66 research outputs found
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
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
Terminal osseous dysplasia with pigmentary defects and cardiomyopathy caused by a novel FLNA variant
Terminal osseous dysplasia with pigmentary defects (TODPD), also known as digitocutaneous dysplasia, is one of the Xâlinked filaminopathies caused by a variety of FLNAâvariants. TODPD is characterized by skeletal defects, skin fibromata and dysmorphic facial features. So far, only a single recurrent variant (c.5217G>A;p.Val1724_Thr1739del) in FLNA has found to be responsible for TODPD. We identified a novel c.5217+5G>C variant in FLNA in a female proband with skeletal defects, skin fibromata, interstitial lung disease, epilepsy, and restrictive cardiomyopathy. This variant causes misâsplicing of exon 31 predicting the production of a FLNAâprotein with an inâframeâdeletion of 16 residues identical to the missâsplicingâeffect of the recurrent TODPD c.5217G>A variant. This misâspliced transcript was explicitly detected in heart tissue, but was absent from blood, skin, and lung. Xâinactivation analyses showed extreme skewing with almost complete inactivation of the mutated allele (>90%) in these tissues, except for heart. The mother of the proband, who also has fibromata and skeletal abnormalities, is also carrier of the FLNAâvariant and was diagnosed with noncompaction cardiomyopathy after cardiac screening. No other relevant variants in cardiomyopathyârelated genes were found. Here we describe a novel variant in FLNA (c.5217+5G>C) as the second pathogenic variant responsible for TODPD. Cardiomyopathy has not been described as a phenotypic feature of TODPD before
Functional Assays Combined with Pre-mRNA-Splicing Analysis Improve Variant Classification and Diagnostics for Individuals with Neurofibromatosis Type 1 and Legius Syndrome
Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by inactivating variants in NF1 and SPRED1. NF1 encodes neurofibromin (NF), a GTPase-activating protein (GAP) for RAS that interacts with the SPRED1 product, Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1 (SPRED1). Obtaining a clinical and molecular diagnosis of NF1 or LS can be challenging due to the phenotypic diversity, the size and complexity of the NF1 and SPRED1 loci, and uncertainty over the effects of some NF1 and SPRED1 variants on pre-mRNA splicing and/or protein expression and function. To improve NF1 and SPRED1 variant classification and establish pathogenicity for NF1 and SPRED1 variants identified in individuals with NF1 or LS, we analyzed patient RNA by RT-PCR and performed in vitro exon trap experiments and estimated NF and SPRED1 protein expression, RAS GAP activity, and interaction. We obtained evidence to support pathogenicity according to American College of Medical Genetics guidelines for 73/114 variants tested, demonstrating the utility of functional approaches for NF1 and SPRED1 variant classification and NF and LS diagnostics.</p
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 a-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
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 5âČ 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
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