Saris, J.J.

Exceptional good cognitive and phenotypic profile in a male carrying a mosaic mutation in the FMR1 gene (Article)

2007-08-01T00:00:00Z

Fragile X (FRAX) syndrome is a commonly inherited form of mental retardation resulting from the lack of expression of the fragile X mental retardation protein (FMRP). It is caused by a stretch of CGG repeats within the fragile X gene, which can be unstable in length as it is transmitted from generation to generation. Once the repeat exceeds a threshold length, the FMR1 gene is methylated and no protein is produced resulting in the fragile X phenotype. The consequences of FMRP absence in the mechanisms underlying mental retardation are unknown. We have identified a male patient in a classical FRAX family without the characteristic FRAX phenotype. His intelligence quotient (IQ) is borderline normal despite the presence of a mosaic pattern of a pre-mutation (25%), full mutation (60%) and a deletion (15%) in the FMR1 gene. The cognitive performance was determined at the age of 28 by the Raven test and his IQ was 81. However, FMRP expression studies in both hair roots and lymphocytes, determined at the same time as the IQ test, were within the affected male range. The percentage of conditioned responses after delay eyeblink conditioning was much higher than the average percentage measured in FRAX studies. Moreover, this patient showed no correlation between FMRP expression and phenotype and no correlation between DNA diagnostics and phenotype.

Prorenin and the heart : the Mannose 6-phosphate connection (Doctoral Thesis)

2002-05-23T00:00:00Z

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.

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

2002-03-04T00:00:00Z

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/L PD123319 to block M6P and AT(1) and AT(2) receptors, respectively. Protein and DNA synthesis were studied by quantifying [(3)H]-leucine and [(3)H]-thymidine incorporation. For comparison, studies with 100 nmol/L Ang II were also performed. Neither prorenin alone, nor angiotensinogen alone, affected protein or DNA synthesis. Prorenin plus angiotensinogen increased [(3)H]-leucine incorporation (+21 +/- 5%, mean +/- SEM, P<0.01), [(3)H]-thymidine incorporation (+29 +/- 6%, P<0.01), and total cellular protein (+14 +/- 3%, P<0.01), whereas Ang II increased DNA synthesis only (+34 +/- 7%, P<0.01). Eprosartan, but not PD123319 or M6P, blocked the effects of prorenin plus angiotensinogen as well as the effects of Ang II. Medium Ang II levels during prorenin and angiotensinogen incubation were <1 nmol/L. In conclusion, prorenin binding to M6PRs on cardiomyocytes per se does not result in enhanced protein or DNA synthesis. However, through Ang II generation, prorenin is capable of inducing myocyte hypertrophy and proliferation. Because this generation occurs independently of M6PRs, it most likely depends on the catalytic activity of intact prorenin in the medium (because of temporal prosegment unfolding) rather than its intracellular activation. Taken together, our results do not support the concept of Ang II generation in cardiomyocytes following intracellular prorenin activation.

Cardiomyocytes bind and activate native human prorenin : role of soluble mannose 6-phosphate receptors (Article)

2001-01-01T00:00:00Z

Cardiomyocytes bind, internalize, and activate recombinant human prorenin through mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptors. To investigate whether this also applies to native human prorenin, neonatal rat myocytes were incubated for 4 hours at 37 degrees C with various prorenin-containing human body fluids. Uptake and activation by M6P/IGFII receptors were observed for plasma prorenin from subjects with renal artery stenosis and/or hypertension and for follicular fluid prorenin. The total amount of cellular renin and prorenin (expressed as percentage of the levels of renin and prorenin in the medium) after 4 hours of incubation was 4 to 10 times lower than after incubation with recombinant human prorenin. Although plasma contains alkaline phosphatases capable of inactivating the M6P label as well as soluble M6P/IGFII receptors that block prorenin binding in a competitive manner and proteins (eg, insulin, IGFII) that increase the number of cell-surface M6P/IGFII receptors, these factors were not responsible for the modest uptake of native human prorenin. Uptake did not occur during incubation of myocytes with plasma prorenin from anephric subjects or with amniotic fluid prorenin, and this was not due to the presence of excessively high levels of M6P/IGFII receptors and/or phosphatase activity in these fluids. In conclusion, myocytes are capable of binding, internalizing, and activating native human prorenin of renal and ovarian origin through M6P/IGFII receptors. Differences in prorenin glycosylation and/or phosphorylation as well as the concentration of soluble M6P/IGFII receptors and growth factors affecting cell-surface M6P/IGFII receptor density determine the amount of prorenin entering the heart and thus cardiac angiotensin II production.

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

2001-01-01T00:00:00Z

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 (Article)

2001-01-01T00:00:00Z

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.

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

2000-01-01T00:00:00Z

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 contractile effects of Ang I and reduced fractional conversion to 1% (0.1% to 8%), thereby excluding a role for Ang I-to-II converting enzymes other than ACE (eg, chymase). The Ang II type 1 receptor antagonist losartan (3 mg. kg(-1). min(-1)) inhibited the vasoconstrictor effects of Ang II. In conclusion, the similar potencies of Ang I and II in the forearm, combined with the fact that only one third of arterially delivered Ang I is converted to Ang II, suggest that in situ-generated Ang II is more important for vasoconstriction than circulating Ang II. Local Ang II generation in the forearm depends on ACE exclusively and results in vasoconstriction via Ang II type 1 receptors.