Helen Imrie

Piezo1 integration of vascular architecture with physiological force

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

A Common and Recurrent 13-bp Deletion in the Autoimmune Regulator Gene in British Kindreds with Autoimmune Polyendocrinopathy Type 1

Autoimmune polyendocrinopathy type 1 (APS1) is an autosomal recessive disorder characterized by autoimmune hypoparathyroidism, autoimmune adrenocortical failure, and mucocutaneous candidiasis. Recently, an autoimmune regulator gene (AIRE-1), which is located on chromosome 21q22.3, has been identified, and mutations in European kindreds with APS1 have been described. We used SSCP analysis and direct DNA sequencing to screen the entire 1,635-bp coding region of AIRE-1 in 12 British families with APS1. A 13-bp deletion (964del13) was found to account for 17 of the 24 possible mutant AIRE-1 alleles, in our kindreds. This mutation was found to occur de novo in one affected subject. A common haplotype spanning the AIRE-1 locus was found in chromosomes that carried the 964del13 mutation, suggesting a founder effect in our population. One of 576 normal subjects was also a heterozygous carrier of the 964del13 mutation. Six other point mutations were found in AIRE-1, including two 1-bp deletions, three missense mutations (R15L, L28P, and Y90C), and a nonsense mutation (R257*). The high frequency of the 964del13 allele and the clustering of the other AIRE-1 mutations may allow rapid molecular screening for APS1 in British kindreds. Furthermore, the prevalence of the 964del13 AIRE-1 mutation may have implications in the pathogenesis of the more common autoimmune endocrinopathies in our population.

Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction

Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance–induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance–mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance–related vascular disease.

Evidence for a New Graves Disease Susceptibility Locus at Chromosome 18q21

Graves disease (GD) is a common autoimmune thyroid disorder that is inherited as a complex multigenic trait. By using a single microsatellite marker at each locus, we screened the type 1 diabetes loci IDDM4, IDDM5, IDDM6, IDDM8, and IDDM10 and the fucosyltransferase-2 locus for linkage in sib pairs with GD. This showed a two-point nonparametric linkage (NPL) score of 1.57 (P=.06) at the IDDM6 marker D18S41, but NPL scores were <1.0 at the other five loci. Thus, the investigation of the IDDM6 locus was extended by genotyping 11 microsatellite markers spanning 48 cM across chromosome 18q12-q22 in 81 sib pairs affected with autoimmune thyroid disease (AITD). Multipoint analysis, designating all AITD sib pairs as affected, showed a peak NPL score of 3.46 (P=.0003), at the marker D18S487. Designation of only GD cases as affected (74 sib pairs) showed a peak NPL score of 3.09 (P=.001). Linkage to this region has been demonstrated in type 1 diabetes (IDDM6), rheumatoid arthritis, and systemic lupus erythematosus, which suggests that this locus may have a role in several forms of autoimmunity.

The Insulin-Like Growth Factor-1 Receptor Is a Negative Regulator of Nitric Oxide Bioavailability and Insulin Sensitivity in the Endothelium

OBJECTIVE In mice, haploinsufficiency of the IGF-1 receptor (IGF-1R+/−), at a whole-body level, increases resistance to inflammation and oxidative stress, but the underlying mechanisms are unclear. We hypothesized that by forming insulin-resistant heterodimers composed of one IGF-1Rαβ and one insulin receptor (IR), IRαβ complex in endothelial cells (ECs), IGF-1R reduces free IR, which reduces EC insulin sensitivity and generation of the antioxidant/anti-inflammatory signaling radical nitric oxide (NO). RESEARCH DESIGN AND METHODS Using a number of complementary gene-modified mice with reduced IGF-1R at a whole-body level and specifically in EC, and complementary studies in EC in vitro, we examined the effect of changing IGF-1R/IR stoichiometry on EC insulin sensitivity and NO bioavailability. RESULTS IGF-1R+/− mice had enhanced insulin-mediated glucose lowering. Aortas from these mice were hypocontractile to phenylephrine (PE) and had increased basal NO generation and augmented insulin-mediated NO release from EC. To dissect EC from whole-body effects we generated mice with EC-specific knockdown of IGF-1R. Aortas from these mice were also hypocontractile to PE and had increased basal NO generation. Whole-body and EC deletion of IGF-1R reduced hybrid receptor formation. By reducing IGF-1R in IR-haploinsufficient mice we reduced hybrid formation, restored insulin-mediated vasorelaxation in aorta, and insulin stimulated NO release in EC. Complementary studies in human umbilical vein EC in which IGF-1R was reduced using siRNA confirmed that reducing IGF-1R has favorable effects on NO bioavailability and EC insulin sensitivity. CONCLUSIONS These data demonstrate that IGF-1R is a critical negative regulator of insulin sensitivity and NO bioavailability in the endothelium.

CTLA4 gene and Graves’ disease: association of Graves’ disease with the CTLA4 exon 1 and intron 1 polymorphisms, but not with the promoter polymorphism

objective Recent studies have shown that Graves’ disease (GD) is linked to and associated with alleles of the cytotoxic T lymphocyte antigen‐4 (CTLA4) locus. However, the true pathogenic polymorphism(s) at this locus remains uncertain. Moreover, the association studies of the promoter CTLA4(−318)C/T polymorphism in white GD populations have produced conflicting results. Therefore, we have analysed three CTLA4 single nucleotide polymorphisms, including promoter CTLA4(−318)C/T, exon 1 CTLA4(49)A/G and intron 1 CTLA4(1822)C/T in our GD cohort from the UK.

Further Evidence for a Susceptibility Locus on Chromosome 20q13.11 in Families with Dominant Transmission of Graves Disease

To the Editor: The susceptibility loci for Graves disease (GD [MIM 275000]), which is a common complex trait (Brix et al. 1998), have been difficult to define (Roman et al. 1992; McLachlan 1993; Davies 1998; Farid 1998; Vaidya et al. 1999). Tomer et al. (1998) recently found evidence for linkage of GD to markers on the long arm of chromosome 20 (MIM 603388), with a peak multipoint LOD score of 3.5 at the marker D20S195. Their linkage analysis was performed by both parametric and nonparametric methods, and their cohort of 53 families with at least two first-degree relatives affected with autoimmune thyroid disease (AITD) was derived from the North American, Italian, Israeli, and British populations (Tomer et al. 1998). We have examined this chromosomal region in a homogeneous cohort of 71 affected GD sib pairs derived from 64 multiplex British GD kindreds (146 subjects with GD, 20 with autoimmune hypothyroidism [MIM 140300], and 72 unaffected). In six families, an additional sibling had autoimmune hypothyroidism, resulting in a total of 77 affected sib pairs with AITD (i.e., either GD or autoimmune hypothyroidism) (table 1). Parents (n=49) and unaffected sibs (n=36) were studied wherever available. All subjects were white, and >95% of the grandparents were from the mainland United Kingdom or were of Irish origin. The clinical definitions of GD and autoimmune hypothyroidism were identical to those described elsewhere (Tomer et al. 1998). Fifty-four (37%) of the patients with GD had significant thyroid-associated orbitopathy (class 3 or worse) (Werner 1977). Background allele frequencies were derived from typing of DNA obtained from local subjects without evidence of autoimmune disease. Nonparametric, parametric, and exclusion-mapping analyses were performed with the use of the GENEHUNTER package, version 2.0 (Kruglyak et al. 1996). For parametric analyses, a population frequency of 1% for GD was assumed, with a nonsusceptibility-genotype penetrance of .005, and allele frequencies were varied, according to Hardy-Weinberg equilibrium, for each susceptibility-genotype penetrance studied. Table 1 Phenotypes of Affected Sib Pairs with AITD Multipoint nonparametric analysis with the use of five microsatellite markers spanning a 21-cM area of 20q13.11 showed no evidence to support linkage in the 71 GD sib pairs, with a peak NPL (nonparametric linkage) score of 0.1 occurring at the marker D20S884 (fig. 1). We were able to formally exclude (LOD score 2.5 from this entire region (fig. 1). Parametric analysis was performed both with and without the assumption of heterogeneity, with both recessive and dominant models. There was no evidence for linkage of GD to this region at disease penetrances of 30%, 60%, or 90%, with either model of inheritance, in the 71 sib pairs (table 2). Figure 1 Linkage analysis in all 64 affected GD kindreds. A, Percentage information content shown at each of the map positions. B, Multipoint nonparametric linkage analysis of kindreds with GD for chromosome 20q13.11 markers. Genotyping was performed by PCR with ... Table 2 Peak Multipoint Parametric LOD Scores for the Chromosome 20q13.11 Markers in the 64 Families with GD and in the Subset of 12 Kindreds with Dominant Transmission of GD The ascertainment strategy (at least two affected sibs with GD) used to recruit families for our study was different from that (at least two affected first-degree relatives with AITD) used by Tomer et al., such that their cohort of families was likely to contain many more affected parent-offspring kindreds (Tomer et al. 1998). We speculated that such affected parent-offspring kindreds might have enriched their cohort for families segregating dominant loci and that this difference in ascertainment might explain the apparent discrepancy between our findings, if the susceptibility locus segregated as a dominant (McCarthy et al. 1998). Therefore, we investigated linkage both in a subgroup of 12 families (38 subjects with GD) who had apparent dominant transmission of GD from parent to offspring and in a subgroup of 28 families with dominant transmission of AITD from parent to offspring (75 subjects with GD and 17 with autoimmune hypothyroid). Multipoint nonparametric analysis in the 12 families with dominant transmission of GD showed a 4-cM plateau suggestive of linkage, with a peak NPL score of 2.02 (P=.023) occurring at the marker D20S106 (fig. 2). This was not observed in the larger subgroup of 28 families with parent-to-offspring transmission of AITD (fig. 2). Parametric analysis in the subgroup with dominant transmission of GD, with the assumption of heterogeneity, showed a peak LOD score of 1.06 occurring at the marker D20S884 with a dominant model (table 2). Figure 2 Linkage analysis of the subset of 12 GD kindreds with dominant transmission of GD, and other groups. A, Percentage information content for the 12 families with dominant transmission of GD is shown at each of the map positions. B, Multipoint nonparametric ... Our study provides some evidence to support the presence of a GD-susceptibility locus in this region of 20q13.11 (Tomer et al. 1998), and we show that this locus appears to be important only in families with dominant inheritance of GD. The small number of such kindreds that we have studied precludes a reliable estimate of the strength of effect of this locus, but our ability to detect the effect using only 12 families with this structure, coupled with the 1:0 allele-sharing ratio of 69% between the sib pairs with GD, suggests that it may have a strong effect. In contrast, our families with affected subjects with GD in only one generation and our families with dominant transmission of AITD do not show evidence of linkage to this locus (figs. 1 and ​and2).2). Analysis of a larger cohort of kindreds with dominant transmission of GD is necessary to confirm the presence of this susceptibility locus for GD. However, the recent mapping of a susceptibility locus for systemic lupus erythematosus (MIM 152700) to this region of chromosome 20 in two different mixed American cohorts (Gaffney et al. 1998; Moser et al. 1998) suggests that this region may harbor a polymorphism(s) that is important in other autoimmune disorders. In addition, our study illustrates that the ascertainment strategies employed in the collection of cohorts of kindreds with complex disorders may have a marked effect on the ability to detect a given susceptibility locus (McCarthy et al. 1998).

Insulin Resistance Impairs Circulating Angiogenic Progenitor Cell Function and Delays Endothelial Regeneration

OBJECTIVE Circulating angiogenic progenitor cells (APCs) participate in endothelial repair after arterial injury. Type 2 diabetes is associated with fewer circulating APCs, APC dysfunction, and impaired endothelial repair. We set out to determine whether insulin resistance adversely affects APCs and endothelial regeneration. RESEARCH DESIGN AND METHODS We quantified APCs and assessed APC mobilization and function in mice hemizygous for knockout of the insulin receptor (IRKO) and wild-type (WT) littermate controls. Endothelial regeneration after femoral artery wire injury was also quantified after APC transfusion. RESULTS IRKO mice, although glucose tolerant, had fewer circulating Sca-1+/Flk-1+ APCs than WT mice. Culture of mononuclear cells demonstrated that IRKO mice had fewer APCs in peripheral blood, but not in bone marrow or spleen, suggestive of a mobilization defect. Defective vascular endothelial growth factor–stimulated APC mobilization was confirmed in IRKO mice, consistent with reduced endothelial nitric oxide synthase (eNOS) expression in bone marrow and impaired vascular eNOS activity. Paracrine angiogenic activity of APCs from IRKO mice was impaired compared with those from WT animals. Endothelial regeneration of the femoral artery after denuding wire injury was delayed in IRKO mice compared with WT. Transfusion of mononuclear cells from WT mice normalized the impaired endothelial regeneration in IRKO mice. Transfusion of c-kit+ bone marrow cells from WT mice also restored endothelial regeneration in IRKO mice. However, transfusion of c-kit+ cells from IRKO mice was less effective at improving endothelial repair. CONCLUSIONS Insulin resistance impairs APC function and delays endothelial regeneration after arterial injury. These findings support the hypothesis that insulin resistance per se is sufficient to jeopardize endogenous vascular repair. Defective endothelial repair may be normalized by transfusion of APCs from insulin-sensitive animals but not from insulin-resistant animals.

A rare variant of the leptin gene has large effects on blood pressure and carotid intima-medial thickness: a study of 1428 individuals in 248 families.

Background: Rare mutations in the leptin (LEP) gene cause severe obesity. Common polymorphisms of LEP have been associated with obesity, but their association with cardiovascular disease has been little studied. We have examined the impact of both common and rare polymorphisms of the LEP gene on blood pressure (BP), subclinical atherosclerosis as measured by carotid intima-medial thickness (CIMT), and body mass index (BMI) in a large family study. Methods: Five polymorphisms spanning LEP were typed in 1428 individuals from 248 nuclear families. BP, CIMT, BMI, and plasma leptin were measured. Results: The polymorphisms typed captured all common haplotypes present at LEP. There was strong association between a rare polymorphism in the 3′ untranslated region of LEP (C538T) and both pulse pressure (p = 0.0001) and CIMT (p = 0.008). C/T heterozygotes had a 22% lower pulse pressure and a 17% lower CIMT than C/C homozygotes. The polymorphism accounted for 3–5% of the population variation in pulse pressure and CIMT. There was no association between any LEP polymorphism and either BMI or plasma leptin level. Conclusions: This large family study shows that the rare T allele at the C538T polymorphism of LEP substantially influences pulse pressure and CIMT, but does not appear to exert this effect through actions on plasma leptin level or BMI. This suggests that autocrine or paracrine effects in vascular tissue may be important physiological functions of leptin. This study also provides evidence that rare polymorphisms of particular genes may have substantial effects within the normal range of certain quantitative traits.

Increasing Circulating IGFBP1 Levels Improves Insulin Sensitivity, Promotes Nitric Oxide Production, Lowers Blood Pressure, and Protects Against Atherosclerosis

Low concentrations of insulin-like growth factor (IGF) binding protein-1 (IGFBP1) are associated with insulin resistance, diabetes, and cardiovascular disease. We investigated whether increasing IGFBP1 levels can prevent the development of these disorders. Metabolic and vascular phenotype were examined in response to human IGFBP1 overexpression in mice with diet-induced obesity, mice heterozygous for deletion of insulin receptors (IR+/−), and ApoE−/− mice. Direct effects of human (h)IGFBP1 on nitric oxide (NO) generation and cellular signaling were studied in isolated vessels and in human endothelial cells. IGFBP1 circulating levels were markedly suppressed in dietary-induced obese mice. Overexpression of hIGFBP1 in obese mice reduced blood pressure, improved insulin sensitivity, and increased insulin-stimulated NO generation. In nonobese IR+/− mice, overexpression of hIGFBP1 reduced blood pressure and improved insulin-stimulated NO generation. hIGFBP1 induced vasodilatation independently of IGF and increased endothelial NO synthase (eNOS) activity in arterial segments ex vivo, while in endothelial cells, hIGFBP1 increased eNOS Ser1177 phosphorylation via phosphatidylinositol 3-kinase signaling. Finally, in ApoE−/− mice, overexpression of hIGFBP1 reduced atherosclerosis. These favorable effects of hIGFBP1 on insulin sensitivity, blood pressure, NO production, and atherosclerosis suggest that increasing IGFBP1 concentration may be a novel approach to prevent cardiovascular disease in the setting of insulin resistance and diabetes.