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of VEGF gene variability in A lesson from the Italian population

Del Bo , Serena Ghezzi , Marina Scarlato , Diego Albani , Daniela Galimberti b b a b Ugo Lucca , Mauro Tettamanti , Elio Scarpini , Gianluigi Forloni , , a c a Nereo Bresolin , Giacomo Pietro Comi

a Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Foundation Ospedale Maggiore Mangiagalli and Regina Elena, Padiglione Ponti, Via F. Sforza 35, 20122 Milano, Italy b Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy c

Abstract

Vascular endothelial growth factor (VEGF) gene polymorphisms have been associated with an increased risk of developing a wide variety of disorders from diabetes to neurodegenerative diseases suggesting functions not confined to its vascular effects originally described. Based on the VEGF protective roles undisclosed in pathological conditions, we evaluate whether VEGF variability might be a determinant also for longevity. Four polymorphisms (−2578C/A, −1190G/A, −1154G/A and −634G/C) within the VEGF gene promoter region in 490 unrelated Italian healthy subjects have been analysed. Significant changes of allele, genotype (−2578/AA versus −2578/CC: OR=2.08, p=0.007; −1190/AA versus −1190/GG: OR=2.01, p=0.011) and haplotype (AAGG: 10.4% versus 14.9%, p=0.03) frequency distributions were observed between young/elderly (25–84 years old) and long-lived (85–99 years old) subjects. These results suggest that VEGF gene variability can be inserted among the genetic factors influencing the lifespan.

Keywords: Aging; Vascular endothelial growth factor; Genetic risk factor; Longevity

1. Introduction

Vascular endothelial growth factor (VEGF) is an impor- tant angiogenesis cytokine that undergoes transcriptional and post-transcriptional induction by hypoxia. It has been implicated in several pathological conditions, from tumour proliferation to inflammatory and ischemic processes (Carmeliet, 2003; Ferrara et al., 2003). VEGF has been originally identified on the basis of its vascular effects, but recently has been recognised as an important sig- nalling molecule in the nervous system, as well as in neurodegenerative disorders and in peripheral neuropathies (Rosenstein and Krum, 2004; Storkebaum and Carmeliet, 2004). An age-dependent decline in VEGF expression has been reported in different tissues, however the molecu- lar alterations responsible for this reduction have not been

Longevity represents a very complex phenomenon due to the interaction among genetic, environmental and lifestyle factors. Identification of genetic and non-genetic factors involved in aging has progressed extensively in the recent years because of increased interest in defining the deter- minants of human life expectancy. It has been suggested that genes and biochemical factors likely to be impli- cated in aging-related disorders may have an important role also in human longevity. Among genetic markers, sev- eral variants in pro- or anti-inflammatory cytokines and in vascular factors have been shown to affect successful

∗ Corresponding author. Tel.: +39 02 55033843; fax: +39 02 50320430. E-mail address: roberto.delbo@unimi.it (R. Del Bo).

2.2. Genetic analysis

elucidated. A number of polymorphisms localised within the VEGF gene promoter region have been linked to increased susceptibility to multiple angiogenesis-dependent diseases and age-related neurological disorders in humans (Del Bo et al., 2005; Lambrechts et al., 2003). Individu- − als homozygous for the haplotypes involving the 2578AA, − − 1154A/G and 634GG variants had lower serum VEGF levels and increased risk of developing amyotrophic lateral − − − sclerosis (ALS). C( 2578)A, C( 1198)T, G( 1190)A and − G( 1154)A polymorphisms have been correlated with the risk of developing Alzheimer’s disease (AD). In addition, the same polymorphisms are able to alter basal and post- stimulation promoter activity in human transfected MCF7 cells (Stevens et al., 2003). Finally, an association has − − been reported between 1154 and 2578 genotypes and VEGF production in the peripheral blood mononucleated cells of healthy subjects (Shahbazi et al., 2002). All these effects are dependent on several sequence polymorphisms, suggesting that VEGF gene variability may contribute to the inherited predisposition to VEGF-mediated pathological conditions.

Total DNA was isolated from peripheral blood accord- ing to standard protocols. Three genomic DNA regions containing portions of the VEGF promoter were amplified by PCR (for primer sequences, see Supplementary data). ◦ Amplification protocol was the following: 5 min at 94 C ◦ for the first cycle, denaturation at 94 C for 30 s, anneal- ◦ ◦ ing at 55–60 C for 30 s, extension at 72 C for 40 s for ◦ the subsequent 35 cycles, and a final extension at 72 C − for 5 min. To detect the polymorphism C( 2578)A, frag- ments were directly electrophoresed on a 3% agarose gel because of an 18-nucleotide insertion always associated with − the 2578A allele, whereas CC homozygotes do not contain − this insertion. The polymorphism G( 634)C was detected − through PCR followed by MwoI digestion. 1190G/A and − 1154G/A variants were detected through direct sequenc- ing analysis of PCR fragments using BigDyeTerminator TM protocol on an automated 3100ABI Prism Genetic Analyzer (Applied Biosystem, Foster City, CA).

All subjects were genotyped for the APOE status, as described (Del Bo et al., 1997).

Aim of the present study is to explore the VEGF gene promoter variability as a genetic determinant for longevity in an Italian population.

2.3. Statistical analysis