Biglycan is required for adaptive remodeling after myocardial infarction.

BACKGROUND: After myocardial infarction (MI), extensive remodeling of extracellular matrix contributes to scar formation and preservation of hemodynamic function. On the other hand, adverse and excessive extracellular matrix remodeling leads to fibrosis and impaired function. The present study investigates the role of the small leucine-rich proteoglycan biglycan during cardiac extracellular matrix remodeling and cardiac hemodynamics after MI. METHODS AND RESULTS: Experimental MI was induced in wild-type (WT) and bgn(-/0) mice by permanent ligation of the left anterior descending coronary artery. Biglycan expression was strongly increased at 3, 7, and 14 days after MI in WT mice. bgn(-/0) mice showed increased mortality rates after MI as a result of frequent left ventricular (LV) ruptures. Furthermore, tensile strength of the LV derived from bgn(-/0) mice 21 days after MI was reduced as measured ex vivo. Collagen matrix organization was severely impaired in bgn(-/0) mice, as shown by birefringence analysis of Sirius red staining and electron microscopy of collagen fibrils. At 21 days after MI, LV hemodynamic parameters were assessed by pressure-volume measurements in vivo to obtain LV end-diastolic pressure, end-diastolic volume, and end-systolic volume. bgn(-/0) mice were characterized by aggravated LV dilation evidenced by increased LV end-diastolic volume (bgn(-/0), 111+/-4.2 microL versus WT, 96+/-4.4 microL; P<0.05) and LV end-diastolic pressure (bgn(-/0), 24+/-2.7 versus WT, 18+/-1.8 mm Hg; P<0.05) and severely impaired LV function (EF, bgn(-/0), 12+/-2% versus WT, 21+/-4%; P<0.05) 21 days after MI. CONCLUSIONS: Biglycan is required for stable collagen matrix formation of infarct scars and for preservation of cardiac hemodynamic function.

Arterial properties in relation to genetic variation in alpha-adducin and the renin-angiotensin system in a White population.

Earlier studies have demonstrated the interaction between ADD1 and ACE in relation to arterial properties. We investigated whether arterial characteristics might also be related to interactions of ADD1 with other renin-angiotensin system genes. Using a family-based sampling frame, we randomly recruited 1064 Flemish subjects (mean age, 43.6 years; 50.4% women). By means of a wall-tracking ultrasound system, we measured the properties of the carotid, femoral and brachial arteries. In multivariate-adjusted analyses, we assessed the multiple gene effects of ADD1 (Gly460Trp), AGT (C-532T and G-6A) and AT1R (A1166C). In ADD1 Trp allele carriers, but not in ADD1 GlyGly homozygotes (P-value for interaction < or =0.014), femoral cross-sectional compliance was significantly higher (0.74 vs 0.65 mm(2) kPa(-1); P=0.020) in carriers of the AT1R C allele than in AT1R AA homozygotes, with a similar trend for femoral distensibility (11.3 vs 10.2 x 10(-3) kPa(-1); P=0.055). These associations were independent of potential confounding factors, including age. Family-based analyses confirmed these results. Brachial diameter (4.35 vs 4.18 mm) and plasma renin activity (PRA) (0.23 vs 0.14 ng ml(-1) h(-1)) were increased (P< or =0.005) in AGT CG haplotype homozygotes compared with non-carriers, whereas the opposite was true for brachial distensibility (12.4 vs 14.4 x 10(-3) kPa(-1); P=0.011). There was no interaction between AGT and any other gene in relation to the measured phenotypes. ADD1 and AT1R interactively determine the elastic properties of the femoral artery. There is a single-gene effect of the AGT promoter haplotypes on brachial properties and PRA.

Urinary mitochondrial DNA copy number identifies renal mitochondrial injury in renovascular hypertensive patients undergoing renal revascularization: A Pilot Study.

AIMS: Patients with renovascular hypertension (RVH) exhibit elevated urinary mtDNA copy numbers, considered to constitute surrogate markers of renal mitochondrial injury. The modest success of percutaneous transluminal renal angioplasty (PTRA) in restoring renal function in RVH has been postulated to be partly attributable to acute reperfusion injury. We hypothesized that mitoprotection during revascularization would ameliorate PTRA-induced renal mitochondrial injury, reflected in elevated urinary mtDNA copy numbers and improve blood pressure and functional outcomes 3 months later. METHODS: We prospectively measured urinary copy number of the mtDNA genes COX3 and ND1 using qPCR in RVH patients before and 24 hrs after PTRA, performed during IV infusion of vehicle (n = 8) or the mitoprotective drug elamipretide (ELAM, 0.05 mg/kg/h, n = 6). Five healthy volunteers (HV) served as controls. Urinary mtDNA levels were also assessed in RVH and normal pigs (n = 7 each), in which renal mitochondrial structure and density were studied ex-vivo. RESULTS: Baseline urinary mtDNA levels were elevated in all RVH patients vs HV and directly correlated with serum creatinine levels. An increase in urinary mtDNA 24 hours after PTRA was blunted in PTRA+ELAM vs PTRA+Placebo. Furthermore, 3-months after PTRA, systolic blood pressure decreased and estimated glomerular filtration rate increased only in ELAM-treated subjects. In RVH pigs, mitochondrial damage was observed using electron microscopy in tubular cells and elevated urinary mtDNA levels correlated inversely with renal mitochondrial density. CONCLUSIONS: PTRA leads to an acute rise in urinary mtDNA, reflecting renal mitochondrial injury that in turn inhibits renal recovery. Mitoprotection might minimize PTRA-associated mitochondrial injury and improve renal outcomes after revascularization.

Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis.

BACKGROUND: Gelatinase B, a matrix metalloproteinase that has proteolytic activity against connective tissue proteins, has been suggested to be important in the connective tissue remodeling processes associated with atherogenesis and plaque rupture. This study tested the hypothesis that sequence variation in the promoter region of the gelatinase B gene influences its expression, predisposing individuals carrying certain genetic variants to more severe atherosclerosis. METHODS AND RESULTS: Single-strand conformation polymorphism analysis was carried out to search the promoter region of the gene encoding gelatinase B for naturally occurring genetic variation. As a result, an unreported common polymorphism was detected, which arose from a cytosine (C) to thymidine (T) transition at position -1562 relative to the start of transcription. Transient transfection experiments and DNA-protein interaction assays indicated that the T allele had a higher promoter activity than the C allele, which appeared to be due to preferential binding of a putative transcription repressor protein to the C allelic promoter. A sample of 584 male patients with myocardial infarction and 645 age-matched male healthy control subjects were genotyped. The allele frequencies were not significantly different between the cases and control subjects. However, in 374 patients with available angiographic data, 26% of those carrying 1 or 2 copies of the T allele had >50% stenosis in 3 coronary arteries, whereas only 15% of C/C homozygotes had triple-vessel disease. CONCLUSIONS: These data suggest that this functional genetic variation influences gelatinase B gene promoter activity in an allele-specific manner and has an effect on atherosclerotic phenotype.

Polymorphisms of the human matrix gla protein (MGP) gene, vascular calcification, and myocardial infarction.

The matrix Gla protein (MGP) is an important inhibitor of vessel and cartilage calcification that is strongly expressed in human calcified, atherosclerotic plaques and could modulate plaque calcification and coronary heart disease risk. Using a genetic approach, we explored this possibility by identifying polymorphisms of the MGP gene and testing their possible association with myocardial infarction (MI) and plaque calcification. Eight polymorphisms were identified in the coding and 5'-flanking sequences of the MGP gene. All polymorphisms were investigated in 607 patients with MI and 667 control subjects recruited into the ECTIM Study (Etude Cas-Témoins de l'Infarctus du Myocarde) and in 717 healthy individuals with echographically assessed arterial calcification and atherosclerosis who were participating in the AXA Study. In the ECTIM Study, alleles and genotypes were distributed similarly in patients and controls in the whole study group; in only 1 subgroup of subjects defined as being at low risk for MI were the concordant A-7 and Ala 83 alleles more frequent in patients with MI than in controls (P<0.003). In the AXA Study among subjects with femoral atherosclerosis, the same alleles were more common in the presence than the absence of plaque calcification (P<0.025). The other MGP polymorphisms were not associated with any investigated clinical phenotype. Transient transfection experiments with allelic promoter-reporter gene constructs and DNA-protein interaction assays were carried out to assess possible in vitro functionality of the promoter variants detected at positions -814, -138, and -7 relative to the start of transcription. When compared with the -138 T allele, the minor -138 C: allele consistently conferred a reduced promoter activity of -20% (P<0.0001) in rat vascular smooth muscle cells and of -50% (P<0.004) in a human fibroblast cell line, whereas the other polymorphisms, including -7, displayed no evidence of in vitro functionality. We conclude that the A-7 or Ala 83 alleles of the MGP gene may confer an increased risk of plaque calcification and MI; however, the observed relationships are weak or limited to subgroups of patients and therefore need confirmation.

Characterization of polymorphic structure of cathepsin G gene: role in cardiovascular and cerebrovascular diseases.

Cathepsin G (CTSG), a serine protease released from activated neutrophils, may cause platelet activation, leading to intravascular thrombosis, thus contributing to cardiovascular and cerebrovascular disease. Applying the candidate gene approach, we screened the 5'-flanking region and the entire coding region of the CTSG gene for genetic variation by using polymerase chain reaction/single-strand conformation polymorphism analysis from 96 patients at high risk for myocardial infarction (MI). We identified 4 polymorphisms in the 5'-flanking region (G-618C, G-315A, C-179T, and C-160T) and 1 polymorphism in the coding region (Asn125Ser) of the gene and genotyped the participants in the Etude Cas-Temoins sur l'Infarctus du Myocarde (ECTIM Study), a case-control study for MI, and in the Etude du Profil Génétique de l'Infarctus Cérébral (GENIC Study), a case-control study for brain infarction (BI), for all identified genetic variants. The potential in vitro functionality of the 4 variants in the 5'-flanking region was investigated with transient transfection analyses in U937 cells with different allelic promoter constructs by using a luciferase assay. Our in vitro analyses did not reveal any differences for the investigated allelic constructs with respect to promoter activity, and none of the polymorphisms in the 5'-flanking region was associated with the available phenotypes in either study. Allele and genotype distributions of all identified polymorphisms did not globally differ between cases and controls in the ECTIM Study. However, in patients from the ECTIM Study, the Ser125 allele was significantly associated with elevated plasma fibrinogen levels (P=0.006), but this effect was not seen in controls (case-control heterogeneity, P=0.04). There was a significant interaction between CTSG Asn125Ser and the beta-fibrinogen gene polymorphism G-455A on plasma fibrinogen levels (P=0.04). In the GENIC Study, the odds ratio for BI associated with CTSG Ser125 carrying was 1.82 (95% CI 1.16 to 2.84, P=0.008) in patients without a history of cardiovascular or cerebrovascular diseases. Our results indicate that the CTSG Ser125 allele is associated with plasma fibrinogen levels in MI patients from the ECTIM Study and with BI in the GENIC Study. Further studies should be carried out to define the underlying mechanisms.

Context-dependency of the relation between left ventricular mass and AGT gene variants.

In the European Project on Genes in Hypertension (EPOGH), we investigated in three populations to what extent in a family-based study, left ventricular mass (LVM) was associated with the C-532T and G-6A polymorphisms in the angiotensinogen (AGT) gene. We randomly recruited 221 nuclear families (384 parents and 440 offspring) in Cracow (Poland), Novosibirsk (Russia), and Mirano (Italy). Echocardiographic LVM was indexed to body surface area, adjusted for covariables, and subjected to multivariate analyses, using generalized estimating equations and quantitative transmission disequilibrium tests in a population-based and family-based approach, respectively. We found significant differences between the two Slavic centres and Mirano in left ventricular mass index (LVMI) (94.9 vs 80.4 g/m2), sodium excretion (229 vs 186 mmol/day), and the prevalence of the AGT -6A (55.7 vs 40.6%) and -532T (16.8 vs 9.4%) alleles. In population-based as well as in family-based analyses, we observed positive associations of LVMI and mean wall thickness (MWT) with the -532T allele in Slavic, but not in Italian male offspring. Furthermore, in Slavic male offspring, LVMI and MWT were significantly higher in carriers of the -532T/-6A haplotype than in those with the -532C/-6G or -532C/-6A allele combinations. In women, LVMI was neither associated with single AGT gene variants nor with the haplotypes (0.19 < P <0.98). In Slavic offspring carrying the AGT -532C/-6G or -532C/-6A haplotypes, LVMI significantly increased with higher sodium excretion (+3.5 g/m2/100 mmol; P=0.003), whereas such association was not present in -532T/-6A haplotype carriers (P-value for interaction 0.04). We found a positive association between LVMI and the AGT -532T allele due to increased MWT. This relation was observed in Slavic male offspring. It was therefore dependent on gender, age and ecogenetic context, and in addition it appeared to be modulated by the trophic effects of salt intake on LVM.

Identification of two polymorphisms in the early growth response protein-1 gene: possible association with lipid variables.

Early growth response factor (EGR)-1 may play an important role in the development of atherosclerosis by inducing the expression of several relevant genes which contribute to the complex modulation of vascular structure and function, leading to vascular occlusive lesions. To investigate the possible role of molecular variants in the human EGR-1 gene for the predisposition to atherosclerosis or coronary heart disease we screened the 5'- and 3'- flanking regions and the entire coding sequence for polymorphisms by polymerase chain reaction/single-strand conformation polymorphism analysis and sequencing. Male patients (n=615) with myocardial infarction and 720 age-matched, male control subjects of the Etude Cas-Témoin de l'Infarctus du Myocarde were genotyped for two newly identified polymorphisms in the 5'- (C-151T) and 3'- (T+861C) flanking region of the EGR-1 gene using hybridization with allele-specific oligonucleotides. Allele and genotype frequencies did not significantly differ between patients with myocardial infarction and control subjects without coronary heart disease. In controls not taking hypolipidemic drugs there was a significant association of the -151T allele with lower plasma levels of total cholesterol (P=0.029), low-density lipoprotein cholesterol (P=0.025) and apolipoprotein B (P=0.038) and a higher ratio of high-density to low-density lipoprotein (P=0.049) than with the C-151 allele. We conclude that the C-151T polymorphism of the EGR-1 gene may contribute to modifications of the lipid metabolism. Our findings need to be replicated in independent studies, and in vitro promoter studies should evaluate the functional consequence of the -151T allele, which disrupts a consensus core sequence for the ubiquitous transcription factor activator protein 4.

Polymorphisms in the genes encoding platelet-derived growth factor A and alpha receptor.

Platelet-derived growth factors (PDGFs) may play an important role in the development of atherosclerosis acting as chemoattractants and mitogens for vascular smooth muscle cells and macrophages. Three dimeric forms of PDGF (AA, AB, BB) have different activities due to distinct binding properties mediated by two types of PDGF receptors (Ralpha, Rbeta). To investigate the possible contribution of molecular variants in the human PDGF-A and PDGF-Ralpha genes to coronary heart disease we screened these genes for polymorphisms by polymerase chain reaction/single-strand conformation polymorphism analysis. A total of 600 men with myocardial infarction and 717 age-matched male controls from four populations in Northern Ireland and France (the ECTIM Study) were gneotyped for newly identified polymorphisms in the genes encoding PDGF-A (C-26IN3T, H69H, C+12IN5T) and PDGF-Ralpha [-1630 I/D (+/-AACTT), A-1506G, C-1390G, G-956A, C-908A, G-793T, +69 I/D (+/-GA)] using allele-specific oligonucleotides. All PDGF-Ralpha polymorphisms, except C-908A, involving a nucleotide change in a common consensus site for GCF and SP-1 transcription factors, were in nearly complete association, generating two major haplotypes. The PDGF-A and PDGF-Ralpha polymorphisms provided a heterozygosity of 0.69 and 0.40, respectively. Genotype and allele frequencies of the PDGF-A and PDGF-Ralpha polymorphisms did not differ between patients with myocardial infarction and controls in either country. None of the polymorphisms investigated was associated with blood pressure, coronary artery stenosis, or any biochemical parameter available in the ECTIM Study.

The 825C/T polymorphism of the G-protein subunit beta3 is not related to hypertension.

A polymorphism at position 825 (C-->T) of the cDNA that encodes the beta3 subunit (GNB3) of the pertussis toxin-sensitive G protein was recently shown to be associated with human hypertension. To verify this finding and to investigate whether this polymorphism could also be associated with coronary heart disease, we analyzed the GNB3 variant in subjects from 2 previously described studies: Projet d'Etude des Gènes de l'hypertension Artérielle Sévère à modérée Essentielle (PEGASE), a case-control study of moderate to severe hypertension (681 cases and 308 controls), and Etude Cas-Témoins de l'Infarctus du Myocarde (ECTIM), a case-control study of myocardial infarction (MI) (564 cases and 633 controls). Genotyping was performed with allele-specific oligonucleotides. Genotype and allele frequencies were in Hardy-Weinberg equilibrium in all groups. Allele and genotype frequencies did not differ significantly between case patients with essential hypertension or MI and control subjects. In the ECTIM study, the 825T allele frequencies in cases and controls from Belfast, Northern Ireland, were 0.31 and 0.30 (P=0.79), respectively; the corresponding frequencies in cases and controls from France were 0.33 and 0.31 (P=0.30), respectively. In the PEGASE study, the 825T allele frequency was 0.35 in female and male cases and 0.31 in male normotensive controls (P=0.12). The odds ratios for hypertension (PEGASE) and MI (ECTIM) associated with T-allele carrying were 1.23 (95% confidence interval, 0.94 to 1.62; P=0.13) and 1.11 (95% confidence interval, 0.88 to 1.39; P=0.37), respectively. There was no association of the GNB3 polymorphism with early onset of hypertension, familial history of hypertension, or blood pressure level. We conclude that the 825C/T polymorphism of the GNB3 gene did not contribute in any important way to the risk of essential hypertension or MI in these studies.

The Gln/Arg polymorphism of human paraoxonase (PON 192) is not related to myocardial infarction in the ECTIM Study.

Paraoxonase is a high-density-lipoprotein associated enzyme capable of hydrolyzing lipid peroxides, which has been suggested to contribute to atherosclerosis and coronary heart disease (CHD). We studied the Gln/Arg polymorphism affecting codon 192 of human paraoxonase (PON 192) to determine whether this polymorphism, which is associated with serum paraoxonase (PON) activity, represents a risk factor for myocardial infarction (MI). The PON 192 polymorphism was analysed in 642 male patients with myocardial infarction and 701 age-matched controls participating in the ECTIM Study (Etude Cas-Témoins de l'Infarctus du Myocarde). The frequency of the Gln allele was 0.69 in cases and 0.70 in controls (ns). The frequency of the PON 192/Arg allele in 405 MI patients who underwent coronary angiography was 0.295, 0.323 and 0.331, respectively in those with 1, 2 or 3 stenosed arteries (stenosis > 50%) (ns). The mean levels of several plasma lipids, lipoproteins and apolipoproteins were compared between the 3 PON genotypes and no difference was observed. The PON 192 polymorphism was unrelated to MI, the severity of coronary atherosclerosis and to plasma levels of several lipid variables.

Effects of three candidate genes on prevalence and incidence of hypertension in a Caucasian population.

BACKGROUND: The genes encoding angiotensin converting enzyme (ACE, I/D), alpha-adducin (ADD, Gly460Trp) and aldosterone synthase (AS, -344C/T) share the potential of influencing blood pressure (BP) via sodium homeostasis. However, most studies in humans focused on single-gene effects and disregarded epistasis, the suppression or potentiation of a gene by other non-allelic genes. METHODS: We studied the singular and combined effects of the aforementioned candidate genes: (1) in relation to BP, plasma renin activity (PRA) and urinary aldosterone in 1461 subjects randomly selected from a Caucasian population; and (2) in relation to the incidence of hypertension in a subgroup of 678 initially normotensive subjects followed up for 9.1 years (median). RESULTS: In cross-sectional analyses, AS/CC homozygosity was associated with slightly lower systolic BP (-1.32 mmHg; P = 0.08). AS/TT homozygotes showed both lower PRA and higher urinary aldosterone excretion (P < or = 0.05). In multiple-gene analyses, compared with the whole study population, ADD/Trp subjects had a higher relative risk of hypertension in the presence of the AS/T allele (1.29; P = 0.05), whereas in combination with AS/CC homozygosity ADD/Trp subjects had the smallest relative risk (0.48; P = 0.003). Hypertension developed in 229 subjects (36.6 cases per 1000 person-years). ACE/DD homozygosity, in comparison with the other ACE genotypes, was associated with increases in the incidence of hypertension, which amounted to 31% (P = 0.005) in single-gene analyses, to 59% (P = 0.004) in carriers of the ADD/Trp allele and to 122% (P = 0.0007) in AS/CC subjects. Among subjects who had both the ADD/Trp allele and the AS/CC genotype, ACE/DD homozygotes manifested a 252% (P = 0.001) higher incidence of hypertension. CONCLUSIONS: Epistatic interactions between the ACE, ADD and AS genes contribute to the prevalence and incidence of hypertension in Caucasians. The clinical relevance of the risk-conferring haplotypes identified in our prospective study was underscored by their positive predictive values, which under the assumption of a 20% life-time risk of hypertension, ranged from 29.8-40.1%.

Structure-based design of substituted diphenyl sulfones and sulfoxides as lipophilic inhibitors of thymidylate synthase.

Six new diphenyl sulfoxide and five new diphenyl sulfones were designed, synthesized, and tested for their inhibition of human and Escherichia coli thymidylate synthase (TS) and of the growth of cells in tissue culture. The best sulfoxide inhibitor of human TS was 3-chloro-N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4- (phenylsulfinyl)-N-(prop-2-ynyl)-aniline (7c) that had a Ki of 27 nM. No sulfone improved on TS inhibition by the previously reported 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2- ynylamino)phenyl phenyl sulfone (Ki = 12 nM). Nevertheless, one sulfone, 4-((2-chlorophenyl)sulfonyl)-N-((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)-N-(prop-2-ynyl)aniline, was selected, on the basis of its inhibition of both TS and cell growth, for antitumor testing; it gave a 61% increase in life span to mice bearing the thymidino kinase-deficient L5178Y (TK-) lymphoma. A crystal structure of N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4-((2- methylphenyl)sulfinyl)-N-(prop-2-ynyl)aniline complexed with E. coli TS was solved and revealed selective binding of one sulfoxide enantiomer. AMBER calculations showed that the enantioselection was due to asymmetric electrostatic effects at the mouth of the active site. In contrast, a similar crystal structure of the sulfoxide 7c, along with AMBER calculations, indicated that both enantiomers bound, but with different affinities. The side chain of Phe176 shifted in order to structurally accommodate the chlorine of the more weakly bound enantiomer.

Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase.

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.

The Leu33/Pro polymorphism (PlA1/PlA2) of the glycoprotein IIIa (GPIIIa) receptor is not related to myocardial infarction in the ECTIM Study. Etude Cas-Temoins de l'Infarctus du Myocarde.

The GPIIb/IIIa receptor complex may contribute to acute coronary syndromes by mediating platelet aggregation. The Leu33/Pro polymorphism (PlA1/PlA2) of the GPIIIa has recently been shown to be associated with CHD in a small case-control study. We have investigated this polymorphism in a large multicenter study of patients with myocardial infarction and controls and found no difference in the distribution of allele and genotype frequencies between cases and controls.

The histidin-rich glycoprotein Pro186/Ser polymorphism is not related to myocardial infarction in the ECTIM study.

The histidin-rich glycoprotein (HRG) may contribute to coronary heart disease as a consequence of its possible thrombophilic properties. To test this hypothesis we have investigated the Pro186/Ser polymorphism of the HRG gene, which is known to strongly affect plasma HRG levels, in a large multicenter case-control study of myocardial infarction (MI). The results failed to demonstrate any association between the polymorphism and MI or angiographically assessed coronary stenosis.

Expression of the progesterone receptor and progesterone- metabolising enzymes in the female and male human kidney.

Due to high binding affinity of progesterone to the human mineralocorticoid receptor (hMR), progesterone competes with the natural ligand aldosterone. In order to analyse how homeostasis can be maintained by mineralocorticoid function of aldosterone at the MR, especially in the presence of elevated progesterone concentrations during the luteal phase and pregnancy, we investigated protective mechanisms such as the decrease of free progesterone by additional binding sites and progesterone metabolism in renal cells. As a prerequisite for sequestration of progesterone by binding to the human progesterone receptor (hPR) we demonstrated the existence of hPR expression in female and male kidney cortex and medulla at the level of transcription and translation. We identified hPR RNA by sequencing the RT-PCR product and characterised the receptor by ligand binding and scatchard plot analysis. The localisation of renal hPR was shown predominantly in individual epithelial cells of distal tubules by immunohistology, and the isoform hPR-B was detected by Western blot analysis. As a precondition for renal progesterone metabolism, we investigated the expression of steroid-metabolising enzymes for conversion of progesterone to metabolites with lower affinity to the hMR. We identified the enzyme 17alpha-hydroxylase for renal 17alpha-hydroxylation of progesterone. For 20alpha-reduction, different hydroxysteroid dehydrogenases (HSDs) such as 20alpha-HSD, 17beta-HSD type 5 (3alpha-HSD type 2) and 3alpha-HSD type 3 were found. Further, we detected the expression of 3beta-HSD type 2 for 3beta-reduction, 5alpha-reductase (Red) type 1 for 5alpha-reduction, and 5beta-Red for 5beta-reduction of progesterone in the human kidney. Therefore metabolism of progesterone and/or binding to hPR could reduce competition with aldosterone at the MR and enable the mineralocorticoid function.

Renal function in relation to three candidate genes.

We recently found that femoral intima media thickness, as well as the incidence of hypertension, is influenced by genes encoding the angiotensin-converting enzyme (ACE; insertion/deletion [I/D]) polymorphism, alpha-adducin (Gly460Trp), and aldosterone synthase (-344C/T). By interfering with blood pressure or sodium homeostasis, these genetic polymorphisms also may change renal function. We therefore investigated serum creatinine level, calculated and measured creatinine clearances, and 24-hour urinary protein excretion in subjects previously genotyped for these three polymorphisms. The 1,454 participants drawn at random from the population (64.3% of those invited) were aged 43.4 years and included 744 women (51.2%). Blood pressure, measured by study nurses at subjects' homes, averaged 123/76 mm Hg. Mean values were 90 micromol/L for serum creatinine; 84 and 88 mL/min/1.73 m(2) for calculated and measured (n = 855) creatinine clearances, respectively; and 90 mg/d of protein for proteinuria (n = 556). The prevalence of mild renal dysfunction (creatinine clearance

Polymorphisms of the endothelin-A and -B receptor genes in relation to blood pressure and myocardial infarction: the Etude Cas-Témoins sur l'Infarctus du Myocarde (ECTIM) Study.

Endothelin-1 is a potent vasoconstrictor that has also mitogenic properties, stimulating the synthesis and secretion of several vasoactive molecules. There is much evidence to suggest that endothelin-1 might be involved in the pathogenesis of hypertension, atherosclerosis, and ischemic heart disease. Endothelin-1 exerts its effects through at least two receptors, ET(A) and ET(B), which are encoded by different genes and have separate tissue distributions and biologic properties. The objective of this study was to identify polymorphisms of the ET(A) and ET(B) receptor genes and to study their association with myocardial infarction (MI) and blood pressure. The coding regions and 1.3 kb upstream of the ET(A) and ET(B) receptor genes were explored by polymerase chain reaction/single strand conformation polymorphism. Six polymorphisms were found in the ET(A) receptor gene and three in the ET(B) receptor gene. Most of these polymorphisms were frequent. Associations between the detected polymorphisms, blood pressure, and MI were examined in the ECTIM study, a multicenter study comparing 652 patients having survived an MI and 773 controls from Belfast (Northern Ireland) and France. Alleles at the different polymorphic sites were similarly distributed in patients with MI and controls. Allele frequencies were similar in both countries, except for the ET(A)/-231 G allele, which appeared more frequently in France than in Belfast (P < .01). The mean systolic and diastolic blood pressure levels did not significantly differ between genotypes. However, a C/T substitution located in the nontranslated part of exon 8 of the ET(A) receptor gene (ET(A)/EX8nt1363) was associated with pulse pressure (P < .005). These results do not support an involvement of the endothelin receptor genes in a predisposition to MI or the determination of blood pressure levels, but suggest that a polymorphism of the ET(A) receptor gene might influence the pulse pressure. This result will have to be confirmed in other studies.

Ambulatory blood pressure and left ventricular structure and function in relation to the G-protein beta3-subunit polymorphism C825T in White Europeans.

The 825T allele of the G-protein beta(3)-subunit is associated with increased intracellular signalling. Its association with hypertension is inconsistent. We, therefore, studied the C825T polymorphism in relation to ambulatory blood pressure as well as left ventricular structure and function in two European populations. We genotyped 248 parents and 318 offspring, enrolled in the European Project on Genes in Hypertension in Cracow, Poland (n=286) and in Novosibirsk, Russian Federation (n=280). The 24-h ambulatory blood pressure was recorded using oscillometric SpaceLabs 90207 monitors. Within each centre, a single observer performed two-dimensionally guided M-mode echocardiography and Doppler sonography to measure left ventricular structure (American Society of Echocardiography conventions) and diastolic function: early (E) and late (A) peak diastolic inflow velocities. We used analysis of covariance and generalized estimating equations to allow for covariables and nonindependence among related subjects. Genotype frequencies were similar (P=0.25) in Cracow and Novosibirsk and amounted to 44.7% for CC, 47.2% for CT, and 8.1% for TT. Among parents (mean age: 51.3 years)-but not among offspring (mean age 25.1 years)-24-h, daytime and night time systolic blood pressures were 5-6 mmHg higher in TT homozygotes than in C allele carriers. In TT homozygous parents (-8.2 cm/sec, P=0.004) as well as in TT homozygous offspring (-7.5 cm/sec, P=0.02), the E-wave was significantly reduced, which in offspring also resulted in a lower E/A ratio (-0.25, P=0.002). Neither in parents nor in offspring, left ventricular mass index was associated with the C825T polymorphism. In conclusion, in TT homozygotes of both generations, early left ventricular relaxation was reduced. In TT homozygous parents, the latter observation might be because of the higher systolic pressure associated with the TT genotype.