Conditional transgenic expression of fibroblast growth factor 9 in the adult mouse heart reduces heart failure mortality after myocardial infarction

BACKGROUND: Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. METHODS AND RESULTS: We used a tetracycline-responsive binary transgene system based on the α-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. CONCLUSIONS: Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI.

Fenofibrato (agonista do receptor ativador da proliferação peroxissomal) modula o sistema renina-angiotensina no coração de camundongos alimentados com dieta hiperlipídica

O objetivo deste trabalho foi estudar a ação do fenofibrato, um agonista do receptor ativador da proliferação peroxissomal alfa, no remodelamento cardíaco e na expressão de componentes do sistema renina-angiotensina (SRA) em um modelo de obesidade induzida por dieta. Camundongos machos C57Bl/6 com três meses de idade foram alimentados durante 11 semanas com dieta controle (grupo C, 3,57 kcal/g de dieta) ou dieta hiperlipídica (grupo HL, 5,40 kcal/g de dieta), em seguida foram separados em quatro grupos e estudados durante cinco semanas: C; HL; C-L (C mais fenofibrato) e HL-F (HL mais fenofibrato). Os animais HL foram mais pesados e apresentaram maior pressão arterial (PA) comparados aos animais C, mas HL-F foram mais leves e tiveram PA menor que HL. A resistência insulínica vista nos camundongos HL foi melhorada com fenofibrato nos camundongos HL-F. Fenofibrato reduziu colesterol total, triglicerídeos e aumentou HDL-c. Os animais HL apresentou um ventrículo esquerdo (VE) mais pesado e com espessura da parede maior, como também cardiomiócitos maiores e uma menor razão cardiomiócito/capilares que os animais C. Fenofibrato foi eficiente em melhorar estas alterações. As expressões cardíacas de Angiotensina II (ANG II) e de seu receptor tipo 1 (AT1R) foram maiores, enquanto que a expressão de seu receptor tipo 2 (AT2R) foi menor nos animais HL que nos animais C, e fenofibrato foi eficiente em atenuar estas diferenças. Como conclusão, a dieta HL lidera para a obesidade, elevação da PA, hipertrofia cardíaca, alterações metabólicas e expressão proteica alterada do SRA em camundongos, sugerindo a participação do SRA nestas alterações. Fenofibrato é eficiente em diminuir a PA e controlar a expressão proteica do SRA, assim como no tratamento da resistência insulínica e do remodelamento cardíaco adverso, diminuindo a hipertrofia dos cardiomiócitos e melhorando a vascularização do miocárdio, desta maneira, diminuindo importantes fatores de risco para doenças cardiovasculares

Tissue-transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats

Cell-based therapies and tissue engineering initiatives are gathering clinical momentum for next-generation treatment of tissue deficiencies. By using gravity-enforced self-assembly of monodispersed primary cells, we have produced adult and neonatal rat cardiomyocyte-based myocardial microtissues that could optionally be vascularized following coating with human umbilical vein endothelial cells (HUVECs). Within myocardial microtissues, individual cardiomyocytes showed native-like cell shape and structure, and established electrochemical coupling via intercalated disks. This resulted in the coordinated beating of microtissues, which was recorded by means of a multi-electrode complementary metal-oxide-semiconductor microchip. Myocardial microtissues (microm3 scale), coated with HUVECs and cast in a custom-shaped agarose mold, assembled to coherent macrotissues (mm3 scale), characterized by an extensive capillary network with typical vessel ultrastructures. Following implantation into chicken embryos, myocardial microtissues recruited the embryo's capillaries to functionally vascularize the rat-derived tissue implant. Similarly, transplantation of rat myocardial microtissues into the pericardium of adult rats resulted in time-dependent integration of myocardial microtissues and co-alignment of implanted and host cardiomyocytes within 7 days. Myocardial microtissues and custom-shaped macrotissues produced by cellular self-assembly exemplify the potential of artificial tissue implants for regenerative medicine.

Design of artificial myocardial microtissues

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1) a linear cell number-microtissue size correlation, (2) intermicrotissue superstructures, (3) retention of key cardiomyocyte-specific cell qualities, (4) a sophisticated extracellular matrix, and (5) a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6), myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.

The biological application of cobalt

This review collects and summarises the biological applications of the element cobalt. Small amounts of the ferromagnetic metal can be found in rock, soil, plants and animals, but is mainly obtained as a by-product of nickel and copper mining, and is separated from the ores (mainly cobaltite, erythrite, glaucodot and skutterudite) using a variety of methods. Compounds of cobalt include several oxides, including: green cobalt(II) (CoO), blue cobalt(II,III) (Co3O4), and black cobalt(III) (Co2O3); four halides including pink cobalt(II) fluoride (CoF2), blue cobalt(II) chloride (CoCl2), green cobalt(II) bromide (CoBr2), and blue-black cobalt(II) iodide (CoI2). The main application of cobalt is in its metal form in cobalt-based super alloys, though other uses include lithium cobalt oxide batteries, chemical reaction catalyst, pigments and colouring, and radioisotopes in medicine. It is known to mimic hypoxia on the cellular level by stabilizing the α subunit of hypoxia inducing factor (HIF), when chemically applied as cobalt chloride (CoCl2). This is seen in many biological research applications, where it has shown to promote angiogenesis, erythropoiesis and anaerobic metabolism through the transcriptional activation of genes such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO), contributing significantly to the pathophysiology of major categories of disease, such as myocardial, renal and cerebral ischaemia, high altitude related maladies and bone defects. As a necessary constituent for the formation of vitamin B12, it is essential to all animals, including humans, however excessive exposure can lead to tissue and cellular toxicity. Cobalt has been shown to provide promising potential in clinical applications, however further studies are necessary to clarify its role in hypoxia-responsive genes and the applications of cobalt-chloride treated tissues.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

Comparative effectiveness of population interventions to improve access to reperfusion for ST-segment–elevation myocardial infarction in Australia

Background Improving timely access to reperfusion is a major goal of ST-segment–elevation myocardial infarction care. We sought to compare the population impact of interventions proposed to improve timely access to reperfusion therapy in Australia. Methods and Results Australian hospitals, population, and road network data were integrated using Geographical Information Systems. Hospitals were classified into those that provided primary percutaneous coronary intervention (PPCI) or fibrinolysis. Population impact of interventions proposed to improve timely access to reperfusion (PPCI, fibrinolysis, or both) were modeled and compared. Timely access to reperfusion was defined as the proportion of the population capable of reaching a fibrinolysis facility ≤60 minutes or a PPCI facility ≤120 minutes from emergency medical services activation. The majority (93.2%) of the Australian population has timely access to reperfusion, mainly (53%) through fibrinolysis. Only 40.2% of the population had timely access to PPCI, and access to PPCI services is particularly limited in regional and nonexistent in remote areas. Optimizing the emergency medical services’ response or increasing PPCI services resulted in marginal improvement in timely access (1.8% and 3.7%, respectively). Direct transport to PPCI facilities and interhospital transfer for PPCI improves timely access to PPCI for 19.4% and 23.5% of the population, respectively. Prehospital fibrinolysis markedly improved access to timely reperfusion in regional and remote Australia. Conclusions Significant gaps in timely provision of reperfusion remain in Australia. Systematic implementation of changes in service delivery has potential to improve timely access to PPCI for a majority of the population and improve access to fibrinolysis to those living in regional and remote areas.

Predictors of physical and mental health-related quality of life outcomes among myocardial infarction patients

Background Health-related quality of life (HRQoL) is an important outcome for patients diagnosed with coronary heart disease. This report describes predictors of physical and mental HRQoL at six months post-hospitalisation for myocardial infarction. Methods Participants were myocardial infarction patients (n=430) admitted to two tertiary referral centres in Brisbane, Australia who completed a six month coronary heart disease secondary prevention trial (ProActive Heart). Outcome variables were HRQoL (Short Form-36) at six months, including a physical and mental summary score. Baseline predictors included demographics and clinical variables, health behaviours, and psychosocial variables. Stepwise forward multiple linear regression analyses were used to identify significant independent predictors of six month HRQoL. Results Physical HRQoL was lower in participants who: were older (p<0.001); were unemployed (p=0.03); had lower baseline physical and mental HRQoL scores (p<0.001); had lower confidence levels in meeting sufficient physical activity recommendations (p<0.001); had no intention to be physically active in the next six months (p<0.001); and were more sedentary (p=0.001). Mental HRQoL was lower in participants who: were younger (p=0.01); had lower baseline mental HRQoL (p<0.001); were more sedentary (p=0.01) were depressed (p<0.001); and had lower social support (p=0.001). Conclusions This study has clinical implications as identification of indicators of lower physical and mental HRQoL outcomes for myocardial infarction patients allows for targeted counselling or coronary heart disease secondary prevention efforts. Trial registration Australian Clinical Trials Registry, Australian New Zealand Clinical Trials Registry, CTRN12607000595415. Keywords: Myocardial infarction; Secondary prevention; Cardiac rehabilitation; Telephone-delivered; Health-related quality of life; Health coaching; Tele-health

Invading edge vs. inner (edvin) patterns of vascularization : an interplay between angiogenic and vascular survival factors defines the clinical behaviour of non-small cell lung cancer

Neo-angiogenesis during neoplastic growth involves endothelial mitogenic and migration stimuli produced by cancer or tumour stromal cells. Although this active angiogenesis takes place in the tumour periphery, the process of vessel growth and survival in inner areas and its clinical role remain largely unexplored. The present study compared the microvessel score (MS) as well as the single endothelial cell score (ECS) in the invading edge and in inner areas of non-small cell lung carcinomas (NSCLCs). Three different patterns of vascular growth were distinguished: the edvin (edge vs. inner) type 1, where a low MS was observed in both peripheral and inner tumour areas; the edvin type 2, where a high MS was noted in the invading front but a low MS in inner areas; and the edvin type 3, where both peripheral and inner tumour areas had a high MS. The ECS was high in the invading edge in edvin type 2 and 3 cases and was sharply decreased in both types in inner areas, suggesting that endothelial cell migration is unlikely to contribute to the angiogenic process in areas away from the tumour front. Expression of the vascular endothelial growth factor (VEGF) and of thymidine phosphorylase (TP) was associated with a high MS in the invading edge. VEGF was associated with a high MS in inner areas (edvin 3), while TP expression was associated with edvin type 2, showing that VEGF (and not TP) contributes to the preservation of the inner vasculature. Both edvin type 2 and 3 cases showed an increased incidence of node metastasis, but edvin type 3 cases had a poorer prognosis, even in the N1-stage group. The present study suggests that tumour factors regulating angiogenesis and vascular survival are not identical. A possible method is reported to quantify these two parameters by comparing the MS in the invading edge and inner areas (edvin types). This observation may contribute to the evaluation of the effectiveness of different therapeutic approaches, namely vascular targeting vs. anti-angiogenesis. Copyright (C) 2000 John Wiley and Sons, Ltd.

Percutaneous coronary intervention with drug-eluting stents or coronary artery bypass surgery in subjects with type 2 diabetes : evaluation of: Farkouth ME, Domanski M, Sleeper LA, et al. Strategies for multivessel revascularization in patients with diabetes. N Engl J Med 2012;367(25):2375-84, and Contini GA, Nicolini F, Fortuna D, et al. Five-year outcomes of surgical or percutaneous myocardial revascularization in diabetic patients. Int J Cardiol 2012. [Epub ahead of print]

There is debate as to whether percutaneous coronary intervention (PCI) with drug-eluting stents or coronary artery bypass surgery (CABG) is the best procedure for subjects with type 2 diabetes and coronary artery disease requiring revascularization. There is some evidence that following these procedures there is less further revascularization with CABG than PCI in subjects with diabetes. Two recent studies; the FREEDOM (Future Revascularization Evaluation in patients with Diabetes mellitus: Optimal Management of Multivessel Disease) trial, and a trial using a real world diabetic population from a Registry, have shown that the benefits of CABG over PCI in subjects with type 2 diabetes extends to lower rates of death and myocardial infarct, in addition to lower rates of revascularization. However, the rates of stroke may be higher with CABG than PCI with drug-eluting stents in this population. Thus, if CABG is going to be preferred to PCI in subjects with type 2 diabetes and multivessel coronary disease, consideration should be given to how to reduce the rates of stroke with CABG.

Comparison of high sensitivity troponin T and I assays in the diagnosis of non-ST elevation acute myocardial infarction in emergency patients with chest pain

Objectives: Concentrations of troponin measured with high sensitivity troponin assays are raised in a number of emergency department (ED) patients; however many are not diagnosed with acute myocardial infarction (AMI). Clinical comparisons between the early use (2 h after presentation) of high sensitivity cardiac troponin T (hs-cTnT) and I (hs-cTnI) assays for the diagnosis of AMI have not been reported. Design and methods: Early (0 h and 2 h) hs-cTnT and hs-cTnI assay results in 1571 ED patients with potential acute coronary syndrome (ACS) without ST elevation on electrocardiograph (ECG) were evaluated. The primary outcome was diagnosis of index MI adjudicated by cardiologists using the local cTnI assay results taken ≥6 h after presentation, ECGs and clinical information. Stored samples were later analysed with hs-cTnT and hs-cTnI assays. Results: The ROC analysis for AMI (204 patients; 13.0%) for hs-cTnT and hs-cTnI after 2 h was 0.95 (95% CI: 0.94–0.97) and 0.98 (95% CI: 0.97–0.99) respectively. The sensitivity, specificity, PLR, and NLR of hs-cTnT and hs-cTnI for AMI after 2 h were 94.1% (95% CI: 90.0–96.6) and 95.6% (95% CI: 91.8–97.7), 79.0% (95% CI: 76.8–81.1) and 92.5% (95% CI: 90.9–93.7), 4.48 (95% CI: 4.02–5.00) and 12.86 (95% CI: 10.51–15.31), and 0.07 (95% CI: 0.04–0.13) and 0.05 (95% CI:0.03–0.09) respectively. Conclusions: Exclusion of AMI 2 h after presentation in emergency patients with possible ACS can be achieved using hs-cTnT or hs-cTnI assays. Significant differences in specificity of these assays are relevant and if using the hs-cTnT assay, further clinical assessment in a larger proportion of patients would be required.