Angiotensin II upregulates renin–angiotensin system in human isolated T lymphocytes

Ang II Upregulation of the T-Lymphocyte Renin-Angiotensin System Is Amplified by Low-Grade Inflammation in Human Hypertension

“…Venous blood were drawn from the antecubital vein, collected in a sterile flask containing 3.8% sodium citrate (9/1, vol/vol) and T-lymphocytes isolation was carried out according to our previous report. 13 Magnetic beads conjugated with antihuman CD 3 (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) were used for positive selection of T-lymphocytes. In each incubation experiment, mRNA expression of ACE, AT1-R, INF-γ and ACE activity and Ang II concentrations were studied in cell pellet.…”

“…ACE activity was also investigated in the supernatants, since ACE can be shed by T-cells. 13 To study the effect of stimulation by Ang II on ACE and AT1-R gene expression, ACE activity and Ang II concentrations, T-lymphocytes were cultured in incubator for 6 or 18 or 24 h with and without 0.1 pmol/l Ang II in addition to the culture medium. Lymphocytes obtained from three hypertensive subjects were incubated in the presence of of 0.1 pmol/l Ang II with or without an AT1-R antagonist (irbesartan) to demonstrate that data obtained could entirely be attributed to Ang II and that the Ang II effects were mediated by AT1-R. 13 Real-time PCR for ACE gene, reverse-transcriptase PCR analysis for AT1-R and INF-γ genes and western blot analysis for AT1-R protein.…”

“…Isolation of total RNA was performed as described previously. 13 FAM-labeled probes and primers for ACE (ACE Hs00174179_m1; Applied Biosystems, Foster City, CA) and for the housekeeping gene β-actin (Invitrogen, Milan, Italy) were used ( Table 2). Relative gene expression was calculated as 2 −ΔCt (ΔC t = C t of the target gene minus C t of β-actin).…”

“…Relative gene expression was calculated as 2 −ΔCt (ΔC t = C t of the target gene minus C t of β-actin). mRNAs for AT1-R and INFγ were semiquantified vs. the housekeeping gene glyceraldeyde-3-phosphate dehydrogenase by reversetranscriptase PCR (Applied Biosystems) 13 using specific primers at annealing temperature and cycle number as reported in Table 2. Western blot analysis was performed as already reported.…”

“…Under physiological conditions, stimulation of T-cells by Ang II upregulated lymphocytic RAS. 13,14 This study was aimed at investigating, whether in hypertensive patients a selective Ang II driven upregulation of lymphocytic RAS occurs and whether T-cell RAS is differently modulated in patients with or without systemic hypertension-related low-grade inflammation. The activity of lymphocytic RAS was investigated in T-cell cultures both under baseline conditions and after Ang II-stimulation.…”