Johanna Kuusisto

Characterization of the early lesion of 'degenerative' valvular aortic stenosis. Histological and immunohistochemical studies.

BackgroundNonrheumatic stenosis of trileaflet aortic valves, often termed senile or calcific valvular aortic stenosis, is considered a “degenerative” process, but little is known about the cellular or molecular factors that mediate its development. Methods and ResultsTo characterize the developing aortic valvular lesion, we performed histological and immunohisto-chemical studies on Formalin-fixed and methanol-Carnoys-fixed parafflin-embedded aortic valve leaflets or on frozen sections obtained at autopsy from 27 adults (age, 46 to 82 years) with normal leaflets (n=6), mild macroscopic leaflet thickening (n= 15), or clinical aortic stenosis (n=6). Focal areas of thickening (“early lesions”) were characterized by (1) subendothelial thickening on the aortic side of the leaflet, between the basement membrane (PAS-positive) and elastic lamina (Verhoeff–van Gieson), (2) the presence of large amounts of intracellular and extracellular neutral lipids (oil red O) and fine, stippled mineralization (von Kossa), and (3) disruption of the basement membrane overlying the lesion. Regions of the fibrosa adjacent to these lesions were characterized by thickening and by protein, lipid, and calcium accumulation. Control valves showed none of these abnormalities. Immunohistochemical studies were performed using monoclonal antibodies directed against macrophages (anti-CD68 or HAM-56), and contractile proteins of smooth muscle cells or myofibroblasts (anti-α-actin and HHF-35) or rabbit polyclonal antiserum against T lymphocytes (anti-CD3). In normal valves, scattered macrophages were present in the fibrosa and ventricularis, and occasional muscle actin-positive cells were detected in the proximal portion of the ventricularis near the leaflet base, but no T lymphocytes were found. In contrast, early lesions were characterized by the presence of an inflammatory infiltrate composed of non-foam cell and foam cell macrophages, occasional T cells, and rare α-actin-positive cells. In stenotic aortic valves, a similar but more advanced lesion was seen. ConclusionsThe early lesion of “degenerative” aortic stenosis is an active inflammatory process with some similarities (lipid deposition, macrophage and T-cell infiltration, and basement membrane disruption) and some dissimilarities (presence of prominent mineralization and small numbers of smooth muscle cells) to atherosclerosis.

Cardiovascular disease risk factors as predictors of Type 2 (non-insulin-dependent) diabetes mellitus in elderly subjects

SummaryRisk factors measured in a cross-sectional study in subjects aged 65–74 years living in eastern Finland were correlated with the risk of developing diabetes 3.5 years later. Sixty-nine of 892 initially non-diabetic subjects developed diabetes during the follow-up. Subjects who subsequently developed diabetes had multiple adverse changes in risk factor levels before the diagnosis of diabetes. Subjects who developed diabetes had higher body mass index and waist-hip ratio as well as higher levels of fasting and 2-h plasma glucose and insulin and higher prevalence of family history of diabetes than those who remained healthy. Furthermore, prevalence of hypertension and levels of diastolic blood pressure and total triglycerides were higher and HDL cholesterol lower among subjects who developed diabetes than among those who remained healthy. The highest risk of developing diabetes was associated with impaired glucose tolerance (World Health Organisation criteria) (odds ratio = 9.8,95% confidence interval = 6.1–5.8). The risk of developing diabetes was 3.7 (3.2–6.1) among subjects in the highest quartile of 2-h insulin distribution, 3.5 (2.0–6.1) in those with triglycerides greater than 2.5 mmol/l,2.7 (1.5–4.6) in those with waist-hip ratio greater than 1.0,2.5 (1.5–4.4) in those with HDL cholesterol less than 1.0 mmol/l, 2.1 (1.2–3.6) in those with body mass index greater than 30 kg/m2, 1.9 (1.1–3.3) among those in the highest quartile of fasting insulin distribution, 1.8 (1.0–3.1) in those having hypertension, and 1.7 (1.0–2.9) in those with a family history of diabetes. The risk of diabetes increased by clustering of risk factors related to insulin resistance (impaired glucose tolerance, triglycerides >2.5 mmol/l, HDL cholesterol <1.0, hypertension) so that the risk was 3.6-fold in those having one risk factor and 59-fold in those having all four risk factors compared to subjects having no risk factors. In conclusion, cardiovascular risk factors related to insulin resistance are predictors of diabetes in the elderly.

Serum proinsulin levels are disproportionately increased in elderly prediabetic subjects

SummaryInsulin resistance and impaired insulin secretion are thought to be the primary defects in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM). Disproportionately increased proinsulin relative to insulin levels are suggested to be an early indicator of a failing pancreas. We examined the relationship of fasting specific insulin, proinsulin, and 32, 33 split proinsulin concentrations, and the proinsulin: insulin ratio to the risk of developing NIDDM 3.5 years later in 65–74-year-old non-diabetic Finnish subjects participating in a populationbased study (n=892) on diabetes and heart disease. Altogether 69 subjects developed NIDDM over a 3.5-year follow-up (cases). The cases were compared to randomly-selected gender-matched control subjects (n=69) and control subjects matched for gender, glucose tolerance status (normal or impaired), and body mass index (n=69). There were no differences in insulin concentrations between cases and random or matched control subjects [median and interquartile range; 123 (77–154), 108 (74–143), 118 (83–145) pmol/l, p=0.271]. Random control subjects had lower proinsulin and 32,33 split proinsulin concentrations and split proinsulin: insulin ratios compared to cases [5.7 (3.8–9.0) vs 7.3 (4.8–10.0) pmol/l, p=0.005; 7.3 (4.5–13.0) vs 10.4 (7.1–18.0) pmol/l, p=0.002; 0.073 (0.057–0.110) vs 0.097 (0.060–0.135), p=0.003]. Matched control subjects had lower proinsulin concentrations and proinsulin: insulin ratios compared to cases [5.9 (4.0–7.7) vs 7.3 (4.8–10.0) pmol/l, p=0.019; 0.048 (0.035–0.071) vs 0.064 (0.045–0.100), p=0.008]. When cases were compared to matched control subjects a 1 SD increase in baseline proinsulin: insulin ratio was associated with a 1.37-fold risk (p=0.020) of developing diabetes. Moreover, this association was independent of fasting glucose concentration at baseline. Thus, in elderly prediabetic subjects disproportionately increased proinsulin concentration, an indicator of defective insulin secretion, is associated with conversion to diabetes over a short time period.

Gender differences in left ventricular function at rest and with exercise in asymptomatic aortic stenosis

In 29 women and 53 men with asymptomatic aortic stenosis, two-dimensional (2-D) and Doppler echocardiography were performed at rest and immediately after treadmill exercise testing to examine gender differences in left ventricular geometry, systolic and diastolic function, functional status, and exercise capacity. Aortic stenosis severity was similar between men and women. Women reported more functional impairment than men (88% +/- 14% vs 95% +/- 7%; p = 0.02). When indexed to body surface area, women had a smaller end-diastolic volume (39 +/- 14 vs 50 +/- 15 ml/m2; p = 0.002), end-systolic volume (13 +/- 6 ml/m2 vs 18 +/- 9 ml/m2; p = 0.01) and left ventricular mass (73 +/- 26 gm/m2 vs 84 +/- 21 gm/m2; p = 0.05), but a higher relative wall thickness in systole (1.5 +/- 0.4 cm vs 1.3 +/- 0.4 cm; p = 0.05), and fractional shortening (43% +/- 7% vs 39% +/- 10%; p = 0.03). Women had higher early and late transmitral velocities than did men (early, 92 +/- 24 cm/sec vs 79 +/- 29 cm/sec; p = 0.05; late, 97 +/- 30 cm/sec vs 68 +/- 23 cm/sec; p < 0.0001), a higher time-velocity integral in early diastole (18.2 +/- 4.8 cm vs 15.1 +/- 4.3 cm; p = 0.006), a significantly shorter exercise duration (4.5 +/- 4.1 minutes vs 8.0 +/- 3.9 minutes; p < 0.0001), a greater degree of functional aerobic impairment (25% +/- 48% vs 2% +/- 33%; p = 0.02), and a smaller increase in cardiac output with exercise (5.4 +/- 3.5 L/min vs 8.0 +/- 4.3 L/min; p = 0.01), in spite of similar peak heart rate and blood pressure responses. In these asymptomatic subjects with aortic stenosis, women had smaller, relatively hypercontractile ventricles, a different diastolic filling profile, more exercise limitation, and poorer functional capacity. These findings demonstrate the importance of gender in the response of the left ventricle to chronic pressure overload.

A role for coding functional variants in HNF4A in type 2 diabetes susceptibility

Aims/hypothesisRare mutations in the gene HNF4A, encoding the transcription factor hepatocyte nuclear factor 4α (HNF-4A), account for ~5% of cases of MODY and more frequent variants in this gene may be involved in multifactorial forms of diabetes. Two low-frequency, non-synonymous variants in HNF4A (V255M, minor allele frequency [MAF] ~0.1%; T130I, MAF ~3.0%)—known to influence downstream HNF-4A target gene expression—are of interest, but previous type 2 diabetes association reports were inconclusive. We aimed to evaluate the contribution of these variants to type 2 diabetes susceptibility through large-scale association analysis.MethodsWe genotyped both variants in at least 5,745 cases and 14,756 population controls from the UK and Denmark. We also undertook an expanded association analysis that included previously reported and novel genotype data obtained in Danish, Finnish, Canadian and Swedish samples. A meta-analysis incorporating all published association studies of the T130I variant was subsequently carried out in a maximum sample size of 14,279 cases and 26,835 controls.ResultsWe found no association between V255M and type 2 diabetes in either the initial (p = 0.28) or the expanded analysis (p = 0.44). However, T130I demonstrated a modest association with type 2 diabetes in the UK and Danish samples (additive per allele OR 1.17 [95% CI 1.08–1.28]; p = 1.5 × 10−4), which was strengthened in the meta-analysis (OR 1.20 [95% CI 1.10–1.30]; p = 2.1 × 10−5).Conclusions/interpretationOur data are consistent with T130I as a low-frequency variant influencing type 2 diabetes risk, but are not conclusive when judged against stringent standards for genome-wide significance. This study exemplifies the difficulties encountered in association testing of low-frequency variants.

New variants in the glycogen synthase gene (Gln71His, Met416Val) in patients with NIDDM from eastern Finland

SummaryImpaired glycogen synthesis after insulin stimulation accounts for most of the insulin resistance in patients with non-insulin-dependent diabetes mellitus (NIDDM). The glycogen synthase gene (GYS1), which encodes the rate-limiting enzyme for glycogen synthesis, is a promising candidate gene for NIDDM. Therefore, we screened all 16 exons of this gene by single-strand conformation polymorphism analysis in 40 patients with NIDDM (age 67 ± 2 years, body mass index 28.2 ± 0.6 kg/m2) from Taipalsaari, eastern Finland. The Gly464Ser variant (exon 11) and a silent polymorphism TTC342TTT (exon 7) have been reported previously. In addition, we found a new variant Gln71His (exon 2) and a new amino acid polymorphism Met416Val (exon 10). An additional sample of 65 patients with NIDDM and 82 normoglycaemic men (age 54 ± 1 years, body mass index 26.3 ±1.4 kg/m2) were screened. The allele frequency of the TTC342TTT silent substitution was 0.29 in both NIDDM and normoglycaemic subjects. The Gln71His and Gly464Ser variants were found in 1 (1 %) and 3 (3 %) subjects, respectively, of the 105 NIDDM patients and in none of the 82 normoglycaemic men. The Met416Val polymorphism was found in 16 (15 %) of the 105 NIDDM patients and in 14 (17 %,) of the 82 control subjects (all heterozygous). The Met416Val polymorphism was not associated with insulin resistance in two groups of normoglycaemic subjects. In conclusion, the new Gln71His and Met416Val substitutions and other variants of the glycogen synthase gene are unlikely to make a major contribution to insulin resistance and NIDDM in diabetic patients from eastern Finland. [Diabetologia (1997) 40: 1313-1319]

The ATF6-Met[67]Val Substitution Is Associated With Increased Plasma Cholesterol Levels

Objective—Activating transcription factor 6 (ATF6) is a sensor of the endoplasmic reticulum stress response and regulates expression of several key lipogenic genes. We used a 2-stage design to investigate whether ATF6 polymorphisms are associated with lipids in subjects at increased risk for cardiovascular disease (CVD). Methods and Results—In stage 1, 13 tag-SNPs were tested for association in Dutch samples ascertained for familial combined hyperlipidemia (FCHL) or increased risk for CVD (CVR). In stage 2, we further investigated the SNP with the strongest association from stage 1, a Methionine/Valine substitution at amino-acid 67, in Finnish FCHL families and in subjects with CVR from METSIM, a Finnish population-based cohort. The combined analysis of both stages reached region-wide significance (P=9×10−4), but this association was not seen in the entire METSIM cohort. Our functional analysis demonstrated that Valine at position 67 augments ATF6 protein and its targets Grp78 and Grp94 as well as increases luciferase expression through Grp78 promoter. Conclusions—A common nonsynonymous variant in ATF6 increases ATF6 protein levels and is associated with cholesterol levels in subjects at increased risk for CVD, but this association was not seen in a population-based cohort. Further replication is needed to confirm the role of this variant in lipids.

PS7 - 3. Predicting Glycated Haemoglobin in the Non-Diabetic General Population: a DIRECT Study

The DETECT-2 risk score is a simple screening tool for prediction of incident type 2 diabetes (based on fasting and/or Oral Glucose Tolerance Test glucose levels). Recently, glycated haemoglobin (HbA1c) levels have been added to diabetes diagnostic criteria. Our study evaluates whether predictors from the DETECT-2 score could be used to predict HbA1c levels after 6 years.