Differential Effects of β-Blockers, Angiotensin II Receptor Blockers, and a Novel AT2R Agonist NP-6A4 on Stress Response of Nutrient-Starved Cardiovascular Cells

Abstract

In order to determine differences in cardiovascular cell response during nutrient stress to different cardiovascular protective drugs, we investigated cell responses of serum starved mouse cardiomyocyte HL-1 cells and primary cultures of human coronary artery vascular smooth muscles (hCAVSMCs) to treatment with β-blockers (atenolol, metoprolol, carvedilol, nebivolol, 3μM each), AT1R blocker losartan (1μM) and AT2R agonists (CGP42112A and novel agonist NP-6A4, 300nM each). Treatment with nebivolol, carvedilol, metoprolol and atenolol suppressed Cell Index (CI) of serum-starved HL-1 cells (≤17%, ≤8%, ≤15% and ≤15% respectively) as measured by the Xcelligence Real-Time Cell Analyzer (RTCA). Conversely, CI was increased by Ang II (≥9.6%), CGP42112A (≥14%), and NP-6A4 (≥25%) respectively and this effect was blocked by AT2R antagonist PD123319, but not by AT1R antagonist losartan. Thus, the CI signature for each drug could be unique. MTS cell proliferation assay showed that NP-6A4, but not other drugs, increased viability (≥20%) of HL-1 and hCAVSMCs. Wheat Germ Agglutinin (WGA) staining showed that nebivolol was most effective in reducing cell sizes of HL-1 and hCAVSMCs. Myeloid Cell Leukemia 1 (MCL-1) is a protein critical for cardiovascular cell survival and implicated in cell adhesion. β-blockers significantly suppressed and NP-6A4 increased MCL-1 expression in HL-1 and hCAVSMCs as determined by immunofluorescence. Thus, reduction in cell size and/or MCL-1 expression might underlie β-blocker-induced reduction in CI of HL-1. Conversely, increase in cell viability and MCL-1 expression by NP-6A4 through AT2R could have resulted in NP-6A4 mediated increase in CI of HL-1. These data show for the first time that activation of the AT2R-MCL-1 axis by NP-6A4 in nutrient-stressed mouse and human cardiovascular cells (mouse HL-1 cells and primary cultures of hCAVSMCs) might underlie improved survival of cells treated by NP-6A4 compared to other drugs tested in this study.

Cardiovascular disease progression in female Zucker Diabetic Fatty rats occurs via unique mechanisms compared to males

“…The X-linked Agtr2 that codes for AT2R is a 30-year old mechanistic target for treatment of CVD 32 – 37 . AT2R activation improves survival of mouse HL-1 cardiomyocytes and human coronary artery vascular smooth muscle cells under conditions of serum starvation 65 and reduces infarct size after myocardial infarction in murine models 35 , 66 . Increased Agtr2 expression in the female vasculature is implicated in the increased female-specific protection from hypertension, vascular injury, and renal function 32 – 34 .…”

Anti-fibrotic Potential of AT2 Receptor Agonists

“…However, AT 2 R stimulation also usually involves an anti-inflammatory effect that may contribute to the beneficial AT 2 R-mediated anti-fibrotic effects. Most data related to chronic AT 2 R stimulation have been obtained using C21, although there are a number of other AT 2 R agonists beginning to emerge in the literature ( Jones et al, 2011 ; Guimond et al, 2014 ; Del Borgo et al, 2015 ; Mahmood and Pulakat, 2015 ) that require rigourous in vivo testing in a similar manner to C21. Such studies will shed further light on the clinical potential of AT 2 R agonists in CVD.…”

Differential Regulation of Cardiac Function and Intracardiac Cytokines by Rapamycin in Healthy and Diabetic Rats

“…We have reported recently that a novel peptide agonist for the angiotensin II type 2 receptor AT2R named NP-6A4 is effective in improving cardiomyocyte and vascular smooth muscle survival under nutrient deficiency stress [64]. We also showed that NP-6A4 could increase MCL-1 expression in cardiovascular cells [64]. We propose that a synergistic treatment with Rapamycin and NP-6A4 would be beneficial in mitigating some of the negative effects of Rapamycin treatment and enhancing cardioprotection in diabetes.…”