Abstract
Activation of the angiotensin (Ang)-converting enzyme (ACE) 2/Ang-(1-7)/MAS receptor pathway of the renin-angiotensin system (RAS) induces protective mechanisms in different diseases. Herein, we describe the cardiovascular phenotype of a new transgenic rat line (TG7371) that expresses an Ang-(1-7)-producing fusion protein. The transgene-specific mRNA and the corresponding protein were shown to be present in all evaluated tissues of TG7371 with the highest expression in aorta and brain. Plasma Ang-(1-7) levels, measured by radioimmunoassay (RIA) were similar to control Sprague-Dawley (SD) rats, however high Ang-(1-7) levels were found in the hypothalamus. TG7371 showed lower baseline mean arterial pressure (MAP), assessed in conscious or anesthetized rats by telemetry or short-term recordings, associated with increased plasma atrial natriuretic peptide (ANP) and higher urinary sodium concentration. Moreover, evaluation of regional blood flow and hemodynamic parameters with fluorescent microspheres showed a significant increase in blood flow in different tissues (kidneys, mesentery, muscle, spleen, brown fat, heart and skin), with a resulting decrease in total peripheral resistance (TPR). TG7371 rats, on the other hand, also presented increased cardiac and global sympathetic tone, increased plasma vasopressin (AVP) levels and decreased free water clearance. Altogether, our data show that expression of an Ang-(1-7)-producing fusion protein induced a hypotensive phenotype due to widespread vasodilation and consequent fall in peripheral resistance. This phenotype was associated with an increase in ANP together with an increase in AVP and sympathetic drive, which did not fully compensate the lower blood pressure (BP). Here we present the hemodynamic impact of long-term increase in tissue expression of an Ang-(1-7)-fusion protein and provide a new tool to investigate this peptide in different pathophysiological conditions.
Highlights
The renin–angiotensin system (RAS) is an important systemic, tissue and cellular modulator of the function of basically all organs
The construct human glial fibrillary acidic protein-Ang-(1-7) used in this study was designed to express a fusion protein, containing: (i) a signal peptide from human renin to ensure the transport of the protein into the endoplasmic reticulum for further secretion of this protein to the extracellular medium; (ii) an immunoglobulin fragment from mouse IgG2b linked to a portion of the human prorenin prosegment which promotes mass and exposes the cleavage site for the endoprotease furin; (iii) a furin cleavage site; (iv) coding sequence for Ang-(1-7) followed by a stop codon inserted by the use of a double-stranded oligonucleotide
Highest mRNA expression for the transgene was found in the aorta, followed by brain areas, such as rostral ventrolateral medulla (RVLM), caudal ventrolateral medulla (CVLM), nucleus tractus solitarii (NTS) in the medulla, as well as hippocampus, periaqueductal gray area (PAG), paraventricular nucleus of the hypothalamus (PVN) and cortex (Figure 1A)
Summary
The renin–angiotensin system (RAS) is an important systemic, tissue and cellular modulator of the function of basically all organs. In rats, a transgenic strain that expresses an Ang-(1-7)-producing fusion protein mainly in testis, TGR(A1-7)-L3292 (TG3292), was previously generated [9]. In these animals, testis functions as an infusion pump releasing Ang-(1-7) into the circulation. No mRNA expression or increase in Ang-(1-7) levels was detected in other tissues of TG3292 rats, such as kidney, adrenal gland, lung, atrium, ventricle, liver, brain and aorta. These animals presented a normotensive phenotype [9]. The same fusion protein was employed to generate transgenic rats and mice overexpressing Ang-(1-7) in the heart [10], which contributed to demonstrate the cardioprotective function of the ACE2/Ang-(1-7)/MAS axis
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