Expression of the mouse putative (pro)renin receptor (RR) was detected in all tissues and cell lines examined including human mesangial cells previously reported to be negative for RR. Mouse RR was also present during development from E9.5. Phylogenic studies revealed RR is highly conserved between species and likely to be important physiologically. The role of RR was investigated in a rat model in which (pro)renin triggers malignant hypertension (MH). TGR(<i>Cyp1al-Ren2) </i>animals carry a mouse renin gene under the control of the <i>Cyp1a1 </i>promoter inducible by dietary indole-3-carbinol. A rapid rise in blood pressure was accompanied by weight loss and polyuria. The animals exhibited microinfarctions, inflammatory cell infiltration and fibrinoid necrosis in the heart and distal tubule hyperplasia and thickening of intra-renal arterial wall without glomerulosclerosis, in the kidney. To investigate the possible role of RR, a prorenin decoy peptide was used to attempt to ameliorate the MH phenotype in TGR(<i>Cyp1a1-Ren2)</i> animals. This peptide which competes with prorenin for binding to RR, has been shown to improve vascular injuries in diabetic nephropathy. In TGR(<i>Cyp1a1-Ren2)</i>, however, no changes in the MH phenotype could be observed, except in the mesentery in which less severe fibrinoid necrosis developed. To complement work on RR, prorenin maturation and renin storage were studied during development. The data showed the complete absence of renin granules in mouse kidneys before birth. Low sodium diet and ACE inhibition triggered (pro)renin granules to be produced in the foetal kidney. Two ACE inhibitors differing in their ability to cross the placenta were used. The data suggest that fetal renin granule formation is under the control of both foetal and maternal RAS. The lack of evidence for regulation of RR in a model of high prorenin and malignant hypertension suggests that the function of this protein may need to be re-assessed.

The kinetics of the permeation of cardiac muscle cells by glucose and the effect of insulin on this process were examined in the isolated perfused rat heart. Previous attempts to determine the parameters of permeation by glucose are considered to be imprecise because endogenous insulin influenced experi¬ mental results, intracellular glucose was inaccurately measured and estimates of glucose utilisation were not associated with defined concentrations of extracellular or intracellular glucose. In principle the parameters of permeation can be deter¬ mined from a steady state relationship between glucose uptake and the intracellular and extracellular concentration of glucose. Alternatively they can be determined from the relationship between the uptake and the extracellular concentration of glucose. if the kinetics of glucose metabolism are taken into account. A novel apparatus for cardiac perfusion was therefore developed in which hearts were brought to a steady state of glucose permeation and utilisation. In the apparatus hearts were perfused with a small volume of perfusate under well controlled conditions. Glucose concentration in perfusates was estimated by rapid and accurate automated methods which met the requirements of the procedure. The utilisation of glucose was determined not only in the steady state but also throughout the approach to that state. The first method for the determination of the parameters of permeation was impracticable because accurate estimates of intracellular glucose could not be made with methods currentlyavailable. Consequently the kinetics of glucose utilisation were compared with the predictions of a mathematical model in which permeation was assumed to be a simple carrier mechanism and the phosphorylation of glucose to be an irreversible enzymecatalysed reaction. Agreement was found. Estimates made of the parameters of permeation in the presence and absence of insulin were in qualitative accord with previous work. The hormone increased the half saturation constant at least 3-fold and the maximum rate of permeation 5-fold. The estimates of the parameters of permeation in the presence of insulin may be inaccurate because in their determination the possibility of a concentration gradient in the extracellular glucose was ignored. However, the estimates provide a reason¬ able explanation of a phenomenon which was observed when the time-course of glucose utilisation was studied in the absence of exogenous insulin. A stimulation of utilisation, presumably by endogenous insulin, occurred consistently only at concentra¬ tions of glucose greater than ImM. It was concluded that the estimates are an improvement on previous values and that the apparatus is a useful addition to the methods available for the study of the metabolism of isolated hearts.

Protein Z (PZ) is a vitamin k-dependent plasma glycoprotein produced mainly in the liver, which circulates as a complex with PZ-dependent protease inhibitor. Human PZ has been reported as having both procoagulant and anticoagulant activity, but its anticoagulant role appears to be more physiologically relevant. PZ levels vary widely among healthy individuals (normal range of 0.6 to 5.7 mug/mL) with the average being higher in men than women. The aim of this study was to investigate the mechanisms by which PZ expression is controlled at the level of transcription, including whether hormonal and/or inflammatory signals modify expression and, therefore, possibly account, at least in part, for the wide normal range. Bioinformatic analysis enabled the identification of two age stable elements (ASEs) in the PZ gene promoter showing close homology to those found in two evolutionarily related proteins, human factor IX and protein C. These regions were found to be bound specifically by potentially the same or a similar protein; but the identity of the protein could not be confirmed as either PEA3 or Ets1, although transactivation data from HepG2 cells suggested PEA3 is important in controlling expression of endogenous PZ.Two further regions, proximal and distal to the PZ transcription start site, were identified by bioinformatic analysis for binding of hepatocyte nuclear factor 4 alpha (HNF4alpha), with the proximal site also reported by Sugawara and colleagues. Although both sites showed specific binding of protein only the protein binding the proximal site was confirmed as being HNF4alpha. The importance of HNF4alpha was further demonstrated by overexpression and siRNA knockdown producing a respective increase and decrease in PZ mRNA levels. Furthermore, the role of steroid hormones and inflammatory signals as potential modifiers of PZ transcription were also assessed in the HepG2 model system. This study has demonstrated, to varying degrees, the importance of tissue-specific factors (HNF4alpha), more ubiquitous factors (PEA3, Ets1), steroid hormones (oestrogen and glucocorticoid) and inflammatory pathways (bacterial LPS induction) on the regulation of PZ gene transcription.

No description available.

No description available.

No description available.

No description available.

Cellular senescence is often defined as an irreversible cell cycle arrest of mitotic cells, however post-mitotic cells, including adipocytes and neurones, have also been shown to display senescent-like characteristics, such as elevated SA-β-Gal activity and increased production of pro-inflammatory cytokines, in response to persistent DNA damage. Our group have shown that a persistent DDR can occur at telomeres independently of length. We investigated the possibility of telomere dysfunction being associated with senescence in a non-rapidly dividing cell type, which is not subject to repeated end-replication problem-associated telomere shortening. We show that telomere damage can be induced in cardiomyocyte cell lines with X-irradiation or oxidative stress in the absence of cell division, with live-cell imaging revealing the presence of persistent DNA damage foci. Endonuclease-mediated telomere-specific double-strand DNA breaks trigger a senescent-like phenotype in cardiomyocytes in vitro, including elevated SA-β-Gal activity, p21 expression, hypertrophy and decrease of proliferation marker Ki-67. We observed an age-dependent increase in telomere dysfunction in both murine and human cardiomyocytes, occurring independently of telomere length. Furthermore, murine cardiomyocytes in vivo are associated with numerous markers of senescence, such as p15, p16 and p21 elevation, along with increased TGF-β expression and increased prevalence of senescence-associated distension of satellites. Increased oxidative stress via MnSOD-/+, Catalase-/-, or MAO-A overexpression (resulting in excess H2O2 production), can drive telomere dysfunction in murine cardiomyocytes in vivo, which correlates with a decrease in heart function, both of which can be rescued with anti-oxidant supplementation. Finally, we show that rapamycin, a drug shown to increase lifespan and delay age-related diseases in numerous organisms, can attenuate the accumulation of TAF in murine cardiomyocytes in vivo, and is associated with a decrease in senescence markers. Our data provide evidence that telomere dysfunction occurs independently of length in cardiomyocytes, and is associated with a senescent-like phenotype.

Kinases comprise universal signalling cascades potentially pivotal to cardiac cell death pathways. Cell death is divided into several modes, chiefly apoptosis and necrosis. Both contribute to the pathophysiology of heart disease, with overlapping signalling pathways of caspase activation and apoptogen release from the mitochondria. This project focuses on the genetic validation of the stress-activated protein kinase, MAP4K4 / HGK, which functions as a control point integrating diverse signals for cardiomyocyte cell death. MAP4K4 and its reported target MAP3K7 / TAK1 appear to operate upstream of JNK in driving cardiac muscle cell death. MAP4K4 is activated in diseased mouse and human myocardium and in cultured rat cardiomyocytes subjected to death signals. Its overexpression in the rodent cardiomyocytes is sufficient to induce cardiomyocyte apoptosis, whereas knockdown of endogenous MAP4K4 using shRNA suppresses it. However, there is no direct or concrete evidence that MAP4K4 has functional importance in a human cardiac muscle cell. Given this unmet need, we deployed shRNAmir-based technology in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) to knockdown MAP4K4 and investigate inhibition of cardiac muscle cell death, in response to death-inducing agents (i.e. H2O2 and c2-ceramide). First, we characterized the purity of the proposed human system by cardiac-specific antibodies and confirmed the cells’ cardiac identity, with a purity of 95% for the most prevalent marker tested. Then, via a series of optimization efforts using assays for caspase-3 and caspase-8 activities, diminished mitochondrial membrane potential and loss of surface membrane integrity, we developed a short-term maintenance protocol for hiPSC-CMs entailing 2 days in defined (transition) medium prior to death induction. Using this method, the cells were refractory to serum-induced proliferation, thus providing us with conditions under which the cell death assays could be carried out without confounding effects on purity. Prior to phenotypic assessment of MAP4K4 knockdown in hiPSC-CMs, we validated the efficacy of multiple shRNAmirs in a heterologous system. Three potent shRNAmirs were selected for virus production and proven to inhibit MAP4K4 (approximately 60-70%) in hiPSC-CMs. We then defined the conditions for provoking cell death with H2O2 and c2-ceramide, and identified the biochemical responses to each of these complementary death signals. Cell death with various H2O2 concentrations at different times was characterized by diminished mitochondrial potential, decreased caspase-8 activity and lack of caspase-3 activation. Cell death with 20μM c2-ceramide was evident at 16h associated with increased caspase-3 activity. Suppression of MAP4K4 in hiPSC-CMs with the most potent shRNAmir resulted in a 2-fold reduction of death (p < 0.001) measured by DRAQ7 and 2-fold reduction (p < 0.05) of cells with diminished mitochondrial potential, compared to the non-silencing control (NSC) upon H2O2 treatment. Interestingly, the reduction of cell death was associated with increased caspase-8 activity. Thus, we prove that MAP4K4 is pro-apoptotic in human cardiac muscle cells, as its knockdown leads to salvage from cell death. This proof-of-concept study will serve as a technical and scientific springboard for later, “higher bandwidth” studies of the entire human kinome or other essential mediators of cardiomyocyte demise.

Vascular endothelial cells, the cells that line blood vessels, play roles both in the development of the circulatory system through providing blood vessel integrity, and its homeostasis through responding to changes in the local environment. During development, the acquisition of functionality of new blood vessels, some of which form from pre-existing ones through the process of angiogenesis, requires generation of endothelial lumens to carry the blood, during a process known as lumenogenesis. Throughout new blood vessel formation, endothelial cells go through coordinated and complex cell shape changes and polarity changes driven by Rho GTPase activity. Rho GTPases control intracellular actin dynamics and therefore affect all stages of new vessel growth. Precise control of Rho GTPase signalling is through a range of upstream activator and downstream effector proteins. Here we have investigated the role of two proteins involved in the antagonistic Rho/ Rac pathways during angiogenesis and lumen formation. Results presented in this thesis describe a role for the Rac guanine nucleotide exchange factor (GEF) DOCK4, and Cdc42 GEF DOCK9, in controlling lumen formation in an organotypic in vitro angiogenesis assay, through both common and distinct cellular mechanisms. The RhoA effector ROCK1/2 controls RhoA- mediated cytoskeletal remodelling and have been shown to control angiogenesis in tissue culture assays. Here we use in vivo knockout mouse model to show that ROCK2 controls actin- based filopodia formation and vessel integrity, as well as other cytoskeletal processes such as ciliogenesis. Further investigation showed that loss of ROCK2 results in aberrant lumen formation. We also highlight a new role for ROCK2 signalling in primary cilia growth and abundance in endothelial cells which may also impact on lumen formation. In summary, this work has identified novel molecular pathways that control lumen formation in endothelial cells. These results build on the current understanding of molecular mechanisms involved in lumen formation, and may also lead to better understanding of the function of ROCK, a potential therapeutic target in therapeutic angiogenesis strategies.