Alessia Lombardi

Modulation of the AT2 subtype receptor gene activation and expression by the AT1 receptor in endothelial cells.

OBJECTIVE To investigate whether angiotensin II type 2 (AT2) receptor (AT2-r) promoter activity and expression are modulated by angiotensin II (Ang II), and whether the AT1 receptor (AT1-r) is involved in this effect. DESIGN AND METHODS Primary endothelial cells obtained from NEONATAL rat aorta, expressing both receptors, were transfected with the rat AT2-r promoter region cloned into a pCAT-reporter vector. The reporter-expression study was performed in a transient transfection assay system. Transfected cells were studied following angiotensin-converting enzyme inhibition to prevent endogenous formation of Ang II. Cells were subsequently stimulated for 6 h with Ang II, either alone or in combination with the AT1-r antagonist DuP753. AT2-r mRNA was assessed by RNase protection assay during the same pharmacological stimuli. RESULTS Stimulation with Ang II caused an increase in promoter activity (+50%, P < 0.05 versus baseline), whereas mRNA expression was reduced by 50% (P < 0.05 versus baseline). Concomitant treatment with DuP753 and Ang II was associated with a 98% increase in promoter activity (P < 0.05 versus baseline). DuP753 also prevented the reduction in mRNA; it actually produced a 100% increase in AT2-r mRNA accumulation (P < 0.01 versus baseline). Studies with the AT2-r antagonist PD123319 indicate that the AT2-r is also involved in the regulation of AT2-r gene promoter activity. CONCLUSIONS These data indicate that Ang II increases AT2-r promoter activity and decreases AT2-r mRNA accumulation in endothelial cells. The AT1 subtype receptor is involved in the modulation of both effects of Ang II. These findings suggest that changes in the expression of AT2 receptors may occur during treatment with AT1-r antagonists, and they indicate the existence of a cross-talk between AT1 and AT2 receptors.

Contribution of Genetic Factors to Renal Lesions in the Stroke-Prone Spontaneously Hypertensive Rat

Abstract—Stroke-prone spontaneously hypertensive rats (SHRSP) develop renal lesions more frequently than the closely related control strain, the stroke-resistant SHR. The aim of this study was to investigate the contribution of genetic factors to the enhanced susceptibility to renal damage of SHRSP in an SHRSP/SHR F2 intercross by means of a genotype/phenotype cosegregation analysis. For this purpose, 154 6-week-old F2-SHRSP/SHR rats (79 male, 75 female) were fed a stroke-permissive Japanese diet for 4 weeks. Systolic blood pressure (SBP) was recorded at the end of the dietary period. Renal damage was scored from 0 to 3, and 274 genetic markers polymorphic between SHR and SHRSP were genotyped. Linkage of genotype markers to the degree of renal disease was determined by &khgr;2 test. Experimental threshold level to declare linkage was calculated by QTL cartographer. SBP was not correlated to renal damage (&rgr; coefficient, 0.201; P =NS). Grade 2 and grade 3 lesions were more frequent in male than in female rats (P =0.01). Two loci, D1Rat238, on chromosome 1 and the IGF receptor-binding protein 4 (Rbp4g) on chromosome 10, were significantly linked to the degree of renal damage, with SHRSP allele being aggressive at D1Rat238 locus and protective at Rbp4g locus. In male rats only, the SHRSP allele at one locus on chromosome 16, D16Mit2, was associated with a more severe degree of renal disease. Our results demonstrate that in this intercross, susceptibility to renal damage is influenced by several genetic loci acting independently from high blood pressure levels and also shows a sexual dimorphism.

Functional cross-talk between angiotensin II and epidermal growth factor receptors in NIH3T3 fibroblasts.

Background The main angiotensin (Ang) II subtype receptors (AT1R and AT2R) are involved in cellular growth processes and exert functionally antagonistic effects. Objective To characterize the mechanisms by which Ang II receptors influence growth, by investigating the interactions between Ang II subtype receptors and epidermal growth factor (EGF) receptors on mitogen-activated protein kinase (MAPK) pathway activation. Design and methods The experiments were performed using a mouse fibroblast cell line, NIH3T3, by transient co-transfection with rat AT1R or AT2R expression vectors, or both. Extracellular-signal-regulated kinase (ERK)1/2 phosphorylation was analysed by western blot and the ERK activity was evaluated using PathDetect, an in-vivo signal transduction pathway trans-reporting system. Selective Ang II receptor antagonists (losartan for AT1R and PD123319 for AT2R) were used to investigate the contributions of each receptor to the response observed. Results Our data show that, in this cellular model, both Ang II receptors phosphorylate ERK1/2. However, in the cells expressing AT1R, the EGF-induced MAPK pathway was enhanced in the presence of Ang II in a synergistic fashion. In contrast, a reduction of EGF-induced MAPK activation was observed in the cells expressing AT2R. In cells expressing both Ang II subtype receptors, Ang II promoted an enhancement of EGF-induced MAPK activation. However, in the presence of the AT1R antagonist, losartan, the effect of EGF was reduced. Conclusion These data indicate the existence of an opposite cross-talk of AT1R and AT2R with EGF receptors, and suggest a complex functional interaction between these pathways in the regulation of cellular growth processes.

Genetic Factors Underlying Impaired Endothelium-Dependent Vasorelaxation in the Stroke-Prone Spontaneously Hypertensive Rat

AbstractBackground: Impaired endothelial-dependent vasorelaxation (IEDV) precedes the development of cerebrovascular lesions in an experimental animal model of inherited stroke, the stroke-prone spontaneously hypertensive rat (SHRSP). We have previously shown that in this experimental model IEDV segregates in the SHRSP/stroke-resistant spontaneously hypertensive rat (SHR) F2 generation independently of blood pressure levels, thus suggesting the existence of a specific genetic basis for this trait. Aim: The aim of this study was to identify the genetic determinants of the occurrence of IEDV in the SHRSP by random marker genome screening. Method: One-hundred and thirty-seven SHR/SHRSP F2 rats (64 males, 73 females) were studied (age 10 weeks). The quantitative phenotypes used for the study were: fractional vasorelaxation to acetylcholine on thoracic aorta rings pre-constricted with norepinephrine, and the integer of the total area under the vasoconstriction and vasodilatation curves. Result: None of the markers tested showed a significant linkage to the phenotype under investigation. A trend toward significance was found for only five of the genetic markers. Conclusion: These results do not allow us to identify specific genetic determinants of IEDV in our experimental model. On a more general perspective, the analysis of genetic factors involved in the pathogenesis of intermediate phenotypes may represent an interesting strategy to gain further insight in to the prevention of cardiovascular diseases.