ageing and had a significant prolongation of lifespan than wild-type littermates (Benigni et al. 2009). In rodents, two isoforms of AT R have been identified, 1 AT R and AT R, while human cells express a single 1A 1B AT receptor (AT R). Angiotensin II (AngII) has been 1 1 involved in organ senescence, given its capacity to robustly stimulate mitochondrial production of reactive oxygen species (ROS) that are crucially implicated in age-related organ damage including heart, kidney and brain (Wilson 1990; de Cavanagh et al. 2007) and contribute to the plethora of clinical phenotypes associ- ating with ageing (Min et al. 2009). Over time, exuber- ant ROS production exceeds the antioxidant defence of the mitochondrial and the nuclear enzymes, so that accumulating damage untimely destroys cells by apo- ptosis or necrosis (Benigni et al. 2010). The protection observed in AT R−/− mice from vascular damage and 1A the associated extension of lifespan were interpreted as a consequence of mitochondrial integrity provided by the expression of survival genes that limited oxidative stress (Benigni et al. 2009). These findings prompted us to investigate the possible role of variations of AGTR1 in influencing human longevity, with the hypothesis that mutations causing reduced AT R expression levels 1 might associate to prolonged lifespan in humans as observed in AT R-deficient mice. Our studies identi- 1A fied two allelic variants in AGTR1 promoter which are significantly overrepresented in Italian centenarians. This association was subsequently replicated in an in- dependent population of Japanese centenarians and controls.

The Japanese replication sample consisted of 589 – centenarians/semi-supercentenarians ageing 99 115 years (mean 104.5±3 years). The gender ratio was 84 % females to 16 % males. Centenarians and semi- supercentenarians were recruited through year 2000 un- til now through four series of screening involving the Tokyo metropolitan area; the centenarians address books published by Ministry of Health, Labor and Wel- fare from 1997 to 2002; all the Japanese nursing homes; the centenarian address lists available at local prefec- tures; and finally through articles published by local newspapers. Younger controls (between 69 and 72 years of age, mean 70.1±0.9 years, 52 % females and 48 % males) were 422 volunteers from Itami City and Asago City, Hyogo prefecture, Japan, who participated in SONIC Study for elderly subjects to investigate factors related to successful ageing in Japan. Written informed consent was obtained from all study participants.

DNA analysis of

In the Italian group AGTR1 exons, their flanking ′ ′ regions, the 5 - and 3 -UTR regions (including ′ 250 bp of the promoter upstream 5 -UTR) were ampli- fied from genomic DNA and sequenced in the two directions on an ABI 3730 DNA analyser (Applied Biosystems). Primers (Online Resource 1) were designed according to AGTR1 genomic reference NC_000003, built 37.1, at National Center for Biotech- nology Information. Variants are referred to transcript variant 4 (NM_031850) RNA reference sequence and numbering begins with the ATG start codon. For quality control purposes, 5 % random sample was sequenced twice. Moreover, subjects carrying very rare genotypes (<5 %) were confirmed by repeating PCR reactions, followed by resequencing of both DNA strands.

Also, we demonstrate that the allelic variants in the promoter of AGTR1 are associated with reduced AT R 1 receptor expression levels in peripheral blood neutro- phils. In addition, screening of the whole coding region of AGTR1 in Italian cohort disclosed in one centenarian a novel mutation leading to amino acid substitution. Finally, we disclose an association between variants in AGTR1 promoter and diastolic blood pressure in centenarians.

Methods

For the replication study in the Japanese group, SNP genotyping was performed by TaqMan SNP assay (Applied Biosystems). The genotyping conditions for

A cohort of 173 Italian centenarians (between 99 and 106 years of age, mean 100.9±1.7 years) living in

Analysis of AT R presence in peripheral leukocytes 1

Fresh blood samples were processed for peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNs) purification as described (Noris et al. 1999; Noris et al. 1996), labelled with an anti-human AT R antibody 1 (Anti AT-1, N-10, Santa Cruz) followed by incubation ’ with fluorescein isothiocyanate goat F(ab )2 fragment anti-rabbit IgG (eBioscience). To identify the various leukocyte subsets, cells were stained with fluorochrome- conjugated mAb antibodies against CD45, CD16, CD14, CD3, CD4 (BD Bioscience and Biolegend). The samples were analysed by fluorescence activated cell sorting (FACS) analysis using FACSAria cytometer (BD Biosci- ence). Results were expressed as percentage of AT R 1 positive cells (percent).

Results