Urinary complement proteins and risk of end-stage renal disease: quantitative urinary proteomics in patients with type 2 diabetes and biopsy-proven diabetic nephropathy.

PURPOSE: To investigate the association between urinary complement proteins and renal outcome in biopsy-proven diabetic nephropathy (DN). METHODS: Untargeted proteomic and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses and targeted proteomic analysis using parallel reaction-monitoring (PRM)-mass spectrometry was performed to determine the abundance of urinary complement proteins in healthy controls, type 2 diabetes mellitus (T2DM) patients, and patients with T2DM and biopsy-proven DN. The abundance of each urinary complement protein was individually included in Cox proportional hazards models for predicting progression to end-stage renal disease (ESRD). RESULTS: Untargeted proteomic and functional analysis using the KEGG showed that differentially expressed urinary proteins were primarily associated with the complement and coagulation cascades. Subsequent urinary complement proteins quantification using PRM showed that urinary abundances of C3, C9, and complement factor H (CFAH) correlated negatively with annual estimated glomerular filtration rate (eGFR) decline, while urinary abundances of C5, decay-accelerating factor (DAF), and CD59 correlated positively with annual rate of eGFR decline. Furthermore, higher urinary abundance of CFAH and lower urinary abundance of DAF were independently associated with greater risk of progression to ESRD. Urinary abundance of CFAH and DAF had a larger area under the curve (AUC) than that of eGFR, proteinuria, or any pathological parameter. Moreover, the model that included CFAH or DAF had a larger AUC than that with only clinical or pathological parameters. CONCLUSION: Urinary abundance of complement proteins was significantly associated with ESRD in patients with T2DM and biopsy-proven DN, indicating that therapeutically targeting the complement pathway may alleviate progression of DN.

Effects of in-utero exposure to selective serotonin reuptake inhibitors and venlafaxine on term and preterm infants.

OBJECTIVE: We hypothesized that in-utero SSRI exposure affects Apgar scores and immediate post-delivery oxygen requirements. STUDY DESIGN: SSRI in-utero exposure was assessed retrospectively in preterm neonates ≥ 28 weeks gestation and term neonates. Primary outcome was Apgar <7 at five minutes and delivery room oxygen requirements. Secondary endpoints included one-minute Apgar, length of stay, birth weight, and NICU admission. RESULTS: Fifty-one preterm and 117 term neonates were exposed to a SSRI; mostly to sertraline. Pre-term SSRI-exposed neonates had 4.1 times higher delivery room oxygen requirements. One minute Apgar <7 was 3.5 times higher and NICU admission 5 times higher 95% CI (1.3-19) in SSRI-exposed term neonates. Higher doses of sertraline had associated adverse outcomes. CONCLUSION: In-utero SSRI exposure was associated with increased neonatal care at birth, differences in Apgar scores compared with controls, and increased NICU admissions. Higher sertraline doses were associated with poorer outcomes.

Dental Decay Phenotype in Nonsyndromic Orofacial Clefting.

Although children with oral clefts have a higher risk for dental anomalies when compared with the general population, prior studies have shown conflicting results regarding their dental decay risk. Also, few studies have assessed dental decay risk in unaffected relatives of children with clefts. Thus, the question of increased risk of dental decay in individuals with oral clefts or their unaffected relatives is still open for empirical investigation. This study characterizes dental decay in the largest international cohort to date of children with nonsyndromic clefts and their relatives, as compared with controls, and it addresses whether families with oral clefts have a significantly increased risk for dental decay versus the general population. A total of 3,326 subjects were included: 639 case probands, 1,549 unaffected relatives, and 1,138 controls. Decay was identified from in-person dental examinations or intraoral photographs. Case-control differences were tested with regression analysis. No significant differences were shown in percentage decayed and filled teeth and decayed teeth in the primary dentition (dft, dt) and permanent dentition (DFT, DT) in cases versus controls. In the cleft region, no significant differences were seen in primary or permanent decay (dt, DT) when compared with controls. No difference was found with regard to cleft type and percentage dft, dt, DFT, and DT in case probands. Nonsignificant differences were found in unaffected siblings and parents versus controls (primary and permanent dentitions). Collectively, these findings indicate that individuals with nonsyndromic oral clefts and their families do not have a higher dental decay risk as compared with the general population. These results suggest that either genetic or environmental factors underlying a higher susceptibility for dental anomalies do not increase caries risk or that the seemingly higher risk for dental decay associated with increased dental anomalies in case probands may be superseded by possible greater access to dental care.

Spectrum of Dental Phenotypes in Nonsyndromic Orofacial Clefting.

Children with oral clefts show a wide range of dental anomalies, adding complexity to understanding the phenotypic spectrum of orofacial clefting. The evidence is mixed, however, on whether the prevalence of dental anomalies is elevated in unaffected relatives and is mostly based on small samples. In the largest international cohort to date of children with nonsyndromic clefts, their relatives, and controls, this study characterizes the spectrum of cleft-related dental anomalies and evaluates whether families with clefting have a significantly higher risk for such anomalies compared with the general population. A total of 3,811 individuals were included: 660 cases with clefts, 1,922 unaffected relatives, and 1,229 controls. Dental anomalies were identified from in-person dental exams or intraoral photographs, and case-control differences were tested using χ(2) statistics. Cases had higher rates of dental anomalies in the maxillary arch than did controls for primary (21% vs. 4%, P = 3 × 10(-8)) and permanent dentitions (51% vs. 8%, P = 4 × 10(-62)) but not in the mandible. Dental anomalies were more prevalent in cleft lip with cleft palate than other cleft types. More anomalies were seen in the ipsilateral side of the cleft. Agenesis and tooth displacements were the most common dental anomalies found in case probands for primary and permanent dentitions. Compared with controls, unaffected siblings (10% vs. 2%, P = 0.003) and parents (13% vs. 7%, P = 0.001) showed a trend for increased anomalies of the maxillary permanent dentition. Yet, these differences were nonsignificant after multiple-testing correction, suggesting genetic heterogeneity in some families carrying susceptibility to both overt clefts and dental anomalies. Collectively, the findings suggest that most affected families do not have higher genetic risk for dental anomalies than the general population and that the higher prevalence of anomalies in cases is primarily a physical consequence of the cleft and surgical interventions.

Pharmacological inhibition of NOX reduces atherosclerotic lesions, vascular ROS and immune-inflammatory responses in diabetic Apoe(-/-) mice.

AIMS/HYPOTHESIS: Enhanced vascular inflammation, immune cell infiltration and elevated production of reactive oxygen species (ROS) contribute significantly to pro-atherogenic responses in diabetes. We assessed the immunomodulatory role of NADPH oxidase (NOX)-derived ROS in diabetes-accelerated atherosclerosis. METHODS: Diabetes was induced in male Apoe(-/-) mice with five daily doses of streptozotocin (55 mg kg(-1) day(-1)). Atherosclerotic plaque size, markers of ROS and immune cell accumulation were assessed in addition to flow cytometric analyses of cells isolated from the adjacent mediastinal lymph nodes (meLNs). The role of NOX-derived ROS was investigated using the NOX inhibitor, GKT137831 (60 mg/kg per day; gavage) administered to diabetic and non-diabetic Apoe(-/-) mice for 10 weeks. RESULTS: Diabetes increased atherosclerotic plaque development in the aortic sinus and this correlated with increased lesional accumulation of T cells and CD11c(+) cells and altered T cell activation in the adjacent meLNs. Diabetic Apoe(-/-) mice demonstrated an elevation in vascular ROS production and expression of the proinflammatory markers monocyte chemoattractant protein 1, vascular adhesion molecule 1 and IFNγ. Blockade of NOX-derived ROS using GKT137831 prevented the diabetes-mediated increase in atherosclerotic plaque area and associated vascular T cell infiltration and also significantly reduced vascular ROS as well as markers of inflammation and plaque necrotic core area. CONCLUSIONS/INTERPRETATION: Diabetes promotes pro-inflammatory immune responses in the aortic sinus and its associated lymphoid tissue. These changes are associated with increased ROS production by NOX. Blockade of NOX-derived ROS using the NOX inhibitor GKT137831 is associated with attenuation of these changes in the immune response and reduces the diabetes-accelerated development of atherosclerotic plaques in Apoe(-/-) mice.

Fine-mapping of 5q12.1-13.3 unveils new genetic contributors to caries.

Caries is a multifactorial disease and little is still known about the host genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified the interval 5q12.1-5q13.3 as linked to low caries susceptibility in Filipino families. Here we fine-mapped this region in order to identify genetic contributors to caries susceptibility. Four hundred and seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. DMFT scores and genotype data of 75 single-nucleotide polymorphisms were evaluated in the Filipino families with the Family-Based Association Test. For replication purposes, a total 1,467 independent subjects from five different populations were analyzed in a case-control format. In the Filipino cohort, statistically significant and borderline associations were found between low caries experience and four genes spanning 13 million base pairs (PART1, ZSWIM6, CCNB1, and BTF3). We were able to replicate these results in some of the populations studied. We detected PART1 and BTF3 expression in whole saliva, and the expression of BTF3 was associated with caries experience. Our results suggest BTF3 may have a functional role in protecting against caries.

Urotensin II receptor antagonism confers vasoprotective effects in diabetes associated atherosclerosis: studies in humans and in a mouse model of diabetes.

AIMS/HYPOTHESIS: The small, highly conserved vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis. However, its effects in diabetes-associated atherosclerosis have not been assessed. METHODS: Endothelial cells were grown in normal- and high-glucose (5 and 25 mmol/l) media with and without UII (10⁻8 mol/l) and/or the UII receptor antagonist, SB-657510 (10⁻8 mol/l). Apoe knockout (KO) mice with or without streptozotocin-induced diabetes were treated with or without SB-657510 (30 mg kg⁻¹ day⁻¹; n = 20 per group) and followed for 20 weeks. Carotid endarterectomy specimens from diabetic and non-diabetic humans were also evaluated. RESULTS: In high (but not normal) glucose medium, UII significantly increased CCL2 (encodes macrophage chemoattractant protein 1 [MCP-1]) gene expression (human aortic endothelial cells) and increased monocyte adhesion (HUVECs). UII receptor antagonism in diabetic Apoe KO mice significantly attenuated diabetes-associated atherosclerosis and aortic staining for MCP-1, F4/80 (macrophage marker), cyclooxygenase-2, nitrotyrosine and UII. UII staining was significantly increased in carotid endarterectomies from diabetic compared with non-diabetic individuals, as was staining for MCP-1. CONCLUSIONS/INTERPRETATION: This is the first report to demonstrate that UII is increased in diabetes-associated atherosclerosis in humans and rodents. Diabetes-associated plaque development was attenuated by UII receptor antagonism in the experimental setting. Thus UII may represent a novel therapeutic target in the treatment of diabetes-associated atherosclerosis.

Polymorphisms in the fetal progesterone receptor and a calcium-activated potassium channel isoform are associated with preterm birth in an Argentinian population.

OBJECTIVE: To investigate genetic etiologies of preterm birth (PTB) in Argentina through evaluation of single-nucleotide polymorphisms (SNPs) in candidate genes and population genetic admixture. STUDY DESIGN: Genotyping was performed in 389 families. Maternal, paternal and fetal effects were studied separately. Mitochondrial DNA (mtDNA) was sequenced in 50 males and 50 females. Y-chromosome anthropological markers were evaluated in 50 males. RESULT: Fetal association with PTB was found in the progesterone receptor (PGR, rs1942836; P=0.004). Maternal association with PTB was found in small conductance calcium activated potassium channel isoform 3 (KCNN3, rs883319; P=0.01). Gestational age associated with PTB in PGR rs1942836 at 32-36 weeks (P=0.0004). MtDNA sequencing determined 88 individuals had Amerindian consistent haplogroups. Two individuals had Amerindian Y-chromosome consistent haplotypes. CONCLUSION: This study replicates single locus fetal associations with PTB in PGR, maternal association in KCNN3, and demonstrates possible effects for divergent racial admixture on PTB.

Defining predictors of cleft lip and palate risk.

Individuals with clefts present considerably more dental anomalies than individuals without clefts. We also have shown that these individuals report cancer in their families more often than do unaffected individuals. We investigated how these conditions correlated with genetic variants associated with clefts to ascertain if specific molecular signatures exist that could help identify individuals at risk for having offspring with these defects. We examined 573 individuals, 158 with clefts, 254 unaffected family members, and 161 non-related controls. Several clinical features, such as laterality, the presence of dental anomalies, medical history, and pregnancy history, were used to assess each individual's cleft status. Then, we performed molecular studies with genes that have been independently associated with oral clefts. We analyzed two datasets: nuclear families and case-control individuals where the case was the child from the family and controls were unrelated non-clefted individuals. In the family data, we confirmed association between clefts and rs987525 on chromosome 8 (p = 0.007) and found an association with rs987525 and tooth agenesis (p = 0.0003). In the case-control data, clefts, supernumerary teeth and familial cancer history were associated with ABCA4-rs481931 on chromosome 1 (p = 2E-19, 0.0007, 2E-06, respectively), and clefts and microdontia were associated with rs1325474 on chromosome 6 (p = 1E-06, 0.0002, respectively).

Association of AXIN2 with non-syndromic oral clefts in multiple populations.

We have previously shown the association of AXIN2 with oral clefts in a US population. Here, we expanded our study to explore the association of 11 AXIN2 markers in 682 cleft families from multiple populations. Alleles for each AXIN2 marker were tested for transmission distortion with clefts by means of the Family-based Association Test. We observed an association with SNP rs7224837 and all clefts in the combined populations (p = 0.001), and with SNP rs3923086 and cleft lip and palate in Asian populations (p = 0.004). We confirmed our association findings in an additional 528 cleft families from the United States (p < 0.009). We tested for gene-gene interaction between AXIN2 and additional cleft susceptibility loci. We assessed and detected Axin2 mRNA and protein expression during murine palatogenesis. In addition, we also observed co-localization of Axin2 with Irf6 proteins, particularly in the epithelium. Our results continue to support a role for AXIN2 in the etiology of human clefting. Additional studies should be performed to improve our understanding of the biological mechanisms linking AXIN2 to oral clefts.

Association of HbA1c levels with vascular complications and death in patients with type 2 diabetes: evidence of glycaemic thresholds.

AIMS/HYPOTHESIS: There is conflicting evidence regarding appropriate glycaemic targets for patients with type 2 diabetes. Here, we investigate the relationship between HbA(1c) and the risks of vascular complications and death in such patients. METHODS: Eleven thousand one hundred and forty patients were randomised to intensive or standard glucose control in the Action in Diabetes and Vascular disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. Glycaemic exposure was assessed as the mean of HbA(1c) measurements during follow-up and prior to the first event. Adjusted risks for each HbA(1c) decile were estimated using Cox models. Possible differences in the association between HbA(1c) and risks at different levels of HbA(1c) were explored using linear spline models. RESULTS: There was a non-linear relationship between mean HbA(1c) during follow-up and the risks of macrovascular events, microvascular events and death. Within the range of HbA(1c) studied (5.5-10.5%), there was evidence of 'thresholds', such that below HbA(1c) levels of 7.0% for macrovascular events and death, and 6.5% for microvascular events, there was no significant change in risks (all p > 0.8). Above these thresholds, the risks increased significantly: every 1% higher HbA(1c) level was associated with a 38% higher risk of a macrovascular event, a 40% higher risk of a microvascular event and a 38% higher risk of death (all p < 0.0001). CONCLUSIONS/INTERPRETATION: In patients with type 2 diabetes, HbA(1c) levels were associated with lower risks of macrovascular events and death down to a threshold of 7.0% and microvascular events down to a threshold of 6.5%. There was no evidence of lower risks below these levels but neither was there clear evidence of harm.

Soluble receptor for AGE (RAGE) is a novel independent predictor of all-cause and cardiovascular mortality in type 1 diabetes.

AIMS/HYPOTHESIS: Activation of the receptor for AGE (RAGE) is implicated in the development and progression of vascular complications of diabetes. In this study, we explore factors and mortality outcomes associated with soluble RAGE (sRAGE) in a multicentre nationwide cohort of Finnish adults with type 1 diabetes. METHODS: Baseline sRAGE concentrations were estimated in 3,100 adults with type 1 diabetes. Clinical and biological variables independently associated with sRAGE were identified using multivariate regression analysis. Independent predictors of mortality were determined using Cox and Fine-Gray proportional-hazards models. RESULTS: The main independent determinants of sRAGE concentrations were estimated glomerular filtration rate, albuminuria, body mass index, age, duration of diabetes, HbA(1c) and insulin dose (all p < 0.05). During a median of 9.1 years of follow-up there were 202 deaths (7.4 per 1,000 patient years). sRAGE was independently associated with all-cause (Cox model: HR 1.03) and cardiovascular mortality (Fine-Gray competing risks model: HR 1.06) such that patients with the highest sRAGE concentrations had the greatest risk of mortality, after adjusting for age, sex, macrovascular disease, HDL-cholesterol, HbA(1c), triacylglycerol, high-sensitivity C-reactive protein (hsCRP) and the presence and severity of chronic kidney disease. Although polymorphisms in the gene coding for RAGE were significantly associated with sRAGE concentrations, none were associated with mortality outcomes. CONCLUSIONS/INTERPRETATION: Increased concentrations of sRAGE are associated with increased all-cause and cardiovascular mortality in type 1 diabetes, potentially reflecting the activation and production of RAGE in the context of accelerated vascular disease. These novel findings highlight the importance of the RAGE activation in the prevention and management of diabetic complications.

Receptor for advanced glycation end-products (RAGE) provides a link between genetic susceptibility and environmental factors in type 1 diabetes.

AIMS/HYPOTHESIS: This group of studies examines human genetic susceptibility conferred by the receptor for advanced glycation end-products (RAGE) in type 1 diabetes and investigates how this may interact with a western environment. METHODS: We analysed the AGER gene, using 13 tag SNPs, in 3,624 Finnish individuals from the FinnDiane study, followed by AGER associations with a high risk HLA genotype (DR3)-DQA1*05-DQB1*02/DRB1*0401-DQB1*0302 (n = 546; HLA-DR3/DR4), matched in healthy newborn infants from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study (n = 373) using allelic analysis. We also studied islets and circulating RAGE in NODLt mice. RESULTS: The rs2070600 and rs17493811 polymorphisms predicted increased risk of type 1 diabetes, whereas the rs9469089 SNP was related to decreased risk, on a high risk HLA background. Children from the DIPP study also showed a decline in circulating soluble RAGE levels, at seroconversion to positivity for type 1 diabetes-associated autoantibodies. Islet RAGE and circulating soluble RAGE levels in prediabetic NODLt mice decreased over time and were prevented by the AGE lowering therapy alagebrium chloride. Alagebrium chloride also decreased the incidence of autoimmune diabetes and restored islet RAGE levels. CONCLUSIONS/INTERPRETATION: These studies suggest that inherited AGER gene polymorphisms may confer susceptibility to environmental insults. Declining circulating levels of soluble RAGE, before the development of overt diabetes, may also be predictive of clinical disease in children with high to medium risk HLA II backgrounds and this possibility warrants further investigation in a larger cohort.

Increased serum potassium affects renal outcomes: a post hoc analysis of the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) trial.

AIMS/HYPOTHESIS: To assess the effect of an angiotensin receptor blocker (ARB) on serum potassium and the effect of a serum potassium change on renal outcomes in patients with type 2 diabetes and nephropathy. METHODS: We performed a post hoc analysis in patients with type 2 diabetes participating in the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) study. Renal outcomes were defined as a composite of doubling of serum creatinine or end-stage renal disease. RESULTS: At month 6, 259 (38.4%) and 73 (10.8%) patients in the losartan group and 151 (22.8%) and 34 (5.1%) patients in the placebo group had serum potassium ≥5.0 mmol/l and ≥5.5 mmol/l, (p < 0.001), respectively. Losartan was an independent predictor for serum potassium ≥5.0 mmol/l at month 6 (OR 2.8; 95% CI 2.0-3.9). Serum potassium at month 6 ≥ 5.0 mmol/l was in turn associated with increased risk for renal events (HR 1.22; 95% CI 1.00-1.50), independent of other risk factors. Adjustment of the overall treatment effects for serum potassium augmented losartan's renoprotective effect from 21% (6-34%) to 35% (20-48%), suggesting that the renoprotective effects of losartan are offset by its effect on serum potassium. CONCLUSIONS/INTERPRETATION: In this study, we found that treatment with the ARB losartan is associated with a high risk of increased serum potassium levels, which is in turn associated with an increased risk of renal outcomes in patients with diabetes and nephropathy. Whether additional management of high serum potassium would further increase the renal protective properties of losartan is an important clinical question.

Delayed intervention with AGE inhibitors attenuates the progression of diabetes-accelerated atherosclerosis in diabetic apolipoprotein E knockout mice.

AIMS/HYPOTHESIS: Formation of AGEs is increased in the diabetic milieu, which contributes to accelerated atherogenesis. We studied whether delayed treatment with AGE-inhibiting compounds, alagebrium chloride and pyridoxamine dihydrochloride, affected established atherosclerosis in experimental diabetes in comparison with the angiotensin-converting enzyme inhibitor, quinapril. METHODS: Streptozotocin-induced diabetic male Apoe (-/-) mice (n = 24 per group) received, by oral gavage, from week 10 to 20 of diabetes: no treatment; alagebrium (1 mg kg(-1) day(-1)); pyridoxamine (1 g/l in drinking water); or quinapril (30 mg kg(-1) day(-1)). Atherosclerotic lesion area (en face analysis) was evaluated for all groups. RESULTS: Delayed intervention with alagebrium decreased plaque area in the diabetic Apoe (-/-) mice compared with untreated mice (total plaque area: alagebrium 10.6 ± 1.6%, untreated, 15.1 ± 1.5%, p < 0.05). This anti-atherosclerotic effect was comparable with that achieved with quinapril (quinapril 8.4 ± 1.4%, vs untreated, p < 0.05). Pyridoxamine also attenuated plaque development in diabetic mice (5.7 ± 1.2% vs untreated 11.9 ± 1.1%, p < 0.05). The anti-atherosclerotic effect conferred by alagebrium and quinapril was associated with a significant reduction in vascular oxidative stress and circulating AGEs and methylglyoxal, although preformed AGEs were not removed from the vascular wall with either delayed intervention. CONCLUSIONS/INTERPRETATION: Inhibition of AGE accumulation, using a delayed intervention with alagebrium or pyridoxamine, significantly attenuated the progression of established diabetes-associated atherosclerosis, similar to results obtained with quinapril. These findings provide further evidence that blockade of AGE-mediated pathways may present a novel therapy for the prevention of atherosclerosis in diabetes.

Large artery biomechanics and diastolic dysfunctionin patients with Type 2 diabetes.

AIMS: To comprehensively characterize large artery biomechanical properties and examine their relationship to cardiac function in patients with Type 2 diabetes mellitus (DM). METHODS: Fifty-five individuals with Type 2 DM were compared with 66 age- and sex-matched healthy control subjects. Arterial biomechanical properties were assessed by systemic arterial compliance (SAC; two-element Windkessel model), carotid-femoral pulse wave velocity (PWVc-f), femoral-dorsalis pedis pulse wave velocity (PWVf-d) and carotid augmentation index. Cardiac structure and function were assessed by echocardiography. RESULTS: Individuals with Type 2 DM had lower SAC and higher PWVc-f when compared with the healthy population. The PWVc-f was significantly lower than the PWVf-d in control individuals, but this difference was not evident in individuals with Type 2 DM due to higher PWVc-f. Augmentation index was similar in both groups, but the time to the first systolic inflection (time to reflection) was shorter in the individuals with Type 2 DM. The individuals with Type 2 DM had a greater prevalence of diastolic abnormalities when compared with the control group. Arterial stiffness indices, including SAC and pulse pressure, correlated with left ventricular filling pressure (defined as peak velocity during early diastolic filling divided by the velocity of movement of the mitral valve annulus in early diastole; r = -0.33 and 0.36 respectively. CONCLUSIONS: Patients with Type 2 DM on standard medication showed preferential stiffening of the large central arteries. However, carotid augmentation index was not different between the two groups and is therefore not a reliable indicator of large artery stiffening in this patient group. Diastolic dysfunction, present in a significant proportion of this population with Type 2 DM, was closely associated with arterial stiffening, suggesting a common aetiology.

Increased risk of cardiovascular disease in Type 1 diabetes: arterial exposure to remnant lipoproteins leads to enhanced deposition of cholesterol and binding to glycated extracellular matrix proteoglycans.

AIMS: To determine fasting and postprandial metabolism of apolipoprotein B48 (apoB48) remnant lipoproteins in subjects with Type 1 diabetes and the relationship to progressive cardiovascular disease, and to investigate the impact of remnant lipoprotein cholesterol accumulation associated with arterial wall biglycan using a rodent model of Type 1 diabetes. METHODS: Normolipidaemic subjects (n = 9) with long-standing Type 1 diabetes (and advanced cardiovascular disease) and seven healthy control subjects were studied. Fasting and postprandial apoB48 concentration was determined following a sequential meal challenge. A rodent model of streptozotocin-induced diabetes was used to investigate the ex vivo retention of fluorescent-conjugated remnants. Binding of remnant lipoproteins to human recombinant biglycan was assessed in vitro. RESULTS: A significantly higher concentration of fasting plasma apoB48 remnants was observed in patients with Type 1 diabetes compared with control subjects. Patients with Type 1 diabetes exhibited a greater total plasma apoB48 area under the curve (AUC) and an increased incremental AUC following a second sequential meal compared with control subjects. The arterial retention of remnants ex vivo and associated cholesterol was increased sevenfold in Type 1 diabetes rats relative to controls. Remnants were shown to bind with significant affinity to human biglycan in vitro and a further 2.3-fold increased binding capacity was observed with glycated biglycan. Remnants were shown to colocalize with both arterial biglycan and glycated matrix proteins in the Type 1 diabetes rodent model. CONCLUSION: Impaired metabolism of remnant lipoproteins associated with enhanced binding to proteoglycans appears to contribute to the arterial cholesterol deposition in Type 1 diabetes. Our findings support the hypothesis that impaired remnant metabolism may contribute to accelerated progression of atherosclerosis in the hyperglycaemic and insulin-deficient state.

Taste genes associated with dental caries.

Dental caries is influenced by a complex interplay of genetic and environmental factors, including dietary habits. Previous reports have characterized the influence of genetic variation on taste preferences and dietary habits. We therefore hypothesized that genetic variation in taste pathway genes (TAS2R38, TAS1R2, GNAT3) may be associated with dental caries risk and/or protection. Families were recruited by the Center for Oral Health Research in Appalachia (COHRA) for collection of biological samples, demographic data, and clinical assessment of oral health, including caries scores. Multiple single-nucleotide polymorphism (SNP) assays for each gene were performed and analyzed by transmission disequilibrium test (TDT) analysis (FBAT software) for three dentition groups: primary, mixed, and permanent. Statistically significant associations were seen in TAS2R38 and TAS1R2 for caries risk and/or protection.

Receptor for AGEs (RAGE) blockade may exert its renoprotective effects in patients with diabetic nephropathy via induction of the angiotensin II type 2 (AT2) receptor.

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) contributes to the development and progression of diabetic nephropathy. In this study, we examined whether the protective effects of RAGE blockade are exerted via modulation of the renal angiotensin II type 2 (AT2) receptor. METHODS: Control and streptozotocin diabetic mice, wild-type or deficient in the AT2 receptor (At2 knockout [KO]) or RAGE (Rage KO), were studied for 24 weeks. Adenoviral overexpression of full-length Rage in primary rat mesangial cells was also used to determine the effects on AT2 production. RESULTS: With diabetes, Rage-deficient mice had less albuminuria, and an attenuation of hyperfiltration and glomerulosclerosis as compared with diabetic wild-type and At2 KO mice. Renal gene and protein expression of RAGE was elevated with diabetes. Diabetic Rage KO mice had a greater increase in renal AT2 receptor protein than was seen in diabetic wild-type mice. Diabetes-induced increases in renal cytosolic and mitochondrial superoxide generation were prevented in diabetic Rage KO mice, but enhanced in all At2 KO mice. Adenoviral overexpression of RAGE or AGE treatment decreased cell surface AT2 expression, in association with increasing superoxide generation; both were reversed using antioxidants N-acetylcysteine and apocynin, and soluble RAGE in primary mesangial cells. CONCLUSIONS/INTERPRETATION: RAGE appears to be a common and key modulator of AT2 receptor expression, a finding that would implicate a newly defined RAGE-AT2 axis in the development and progression of diabetic nephropathy.

Angiotensin II subtype 2 receptor blockade and deficiency attenuate the development of atherosclerosis in an apolipoprotein E-deficient mouse model of diabetes.

AIMS/HYPOTHESIS: Most of the known actions of angiotensin II have been considered primarily to be the result of angiotensin II subtype 1 receptor activation. However, recent data suggest that the angiotensin II subtype 2 receptor (AT(2)R) may modulate key processes linked to atherosclerosis. The aim of this study was to investigate the role of AT(2)R in diabetes-associated atherosclerosis using pharmacological blockade and genetic deficiency. METHODS: Aortic plaque deposition was assessed in streptozotocin-induced diabetic apolipoprotein E (Apoe) knockout (KO) and At ( 2 ) r (also known as Agtr2)/Apoe double-KO (DKO) mice. Control and diabetic Apoe-KO mice received an AT(2)R antagonist PD123319 (5 mg kg(-1) day(-1)) via osmotic minipump for 20 weeks (n = 7-8 per group). RESULTS: Diabetes was associated with a sixfold increase in plaque area (diabetic Apoe-KO: 12.7 +/- 1.4% vs control Apoe-KO: 2.3 +/- 0.4%, p < 0.001) as well as a significant increase in aortic expression of the gene At ( 2 ) r (also known as Agtr2). The increase in plaque area with diabetes was attenuated in AT(2)R antagonist-treated diabetic Apoe-KO mice (7.1 +/- 0.5%, p < 0.05) and in diabetic At ( 2 ) r/Apoe DKO mice (9.2 +/- 1.3%, p < 0.05). These benefits occurred independently of glycaemic control or BP, and were associated with downregulation of a range of pro-inflammatory cytokines, adhesion molecules, chemokines and various extracellular matrix proteins. CONCLUSIONS/INTERPRETATION: This study provides evidence for AT(2)R playing a role in the development of diabetes-associated atherosclerosis. These findings suggest a potential utility of AT(2)R blockers in the prevention and treatment of diabetic macrovascular complications.