver function tests had been normal before starting hydralazine. Other etiologies, including viruses, common toxins, drugs, autoimmune, and copper-induced hepatitis, were excluded. Abdominal imaging studies did not show any evidence of intrahepatic or extrahepatic biliary ductal dilatation, and no pathologies were seen in the liver and pancreas. The patient's liver biopsy revealed extensive lobular hepatitis, significant necrosis, mixed inflammatory infiltrate, and no significant fibrosis, supporting a diagnosis of drug-induced liver injury. Hydralazine was immediately discontinued. She showed improvement of clinical and laboratory abnormalities within 5 days after discontinuation of hydralazine. To establish the diagnosis of hydralazine-induced liver injury, we used assessment tool outlined by the Council for International Organization of Medical Sciences (CIOMS) scale that led to "high probable" relationship. Although rare, clinically significant, and potentially life-threatening liver injury can result from use of hydralazine. Both clinical and histological presentations in our patient suggest acute liver injury. The hydralazine-induced hepatitis seems to be reversible as discontinuation of the drug improves clinical outcomes. We highly recommend monitoring of the liver function during hydralazine treatment.

A role for versican in the development of leiomyosarcoma.

Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an extracellular matrix proteoglycan, increases in LMS. Microarray analyses of 80 LMSs and 24 leiomyomas showed a significant elevated expression of versican in human LMS versus benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knock down versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the extracellular matrix macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and had reduced levels of mitosis as compared with controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.

Shared genetic factors for age at natural menopause in Iranian and European women.

STUDY QUESTION: Do differences in heritable genetic factors explain some of the difference in age at natural menopause (ANM) among populations? SUMMARY ANSWER: One single nucleotide polymorphism (SNP)-ANM association (rs16991615) detected in European women was replicated in Iranian women. WHAT IS KNOWN ALREADY: Genetics plays an important role in ANM, and well-powered genome-wide association studies (GWAS) of ANM performed in European women have discovered many statistically significant SNP-ANM associations. Average ANM varies by ethnicity, and population-specific differences in ANM-associated alleles may in part explain these differences. STUDY DESIGN, SIZE, DURATION: After quality control procedures, 97 SNPs were analyzed in genotype data of 828 Iranian women who experienced natural menopause. SNP genotyping data were used to perform linear regression analyses with ANM as a quantitative trait. Study participants were drawn from the population-based Tehran Lipid and Glucose Study based in Tehran, Iran. This study was performed between February 2009 and March 2012. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Based on an ANM-GWAS literature review, eight SNPs at four loci previously associated with ANM in European women were tested for replication in Iranian women. Linear regression analyses were performed including (n = 828) and excluding (n = 783) women who experience premature ovarian failure (ANM before 40 years of age). In addition, to search for novel population-specific ANM risk alleles, a pool-based GWAS was performed using this collection of Iranian women. Two DNA pools were constructed and compared: an 'early' ANM pool (lower 20(th) percentile of menopause ages, 40-45 years, n = 165) and a 'late' ANM pool (upper 20(th) percentile of menopause ages, 54-65 years, n = 187). Each DNA pool was assayed on four Illumina Human1M-Duo arrays, and allele-based tests of association were used to rank SNPs. One hundred and two highly ranked SNPs were chosen for individual genotyping by Sequenom MassARRAY and association analysis in the Iranian women. MAIN RESULTS AND THE ROLE OF CHANCE: One SNP-ANM association previously detected in European women was replicated in Iranian women (rs16991615; ß = 1.07, standard error (SE): 0.49, P = 0.02). SNPs at the previously reported 19q13.42 and 6p24.2 loci also approached statistical significance and had consistent SNP effects (magnitude and direction) in Iranian women (rs1172822; ß = -0.39, SE: 0.22, P = 0.08; and rs2153157, ß = 0.41, SE: 0.21, P = 0.05). We found little evidence for novel SNP-ANM associations in Iranian women; no SNP selected based on the pool-based GWAS achieved genome-wide significance. LIMITATIONS, REASONS FOR CAUTION: Due to small sample size this study was powered to reliably detect only moderate-to-large SNP effect sizes. This limited our ability to replicate many of the previously reported SNP-ANM risk alleles and to discover novel SNP-ANM associations' specific to the Iranian population. In performing our pool-based GWAS, a reduction in power was introduced relative to a conventional GWAS. WIDER IMPLICATIONS OF THE FINDINGS: Our results imply that European and Iranian women share ANM-associated genetic variants. Our study was underpowered but for all SNPs tested the direction of the effect was consistent with data from the European study. Therefore, we anticipate that many (if not all) of the ANM-associated SNPs discovered in European women will replicate in Iranian women upon genotyping a sufficient number of women. Our data do not support the hypothesis that population-specific SNP-ANM associations explain population-specific differences in the mean ANM.

Mapping of the Wnt/ß-catenin/TCF response elements in the human versican promoter.

Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and is considered to be crucial to several key cellular processes involved in development and disease. There is differential temporal and spatial expression of versican by multiple cell types and in different developmental and pathological timeframes. In order to fully appreciate the functional roles of versican as it relates to changing patterns of expression in development and disease, an in-depth knowledge of versican's biosynthetic processing is necessary. We have recently shown that ß-catenin/T-cell factor (TCF) complex formation at the versican promoter site is essential for activation of versican transcription. The transcriptional activator ß-catenin is the key mediator of the canonical Wnt signaling pathway. However, ß-catenin does not itself bind DNA and thus functions via interaction with TCF/Lymphoid-enhancing factor (LEF) transcription factors. These proteins contain a high-mobility group (HMG) box that binds DNA in a sequence-specific manner. Thus, in the case of active Wnt signaling, ß-catenin activates, in cooperation with proteins of the TCF/LEF family, the expression of a wide variety of target genes. The goal of this chapter is to describe the techniques used to elucidate the transcriptional control of versican by the ß-catenin/TCF response elements in its promoter site and to demonstrate how this signaling may be assayed experimentally. These approaches provide insight into the transcriptional regulation of the versican gene and provide the basis for the identification of novel Wnt/ß-catenin/TCF-regulated genes that are part of the signaling machinery regulating early embryogenesis, neoplasia, and cardiovascular remodeling.

Estimates of array and pool-construction variance for planning efficient DNA-pooling genome wide association studies.

BACKGROUND: Until recently, genome-wide association studies (GWAS) have been restricted to research groups with the budget necessary to genotype hundreds, if not thousands, of samples. Replacing individual genotyping with genotyping of DNA pools in Phase I of a GWAS has proven successful, and dramatically altered the financial feasibility of this approach. When conducting a pool-based GWAS, how well SNP allele frequency is estimated from a DNA pool will influence a study's power to detect associations. Here we address how to control the variance in allele frequency estimation when DNAs are pooled, and how to plan and conduct the most efficient well-powered pool-based GWAS. METHODS: By examining the variation in allele frequency estimation on SNP arrays between and within DNA pools we determine how array variance [var(e(array))] and pool-construction variance [var(e(construction))] contribute to the total variance of allele frequency estimation. This information is useful in deciding whether replicate arrays or replicate pools are most useful in reducing variance. Our analysis is based on 27 DNA pools ranging in size from 74 to 446 individual samples, genotyped on a collective total of 128 Illumina beadarrays: 24 1M-Single, 32 1M-Duo, and 72 660-Quad. RESULTS: For all three Illumina SNP array types our estimates of var(e(array)) were similar, between 3-4 × 10-4 for normalized data. Var(e(construction)) accounted for between 20-40% of pooling variance across 27 pools in normalized data. CONCLUSIONS: We conclude that relative to var(e(array)), var(e(construction)) is of less importance in reducing the variance in allele frequency estimation from DNA pools; however, our data suggests that on average it may be more important than previously thought. We have prepared a simple online tool, PoolingPlanner (available at http://www.kchew.ca/PoolingPlanner/), which calculates the effective sample size (ESS) of a DNA pool given a range of replicate array values. ESS can be used in a power calculator to perform pool-adjusted calculations. This allows one to quickly calculate the loss of power associated with a pooling experiment to make an informed decision on whether a pool-based GWAS is worth pursuing.

Vascular endothelial growth factor increases human cardiac microvascular endothelial cell permeability to low-density lipoproteins.

BACKGROUND: Endothelial cell hyperpermeability is a proposed mechanism of increased lipid insudation into the vessel walls of allografts. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability and its expression is upregulated in human heart allografts. The goal of these experiments was to investigate the effects of VEGF on low-density lipoprotein (LDL) permeability through confluent monolayers of human cardiac microvascular endothelial cells (HCMEC) in vitro. METHODS: VEGF mRNA and protein expression was characterized in coronary arteries from cardiac allograft vasculopathy patients as compared with healthy controls using in situ hybridization and immunohistochemical staining of sub-adjacent sections. HCMEC were grown to confluence and treated with VEGF-A(121) or VEGF-A(165). Permeability of LDL in confluent endothelial monolayers was measured using fluorometry. Transendothelial electrical resistance (TER) measurements were used to indirectly measure the tight junctional status. Immunocytochemical staining was performed to visualize changes in CD31 and zonula occludens-1. RESULTS: We observed significant increases in VEGF expression within the superficial and deep intima and media of coronaries from allografts, as compared with controls. In vitro treatment with VEGF-A(121) and VEGF-A(165) significantly increased LDL passage through endothelial monolayers. We further showed that VEGF-A(121) and VEGF-A(165) caused significant decreases in TER at 2 to 4 hours post-treatment. Also, VEGF induced disruption of tight junctions, resulting in an increase in the intercellular gap formation. CONCLUSIONS: These results demonstrate that VEGF increases low-density lipoprotein permeability through endothelial monolayers, and this effect is correlated with VEGF-induced disruption of endothelial tight junctions resulting in the formation of intercellular gaps.

ATP-binding cassette transporter A1 expression and apolipoprotein A-I binding are impaired in intima-type arterial smooth muscle cells.

BACKGROUND: Accumulation of excess cholesterol by intimal arterial smooth muscle cells (SMCs) contributes to the formation of foam cells in atherosclerotic lesions. The purpose of this study was to examine the expression and activity of ATP-binding cassette transporter A1 (ABCA1) in model intimal and medial arterial SMCs, in human atherosclerotic coronary artery intimal and medial layers, and in human intimal and medial SMCs. METHODS AND RESULTS: Model intimal arterial SMCs showed increased cholesteryl ester accumulation, absence of apolipoprotein A-I-mediated lipid efflux, markedly diminished ABCA1 expression, and poor apoA-I binding compared with medial-layer SMCs. Total ABCA1 mRNA and SMC-specific ABCA1 protein levels were diminished in the intimal layer compared with the medial layer of atherosclerotic human coronary arteries. Increased expression of ABCA1 by liver X receptor agonist treatment or gene transfection failed to correct apolipoprotein A-I binding, lipid efflux, or high-density lipoprotein particle formation by intima-type SMCs. In addition to impaired ABCA1 expression, intima-type SMCs appear to lack a critical binding factor or factors required for the apolipoprotein A-I-ABCA1 interaction, cholesterol efflux, and high-density lipoprotein particle formation. CONCLUSIONS: ABCA1 expression is reduced in cultured model intimal and human atherosclerotic lesion SMCs, suggesting that reduced ABCA1 activity contributes to smooth muscle foam cell formation in the intima.

Nonlinear association between serum testosterone levels and coronary artery disease in Iranian men.

Previous studies have shown controversial results about the role of androgens in coronary artery disease (CAD). We performed this study to examine and compare the relationship between androgenic hormones and CAD using conventional linear statistical techniques as well as novel non-linear approaches. The study was conducted on 502 consecutive men who were referred for selective coronary angiography at Tehran Heart Center due to different indications. We studied the relationship between androgenic hormones and CAD by using the generalized linear models, generalized additive models, and neural networks. Free testosterone (fT), total testosterone (tT) and dehydroepiandrosterone sulfate levels in patients with significant CAD versus normal individuals were 6.69 +/- 3.20 pg/ml, 16.60 +/- 6.66 nm/l, and 113.38 +/- 72.9 microg/dl versus 7.12 +/- 3.58 pg/ml, 15.82 +/- 7.26 nm/l, and 109.03 +/- 68.19 microg/dl, respectively (P > 0.05). The Generalized linear models was unable to show any significant relationship between androgenic hormones and CAD, while generalized additive model and neural networks supported the significant effect of androgenic hormones on CAD. This finding suggests a nonlinear association of tT levels with CAD: lower levels have a preventive effect on CAD, whereas higher values increase the risk of CAD. Emphasizing the non-linearity of the variables may provide new insight into the possible explanation of the effect of androgenic hormones on CAD.

Ablation of matrix metalloproteinase-9 increases severity of viral myocarditis in mice.

BACKGROUND: Coxsackievirus B3 (CVB3) causes human myocarditis, which can result in cardiac damage, maladaptive remodeling, and heart failure. Matrix metalloproteinases (MMP)-8 and -9 have been identified in virus-infected myocardium, but their particular roles and underlying mechanisms of effect are unknown. For the first time, we examine the severity of CVB3-induced myocarditis in MMP-8-and MMP-9-deficient mice. METHODS AND RESULTS: CVB3-infected MMP-8 and MMP-9 knockout (KO) mice and corresponding wild-type (WT) mice were euthanized and harvested at 9 days after infection. Expression of MMP-2, -8, -12, and -13 and tissue inhibitors of MMPs was assessed by zymography or immunoblotting on harvested hearts, and in situ hybridization was performed to detect active infection. Infected MMP-9 KO mice had greater myocardial injury and foci of infection than WT mice despite similar pancreatic infection. Increased fibrosis (10.6+/-2.7% versus 7.1+/-2.6%, P=0.04), viral titer, as well as decreased cardiac output, were evident in MMP-9 KO compared with WT mice as assessed by picrosirius red staining, plaque assay, and echocardiography, respectively. Immune infiltration was also greatly increased in MMP-9 KO compared with WT mice (15.2+/-12.6% versus 2.0+/-3.0%, P<0.002). Myocardial interferon-beta1, interferon-gamma, interleukin-6, interleukin-10, and macrophage inflammatory protein-1alpha expression was elevated in MMP-9 KO mice as measured by quantitative real-time polymerase chain reaction and ELISA. In contrast, MMP-8 KO mice had the same degree of cardiac injury, fibrosis, and viral infection as their WT counterparts. CONCLUSIONS: During acute CVB3 infection, MMP-9 appears necessary to halt virus propagation in the heart, promote proper immune infiltration and remodeling, and preserve cardiac output.

Coxsackievirus B3 activates nuclear factor kappa B transcription factor via a phosphatidylinositol-3 kinase/protein kinase B-dependent pathway to improve host cell viability.

Coxsackievirus B3 (CVB3) is the most common viral infectant of heart muscle. CVB3 directly injures cardiomyocytes. We have previously reported on a regulatory role for the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) pathway during CVB3 infection. Yet, the mechanism underlying this regulatory role has not been elucidated. The PI3K/Akt pathway is involved in various cellular processes and exerts its function through the activation of several downstream effectors. Among them, nuclear factor kappa B (NFkappaB) transcription factor is involved in inflammation, survival and apoptosis. In this study, we investigated the role of NFkappaB as a potential downstream mediator of signals through the PI3K/Akt cascade, in regulating CVB3-induced cellular injury. We report that CVB3 infection induces the translocation of NFkappaB into the nucleus of infected cells. Inhibition of the PI3K/Akt pathway markedly decreases virus-induced NFkappaB activation. Further, NFkappaB inhibition significantly suppresses host viability, suggesting a pro-survival role for NFkappaB. Short-term treatment of cells with tumour necrosis factor-alpha (TNF-alpha), a potent activator of NFkappaB, promotes host cell viability without affecting virus replication. However, a prolonged treatment has a detrimental effect on cells, indicating the existence of a delicate balance between the anti- and pro-apoptotic roles of TNF-alpha in the setting of CVB3 infection.

Androgen receptor regulation of the versican gene through an androgen response element in the proximal promoter.

Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.

Allograft vasculopathy versus atherosclerosis.

Over the last 4 decades, heart transplantation (HTx) has evolved as a mainstream therapy for heart failure. Approximately half of patients needing HTx have organ failure consequent to atherosclerosis. Despite advances in immunosuppressive drugs, long-term success of HTx is limited by the development of a particular type of coronary atherosclerosis, referred to as cardiac allograft vasculopathy (CAV). Although the exact pathogenesis of CAV remains to be established, there is strong evidence that CAV involves immunologic mechanisms operating in a milieu of nonimmunologic risk factors. The immunologic events constitute the principal initiating stimuli, resulting in endothelial injury and dysfunction, altered endothelial permeability, with consequent myointimal hyperplasia and extracellular matrix synthesis. Lipid accumulation in allograft arteries is prominent, with lipoprotein entrapment in the subendothelial tissue, through interactions with proteoglycans. The apparent endothelial "intactness" in human coronary arteries of the transplanted heart suggest that permeability and function of the endothelial barrier altered. Various insults to the vascular bed result in vascular smooth muscle cell (SMC) activation. Activated SMCs migrate from the media into the intima, proliferate, and elaborate cytokines and extracellular matrix proteins, resulting in luminal narrowing and impaired vascular function. Arteriosclerosis is a broad term that is used to encompass all diseases that lead to arterial hardening, including native atherosclerosis, postangioplasty restenosis, vein bypass graft occlusion, and CAV. These diseases exhibit many similarities; however, they are distinct from one another in numerous ways as well. The present review summarizes the current understanding of the risk factors and the pathophysiological similarities and differences between CAV and atherosclerosis.

Versican: signaling to transcriptional control pathways.

Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix, which provides a loose and hydrated matrix during key events in development and disease. Versican participates in cell adhesion, proliferation, migration, and angiogenesis, and hence plays a central role in tissue morphogenesis and maintenance. In addition, versican contributes to the development of a number of pathologic processes including atherosclerotic vascular diseases, cancer, tendon remodeling, hair follicle cycling, central nervous system injury, and neurite outgrowth. Versican is a complex molecule consisting of modular core protein domains and glycosaminoglycan side chains, and there are various steps of synthesis and processes regulating them. Also, there is differential temporal and spatial expression of versican by multiple cell types and in different developmental and pathological time frames. To fully appreciate the functional roles of versican as it relates to changing patterns of expression in development and disease, an in depth knowledge of versican's biosynthetic processing is necessary. The goal of this review is to evaluate the current status of our knowledge regarding the transcriptional control of versican gene regulation. We will be focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans-factors that have been characterized.

Novel role for integrin-linked kinase in modulation of coxsackievirus B3 replication and virus-induced cardiomyocyte injury.

Viral myocarditis is a major cause of sudden cardiac death in children and young adults. Among viruses, coxsackievirus B3 (CVB3) is the most common agent for myocarditis. Recently, more consideration has been given to the role of signaling pathways in pathogenesis of enteroviral myocarditis, providing new platform for identifying a new potential therapeutic target for this, so far, incurable disease. Previously, we reported on the role of the protein kinase-B/Akt in CVB3 replication and virus-induced cell injury. Here, we report on regulation of virus-induced Akt activation by the integrin-linked kinase in infected mouse cardiomyocytes and HeLa cells. This study also presents the first observation that inhibition of ILK in CVB3-infected cells significantly improves the viability of infected cells, while blocking viral replication and virus release. Complementary experiments using a constitutively active form of Akt1 revealed that the observed protective effect of ILK inhibition is dependent on the associated downregulation of virus-induced Akt activation. To our knowledge, this is the first report of such beneficial effects of ILK inhibition in a viral infection model and conveys new insights in our efforts to characterize a novel therapeutic target for treatment of enteroviral myocarditis.

Progress in heart transplantation.

The field of heart transplantation was built upon the discoveries of immunity and tolerance by Landsteiner, Medawar, Burnet, and others, as well as technical advancements in surgical technique by Carrel. Since the first successful human heart transplant performed by Christiaan Barnard in 1967, there has been substantial progress in the field of heart transplantation, especially over the last several decades. With advances in immunosuppression and surgical techniques, the rates of acute rejection and infection leading to graft failure have declined. However, the detection of acute and chronic allograft rejection remains one of the most important yet unsettled matters. As such, many new horizons exist for further advancement of the field of heart transplantation and for improving the outcomes of the patients we serve.

Detection of cardiac signaling in the injured and hypertrophied heart.

Cardiac hypertrophy is a compensatory response to a variety of physiological or pathological stimuli. However, prolonged hypertrophic responses may eventually lead to heart failure, arrhythmia, and sudden death. A number of intracellular signaling pathways have been implicated to play a critical role in the regulation of cardiac hypertrophy. In this chapter, the mitogen-activated protein kinase signaling pathway is used to illustrate conventional assays to detect the expression, phosphorylation, and activation of signaling proteins during cardiac hypertrophy, including Western blot, immunohistochemical staining, and immune complex kinase assays. Newly emerging techniques for analyzing cell signaling are also discussed in this chapter. Identifying and characterizing the expression and activation of these signaling proteins will provide important insights into the mechanisms that regulate hypertrophic cell growth and assist in development of new therapeutic approaches to limit cardiac hypertrophy.

Regulation of the versican promoter by the beta-catenin-T-cell factor complex in vascular smooth muscle cells.

The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3beta, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3beta activity by LiCl augmented accumulation of beta-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. Beta-catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of beta-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and beta-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of beta-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3beta pathway via the beta-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events.

Transplant vascular disease: role of lipids and proteoglycans.

The long-term success of cardiac allograft transplantation is limited by the development of a particular type of coronary atherosclerosis referred to as transplant vascular disease (TVD). Although the exact pathogenesis of TVD remains to be established, there is growing evidence that TVD involves immunological mechanisms operating in a milieu of nonimmunological risk factors. These immunological events constitute the principal initiating stimuli, resulting in endothelial injury with consequent myointimal hyperplasia, extracellular matrix synthesis and invocation of proteoglycan (PG)-lipoprotein interactions, leading, ultimately, to lipid retention in the vessel wall. The profound early 'insudation' of apolipoproteins along with uncertain endothelial 'intactness' in human coronary arteries in the transplanted heart, suggest that permeability of these vessel walls must be altered. Further, frequent and typically diffuse intracellular and extracellular accumulation of lipids and PGs in both the intimal and medial layers of cardiac allograft arteries has affirmed that the alloimmune environment accompanied with aberrant expression of extracellular matrix components, especially PGs, may strongly promote lipid imbibition in the allograft vascular bed, leading to TVD. In summary, the cumulative data support the view that profound lipid accumulation occurs in allograft arteries beginning very early post-transplantation, contributing to intimal thickening; that lipoproteins enter and are trapped in the subendothelial tissue, apparently through interactions with PGs; that with direct glycosaminoglycans, apolipoprotein interactions may occur, or they may occur through bridging molecules like phospholipase A2 and lipoprotein lipase; and that prolonged residence in the intima leads to lipoprotein modification, with subsequent modulation of biological processes that promote atherogenesis.