Rubattu et al Fig. 1 - Schematic representation of the major pathological effects induced by the ANP gene alterations, i.e. structural mutations, ab- lation and overexpression. The modulation of cardiac mass described in both knock out and transgenic mice is independent of BP levels. and functional alterations of this gene in the disease prone rat, but not in its related control, the stroke-resis- tant rat (8). In fact, presence of a promoter gene muta- tion was associated with a significantly reduced tran- scription of ANP in the stroke-prone rat (9). Moreover, a coding mutation, responsible for a Gly-Ser transposi- tion within the exon 2 of the SHRsp gene, was associat- ed with a significant reduction of endothelial cell prolif- eration and of the formation of new vessels (10). Based on robust experimental evidence, investiga- tion of the role of ANP gene into the predisposition to develop stroke in humans was also carried out. Thus, it was demonstrated that carrier status for two coding mutations, an exon 1 and an exon 3 variants, were associated with an increased risk of cerebrovas- cular accidents in distinct human populations. A Val- Met transposition within exon 1 was associated with a two-fold increased risk of stroke in the Physician’s Health Study (11), whereas a stop codon mutation lead- ing to a 30 rather than 28 aa long alpha-ANP was asso- ciated with a four-fold increased risk of primary stroke and with a three-fold increased risk of secondary stroke in the Sardinian Stroke Study (12). The contributory ef- fect of ANP gene molecular variants to human stroke was independent from all conventional risk factors, such as hypertension, diabetes, obesity, hypercholes- terolemia, and even atrial fibrillation. Current studies are exploring the possible functional implications of the ANP gene molecular variants and the putative disease mechanisms underlying the increased predisposition to develop vascular lesions in carriers of such peptide variants. In this regard, in vitro studies have previously docu- mented an antiproliferative effect of regular ANP on rat cultured vascular cells (13). In fact, exposure of rat en- dothelial cells to ANP stimulated the process of apopto- sis through the induction of p53 and the inhibition of Bcl2 proteins (14). The proapoptotic effect of ANP was inhibited by specific blockade of the NPR-A receptor. Consistent results were obtained in myocardiocytes (15). Of interest, parallel epidemiological observations have reinforced the etiopathogenetic contribution of ANP to cardiovascular events. Results from the Fram- ingham population have underscored a predictive role of high circulating levels of natriuretic peptides towards an increased mortality due to cardiovascular causes (16). Moreover, high plasma levels of natriuretic pep- tides at the time of either a stroke or a myocardial in- farction have been correlated with a negative prognos- tic value for the patient, since they are associated with an increased mortality rate within the following five years (17). ANP and hypertension Convincing evidence in favour of a contributory role of ANP in the development of hypertension has been provided only recently through genetic approaches in experimental animal models (18-20). In fact, ablation of ANP gene in mice was associated with the development of salt-sensitive hypertension (18), whereas the overex- pression of the ANP gene in transgenic mice was asso- ciated with reduced blood pressure levels (19). On the other hand, ablation of the ANP receptor (NPR-A) gene caused hypertension as well, although not of the salt- sensitive type (20). Testing of ANP gene, through the candidate gene ap- proach, in human hypertensive populations has led so far to evidence of a significant association only in cer- tain ethnic groups, such as African Americans (21). Some positive results were also obtained with the char- acterization of NPR-A gene (22). However, a more direct demonstration of a causal role of both ANP and NPR-A genes in the predisposition to develop hypertension in humans is still lacking. Current studies are trying to clar- ify this issue (unpublished observations from our group). 79