Prorenin and the heart : the Mannose 6-phosphate connection

The knowledge concerning the formation of angiotensins at cardiac tissue sites in relation to the presence and origin of cardiac renin, angiotensinogen and ACE is evaluated in chapter 2. To gain insight in the functional importance of locally generated angiotensin 11, the response of human forearm blood flow to infusion of either angiotensin I or angiotensin 11 was investigated (Chapter 3). To extend our results in the perfused isolated rat heart,31 experiments were performed to detect de novo synthesis of RAS components by neonatal rat cardiomyocytes and -fibroblasts under basal conditions and after stretch (Chapter 4). In addition, we characterized the binding and activation of human recombinant prorenin via mannose 6- phosphate/IGF11 receptors on the surface of human endothelial cells, and neonatal rat cardiomyocytes and -fibroblasts (Chapters 5 and 6). To validate our results obtained with human recombinant prorenin, neonatal rat cardiomyocytes were also incubated with human (pro)renin- containing body fluids (Chapter 7). The latter studies also addressed the importance of soluble mannose 6-phosphate/IGF11 receptors. Finally, since 1) under certain conditions man nose 6-phosphate/IGF11 receptor activation initiates transcellu\ar signaling pathways,'2 and 2) renin binding to glomerular mesangial cells leads to plasminogen activator inhibitor type-1 release and an increase in 3H-thymidine incorporation,25 we investigated whether prorenin binding and/or uptake by rat cardiomyocytes, in the presence or absence of angiotensinogen, resulted in a cellular response (Chapter 8). In these latter studies we also investigated intra- and extracellular angiotensin 11 generation and compared the effects of prorenin with those obtained with angiotensin II in parallel experiments

Stability of Life-Satisfaction Over Time: analysis of change in ranks in a national population

This paper is about constancy of differences in life-satisfaction in society. It analyzes data of a large panel study in Germany, which involved yearly interviews between 1984 and 1994. Year-to-year correlation started at +.45 and increased gradually to +.54. The correlation between the first and later reports declined through the years, the correlation between the 1st and the 11th report was only +.29. Observed overtime correlation may result from six effects: Firstly the correlation is attenuated by error: 1) common measurement error, such as haphazard responding, and 2) error in estimating general satisfaction due to passing uplifts and hassles. Both errors may shrink as 3) respondents get experienced in answering questions about life-satisfaction and 4) when they mature. Next, 'true' correlation will depend on: 5) mayor changes in life, such as loss of job or getting married, and 6) stable stocks, such as personal capabilities and social relations. This paper develops models to disentangle these effects. The best fitting model suggests that almost half of the initial variance in life-satisfaction was due to error: 23% to error in responding (effect 1) and 19% to error in estimating one's satisfaction with life (effect 2). In 10 years the error component shrinks by 10%, largely due to learning effect 3) and partly due to aging (effect 4). In the end, life-changes explained 30% of the varianc

Functional importance of angiotensin-converting enzyme-dependent in situ angiotensin II generation in the human forearm

To assess the importance for vasoconstriction of in situ angiotensin (Ang) II generation, as opposed to Ang II delivery via the circulation, we determined forearm vasoconstriction in response to Ang I (0.1 to 10 ng. kg(-1). min(-1)) and Ang II (0.1 to 5 ng. kg(-1). min(-1)) in 14 normotensive male volunteers (age 18 to 67 years). Changes in forearm blood flow (FBF) were registered with venous occlusion plethysmography. Arterial and venous blood samples were collected under steady-state conditions to quantify forearm fractional Ang I-to-II conversion. Ang I and II exerted the same maximal effect (mean+/-SEM 71+/-4% and 75+/-4% decrease in FBF, respectively), with similar potencies (mean EC(50) [range] 5.6 [0.30 to 12.0] nmol/L for Ang I and 3.6 [0.37 to 7.1] nmol/L for Ang II). Forearm fractional Ang I-to-II conversion was 36% (range 18% to 57%). The angiotensin-converting enzyme (ACE) inhibitor enalaprilat (80 ng. kg(-1). min(-1)) inhibited the contra

Prorenin-induced myocyte proliferation: no role for intracellular angiotensin II

Cardiomyocytes bind, internalize, and activate prorenin, the inactive precursor of renin, via a mannose 6-phosphate receptor (M6PR)--dependent mechanism. M6PRs couple directly to G-proteins. To investigate whether prorenin binding to cardiomyocytes elicits a response, and if so, whether this response depends on angiotensin (Ang) II, we incubated neonatal rat cardiomyocytes with 2 nmol/L prorenin and/or 150 nmol/L angiotensinogen, with or without 10 mmol/L M6P, 1 micromol/L eprosartan, or 1 micromol/

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin

Prorenin accumulation and activation in human endothelial cells: importance of mannose 6-phosphate receptors

ACE inhibitors improve endothelial dysfunction, possibly by blocking endothelial angiotensin production. Prorenin, through its binding and activation by endothelial mannose 6-phosphate (M6P) receptors, may contribute to this production. Here, we investigated this possibility as well as prorenin activation kinetics, the nature of the prorenin-activating enzyme, and M6P receptor-independent prorenin binding. Human umbilical vein endothelial cells (HUVECs) were incubated with wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala, thereby preventing cleavage by known proteases), M6P-free prorenin, and nonglycosylated prorenin, with or without M6P, protease inhibitors, or angiotensinogen. HUVECs bound only M6P-containing prorenin (K(d) 0.9+/-0.1 nmol/L, maximum number of binding sites [B(max)] 1010+/-50 receptors/cell). At 37 degrees C, because of M6P receptor recycling, the amount of prorenin internalized via M6P receptors was >25 times B(max). Inside the cells, wild-type and K/A-2 prorenin were proteolytically activated to renin. Renin was subsequently degraded. Protease inhibitors interfered with the latter but not with prorenin activation, thereby indicating that the activating enzyme is different from any of the known prorenin-activating enzymes. Incubation with angiotensinogen did not lead to endothelial angiotensin generation, inasmuch as HUVECs were unable to internalize angiotensinogen. Most likely, therefore, in the absence of angiotensinogen synthesis or endocytosis, M6P receptor-mediated prorenin internalization by endothelial cells represents prorenin clearance

Inhibition of dendritic cell activation and modulation of T cell polarization by the platelet secretome

Platelet transfusions are a frequently administered therapy for especially hemato-oncological patients with thrombocytopenia. Next to their primary function in hemostasis, currently there is increased attention for the capacity of platelets to affect the function of various cells of the immune system. Here, we investigate the capacity of platelets to immuno-modulate monocyte-derived dendritic cells (moDC) as well as primary dendritic cells and effects on subsequent T cell responses. Platelets significantly inhibited pro-inflammatory (IL-12, IL-6, TNF alpha) and increased anti-inflammatory (IL-10) cytokine production of moDCs primed with toll-like receptor (TLR)-dependent and TLR-independent stimuli. Transwell assays and ultracentrifugation revealed that a soluble factor secreted by platelets, but not microvesicles, inhibited DC activation. Interestingly, platelet-derived soluble mediators also inhibited cytokine production by human ex vivo stimulated myeloid CD1c+ conventional DC2. Moreover, platelets and platelet-derived soluble mediators inhibited T cell priming and T helper differentiation toward an IFN gamma+ Th1 phenotype by moDCs. Overall, these results show that platelets are able to inhibit the pro-inflammatory properties of DCs, and may even induce an anti-inflammatory DC phenotype, with decreased T cell priming capacity by the DC. The results of this study provide more insight in the potential role of platelets in immune modulation, especially in the context of platelet transfusions.Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Storage-Induced Platelet Apoptosis Is a Potential Risk Factor for Alloimmunization Upon Platelet Transfusion

Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Determinants of human adipose tissue gene expression: impact of diet, sex, metabolic status, and cis genetic regulation

Weight control diets favorably affect parameters of the metabolic syndrome and delay the onset of diabetic complications. The adaptations occurring in adipose tissue (AT) are likely to have a profound impact on the whole body response as AT is a key target of dietary intervention. Identification of environmental and individual factors controlling AT adaptation is therefore essential. Here, expression of 271 transcripts, selected for regulation according to obesity and weight changes, was determined in 515 individuals before, after 8-week low-calorie diet-induced weight loss, and after 26-week ad libitum weight maintenance diets. For 175 genes, opposite regulation was observed during calorie restriction and weight maintenance phases, independently of variations in body weight. Metabolism and immunity genes showed inverse profiles. During the dietary intervention, network-based analyses revealed strong interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently controlled AT gene expression. These analyses help understanding the relative importance of environmental and individual factors that control the expression of human AT genes and therefore may foster strategies aimed at improving AT function in metabolic diseases

Novel GAA Variants and Mosaicism in Pompe Disease Identified by Extended Analyses of Patients with an Incomplete DNA Diagnosis

Pompe disease is a metabolic disorder caused by a deficiency of the glycogen-hydrolyzing lysosomal enzyme acid α-glucosidase (GAA), which leads to progressive muscle wasting. This autosomal-recessive disorder is the result of disease-associated variants located in the GAA gene. In the present study, we performed extended molecular diagnostic analysis to identify novel disease-associated variants in six suspected Pompe patients from four different families for which conventional diagnostic assays were insufficient. Additional assays, such as a generic-splicing assay, minigene analysis, SNP array analysis, and targeted Sanger sequencing, allowed the identification of an exonic deletion, a promoter deletion, and a novel splicing variant located in the 5′ UTR. Furthermore, we describe the diagnostic process for an infantile patient with an atypical phenotype, consisting of left ventricular hypertrophy but no signs of muscle weakness or motor problems. This led to the identification of a genetic mosaicism for a very severe GAA variant caused by a segmental uniparental isodisomy (UPD). With this study, we aim to emphasize the need for additional analyses to detect new disease-associated GAA variants and non-Mendelian genotypes in Pompe disease where conventional DNA diagnostic assays are insufficient