Osteopontin Expression in Cardiomyocytes Is Increased in Pediatric Patients With Sepsis or Pneumonia

Sepsis and pneumonia are major causes of death in the United States, and their pathophysiology includes infection with inflammation and immune dysfunction. Both sepsis and pneumonia cause cardiovascular dysfunction. The expression of Osteopontin (OPN) in cardiomyocytes of patients with sepsis or pneumonia, and its role the induced cardiac dysfunction have not been thoroughly investigated. OPN is a matricellular protein synthesized by multiple diseased tissues and cells including cardiomyocytes. Here, we studied the expression of OPN protein using immunofluorescence in human myocardial autopsy tissues from pediatric and mid age or elderly patients with sepsis and/or pneumonia. Fourteen human myocardial tissues from six pediatric patients and eight mid-age or elderly patients were studied. Immunofluorescence was used to investigate the expression of OPN in paraffin-embedded heart sections co-stained with the myocyte markers Actin Alpha 1 (ACTA1) and Myosin Light Chain 2 (MLC2). A quantitative analysis was performed to determine the number of ACTA1 and MLC2 positive cardiomyocytes that express OPN. The results showed that OPN expression was significantly increased in cardiomyocytes in the hearts from pediatric patients with sepsis and/or pneumonia (N = 3) relative to pediatric patients without sepsis/pneumonia (N = 3), or adult to elderly patients with sepsis/pneumonia (N = 5). Among the older septic hearts, higher levels of cardiomyocyte OPN expression was seen only in conjunction with severe coronary arterial occlusion. This is the first study to document increased OPN expression in cardiomyocytes of pediatric subjects with sepsis or pneumonia. Our findings highlight a potentially important role for OPN in sepsis- or pneumonia-mediated cardiac dysfunction in pediatric patients

Age-related molecular genetic changes of murine bone marrow mesenchymal stem cells

<p>Abstract</p> <p>Background</p> <p>Mesenchymal stem cells (MSC) are pluripotent cells, present in the bone marrow and other tissues that can differentiate into cells of all germ layers and may be involved in tissue maintenance and repair in adult organisms. Because of their plasticity and accessibility these cells are also prime candidates for regenerative medicine. The contribution of stem cell aging to organismal aging is under debate and one theory is that reparative processes deteriorate as a consequence of stem cell aging and/or decrease in number. Age has been linked with changes in osteogenic and adipogenic potential of MSCs.</p> <p>Results</p> <p>Here we report on changes in global gene expression of cultured MSCs isolated from the bone marrow of mice at ages 2, 8, and 26-months. Microarray analyses revealed significant changes in the expression of more than 8000 genes with stage-specific changes of multiple differentiation, cell cycle and growth factor genes. Key markers of adipogenesis including lipoprotein lipase, FABP4, and Itm2a displayed age-dependent declines. Expression of the master cell cycle regulators p53 and p21 and growth factors HGF and VEGF also declined significantly at 26 months. These changes were evident despite multiple cell divisions in vitro after bone marrow isolation.</p> <p>Conclusions</p> <p>The results suggest that MSCs are subject to molecular genetic changes during aging that are conserved during passage in culture. These changes may affect the physiological functions and the potential of autologous MSCs for stem cell therapy.</p

Dynamic Regulation of Vascular Myosin Light Chain (MYL9) with Injury and Aging

Aging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels.We studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p<0.05) in the smooth muscle cell layers. We confirmed this finding on an aging aortic microarray experiment available through NCBI's GEO database. We found that Myl9 was consistently upregulated with age in healthy rat aortas. To determine the arterial localization of Myl9 with age and injury, we performed immunohistochemistry for Myl9 in rat iliac arteries and found that in healthy and injured (30 days post injury) arteries, Myl9 expression increased with age in the endothelial layers.The consistent upregulation of the myosin light chain protein (Myl9) with age and injury in arterial tissue draws attention to the increased vascular permeability and to the age-caused predisposition to arterial constriction after balloon angioplasty

SRRM2, a Potential Blood Biomarker Revealing High Alternative Splicing in Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects about five million people worldwide. Diagnosis remains clinical, based on phenotypic patterns. The discovery of laboratory markers that will enhance diagnostic accuracy, allow pre-clinical detection and tracking of disease progression is critically needed. These biomarkers may include transcripts with different isoforms.We performed extensive analysis on 3 PD microarray experiments available through GEO and found that the RNA splicing gene SRRM2 (or SRm300), sereine/arginine repetitive matrix 2, was the only gene differentially upregulated among all the three PD experiments. SRRM2 expression was not changed in the blood of other neurological diseased patients versus the healthy controls. Using real-time PCR, we report that the shorter transcript of SRRM2 was 1.7 fold (p = 0.008) upregulated in the substantia nigra of PDs vs controls while the longer transcript was 0.4 downregulated in both the substantia nigra (p = 0.03) and amygdala (p = 0.003). To validate our results and test for the possibility of alternative splicing in PD, we performed independent microarray scans, using Affymetrix Exon_ST1 arrays, from peripheral blood of 28 individuals (17 PDs and 11 Ctrls) and found a significant upregulation of the upstream (5') exons of SRRM2 and a downregulation of the downstream exons, causing a total of 0.7 fold down regulation (p = 0.04) of the long isoform. In addition, we report novel information about hundreds of genes with significant alternative splicing (differential exonic expression) in PD blood versus controls.The consistent dysregulation of the RNA splicing factor SRRM2 in two different PD neuronal sources and in PD blood but not in blood of other neurologically diseased patients makes SRRM2 a strong candidate gene for PD and draws attention to the role of RNA splicing in the disease

A Genomic Pathway Approach to a Complex Disease: Axon Guidance and Parkinson Disease

While major inroads have been made in identifying the genetic causes of rare Mendelian disorders, little progress has been made in the discovery of common gene variations that predispose to complex diseases. The single gene variants that have been shown to associate reproducibly with complex diseases typically have small effect sizes or attributable risks. However, the joint actions of common gene variants within pathways may play a major role in predisposing to complex diseases (the paradigm of complex genetics). The goal of this study was to determine whether polymorphism in a candidate pathway (axon guidance) predisposed to a complex disease (Parkinson disease [PD]). We mined a whole-genome association dataset and identified single nucleotide polymorphisms (SNPs) that were within axon-guidance pathway genes. We then constructed models of axon-guidance pathway SNPs that predicted three outcomes: PD susceptibility (odds ratio = 90.8, p = 4.64 × 10−38), survival free of PD (hazards ratio = 19.0, p = 5.43 × 10−48), and PD age at onset (R2 = 0.68, p = 1.68 × 10−51). By contrast, models constructed from thousands of random selections of genomic SNPs predicted the three PD outcomes poorly. Mining of a second whole-genome association dataset and mining of an expression profiling dataset also supported a role for many axon-guidance pathway genes in PD. These findings could have important implications regarding the pathogenesis of PD. This genomic pathway approach may also offer insights into other complex diseases such as Alzheimer disease, diabetes mellitus, nicotine and alcohol dependence, and several cancers

A conceptual framework toward identifying and analyzing challenges to the advancement of pharmacy

Background: Pharmacists and healthcare professionals are faced with increasing and changing health care needs around the world. In order to meet these demands, they are required to continuously upgrade and develop their professions. Reprofessionalization drives are therefore crucial to the successful delivery of health services, but traditional theories of the professions provide little practical guidance to evaluating the overall status of a profession. Objective: This study proposes a new conceptual framework of three interrelated professional sectors: education, regulation and practice, and uses it to identify and analyse challenges facing the pharmacy profession in Jordan. Methods: A multiple-method qualitative study comprised of semi-structured interviews and focus groups was conducted in Amman, Jordan. To explore and identify the challenges, apurposively recruited cross-sector sample of fifty-three key informants, stakeholders and pharmacists were interviewed. Interview transcripts were translated and then analysed using QSR NVivo 10. Thematic analysis identified eight main challenges facing pharmacy in Jordan. The original participants were then invited to participate in focus groups, the purpose of which was to validate the interview findings, map them against the conceptual framework and discuss recommendations for development. Results:The eight validated challenges span the following areas: graduates preparedness for practice, pharmacy education accreditation and quality assurance, pre-registration requirements, workforce development, workforce planning, remuneration and wage rate, pharmacy assistants, and PharmD pharmacists. Focus group participants used the framework to map each of the challenges to the primary sector-to-sector disconnect that they perceived to explain it. A list of recommendations addressing each of the challenges was also devised. Conclusions: The framework was found to offer valuable insight as an explanatory and diagnostic tool in policy-relevant research. By emphasizing the processual and contextual nature of reprofessionalization, the framework presents an alternative approach to traditional theories. This study also raises important questions regarding the status of pharmacy in Jordan and aims to provide guidance for local development and much-needed reprofessionalization drives

Optimizing human post-mortem brain tissue gene expression profiling in Parkinson's disease and other neurodegenerative disorders: from target "fishing" to translational breakthroughs

Insights on the etiopathogenesis of common neurodegenerative disorders such as Parkinson's disease (PD) and Alzheimer's disease (AD) have been largely based on the discovery of gene mutations in genetically determined forms. Although these discoveries have been helpful in elucidating the basic molecular pathogenesis of familial forms, they represent a small fraction of cases, leaving the large majority classified as idiopathic. In the postgenomic era, brain tissue gene expression profiling has allowed relative quantitative assessment of thousands of genes simultaneously from one tissue sample, providing clues for novel candidate genes and processes implicated in neurodegenerative disorders. Some remain critical of "fishing expedition" science, but gene expression profiling is a discovery-based procedure well suited for the study of largely idiopathic and multifactorial diseases. However, the technology is still under development, and many methodological and biological aspects contribute to the heterogeneous results obtained from gene expression profiling. In this Review, we discuss the advantages and limitations of this technology in simple terms and identify the key variables that influence/limit gene expression profiling-derived translational breakthroughs in neurodegenerative diseases

Abstract 285: Osteopontin Deficiency Ameliorates Heart Failure with Preserved Ejection Fraction Pathology by Upregulating Mitochondrial 2-Oxoglutarate Dehydrogenase Like Enzyme

HFpEF is an increasingly prevalent syndrome associated with impaired myocardial energetics, for which no etiologic therapy is available. Osteopontin (OPN) is a matricellular protein that is upregulated in the circulation of HFpEF patients, and reported to induce mitochondrial stress in rodent cardiomyocytes. Here we evaluate the role of circulating OPN in regulating myocardial function in the nephrotic Col4a3 -/- mouse model of HFpEF. We performed extensive cardiac, biochemical and mitochondrial analyses of the Col4a3 -/- mouse and found a striking HFpEF phenotype. We showed OPN levels were elevated in Col4a3 -/- mice (FC=2.1, n=6; p<.01). Col4a3 -/- mice were hypertensive, had diastolic dysfunction, myocyte hypertrophy and interstitial fibrosis - all of which were ameliorated in Col4a3 -/- OPN -/- mice (n=5-20; p<.05). Col4a3 -/- hearts had dysmorphic mitochondria (EM), lowered antioxidant capacity as a 50% reduction in GSH/GSSG ratio (n=6; p<.05) and lower protein levels of mitochondrial respiratory complexes I, II and IV (p<.05). Flux assay in adult cardiomyocytes showed that maximal respiration was reduced in Col4a3 -/- hearts (575.84±37.6 vs 322.34±25.48 pmol/min in WT, n=9; p<.0001). Microarray data (validated by mitochondrial blot) implicated OGDHL as decreased in Col4a3 -/- hearts but increased in double knockout Col4a3 -/- OPN -/- hearts compared to WT (n=3; p<.05). OGDH activity was also lower in Col4a3 -/- hearts (17.1±7.3 vs 2.5±1.1 mU/mg in WT; n=6; p<.05). In Col4a3 -/- mice, heart-specific AAV9-mediated overexpression of OGDHL, similar to global OPN KO, improved survival by ~50-100% (p<.0001). Isovolumetric relaxation time, a marker of diastolic dysfunction, which is prolonged in Col4a3 -/- mice (26.17 vs 15.30±1 ms, n=26; p<.001) was decreased in Col4a3 -/- OPN -/- mice (18.1±1 ms, n=37; p<.01) as well as in AAV9-cTnT-OGDHL-treated Col4a3 -/- mice (16.7±2.5 ms, n=8; p<.05). In conclusion, we present a new mouse model for HFpEF in which diastolic function and lifespan can be improved by genetic deletion of OPN or cardiac OGDHL gene therapy. Our results elucidate for the first time the pivotal roles of circulating OPN and cardiac OGDHL in HFpEF pathophysiology and present two related potential therapeutic targets for HFpEF

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes