Angiotensinogen promotor haplotype dependent regulation of angiotensinogen gene expression

Microangiopathy related cerebral damage (MARCD) is a common MRI observation in the elderly. Its clinical manifestation includes cognitive impairment, depression and gait disturbances, common causes of dysability in older individuals. The only known risk factors are age and hypertension. The heritability of MARCD was reported to be 73%. In an effort to identify specific genetic factors responsible for this suprisingly high heritability we investigated the role of the angiotensinogen (AGT) gene, a major determinant of blood pressure. We showed that the AGT gene encompasses 5 unique haplotypes (A,B,C,D and E) determined by 4 polymorphisms at positions - 6:g/a, -20:a/c, -153:g/a, and -218:g/a in its regulatory (promoter) region. We observed a highly significant association between the B haplotype (-6:a, -20:c, -153:g, -218:g) and MARCD (p=0.0003). Interestingly, the association was independent of hypertension, suggesting that the effect of the haplotype is mediated by the local and not by the systemic renin-angiotensin system (RAS). The B haplotype carries the -6:a and the -20:c mutations, which were previously indicated as functionally important. We found however, that none of these single nucleotide mutations was as strongly associated with microangiopathy as the B haplotype. We hypothesize that the positive association between AGT gene variants and MARCD is due to an altered expression of AGT in astrocytes, which are the major producers of AGT in the brain. Since AGT is a potent regulator of smooth muscle cell growth and contractibility, matrix protein syntheses and the oxidative state of the vessel wall, alterations in the local availability of AGT may causally be related to microangiopathy. In our preliminary experiments the B haplotype was associated with an about 6 fold increased promoter activity. The aim of the present project is to further elucidate the role of the haplotype in AGT expression. First, we will explore which combination of nucleotide changes are sufficient to alter promoter activity. The sequence elements mediating the altered promoter activity will be then elaborated by deletional and substitutional mutants. Identification of nuclear proteins binding to these elements will be initiated. The results of this study may not only extend our etiologic understanding of cerebral microangiopathy but might also point to possible favorable effects of drugs acting on the RAS beyond those expected from lowering blood pressure alone.

Cerebral small vessel disease (cSVD) is endemic in elderly populations. Its morphologic correlates include white matter lesions and lacunes in the brain. In population-based MRI studies the prevalence of such lesions is upto 88% at the age of 55-years and 92% at the age of 65-years. Lacunes are seen with a frequency of 20% above the age of 60-years. cSVD enhances the risk for stroke, cognitive impairment, gait disturbances and falls in the elderly, and is an important cause for invalidity in this age group. Its socioeconomic impact is significant. Inspite of intensive research there is still no effective treatment available to stop or at least to slow down progression of this disease. The only well-documented risk-factors for cSVD are age and hypertension, although only half of the patients with severe lesions has higher blood pressure. Recently twin studies had shown the relevance of heritability in cSVD. Association studies including our own investigations identified genetic factors such as genetic variants at the Renin-Angiotensin System (RAS), which enhance the risk for cSVD. We found a strong and significant increased risk for cSVD in persons, who carry a genetic variant of the Angiotensinogen gene promoter. In the present project we aimed to evaluate the functional relevance of this promoter variant in relation to cSVD. In cell experiments we showed that the promoter variant has an increased transcriptional activity in astrocytes. This may lead to an increased RAS activity in the brain. Increased RAS activity has multiple effects on the vessel wall, such as vasoconstriction, thickening of the vessel wall or impaired permeability of the blood-brain barrier. All of these processes might be involved in the development of cSVD. A further though indirect support for the functional relevance of angiotensinogen variant comes from our genetic epidemiological study showing that this variant represents an evolutionary ancient AGT-promoter. We expect that the results of this project will pinpoint to novel therapeutic strategies for cSVD. Our findings suggest that RAS inhibitors, which are already widely used as save and efficient antihypertensive agents, may also positively influence the course of cSVD.