Objective The angiotensin II type 1 receptor (AT1R) can be activated under conditions of mechanical stretch in some cellular systems. Whether this activity influences signaling within the abdominal aorta to promote to abdominal aortic aneurysm (AAA) development remains unknown. We evaluated the hypothesis that mechanical AT1R activation can occur under conditions of hypertension (HTN) and contribute to AAA formation. Methods BPH/2 mice, which demonstrate spontaneous neurogenic, low-renin HTN, and normotensive BPN/3 mice underwent AAA induction via the calcium chloride model, with or without an osmotic minipump delivering 30 mg/kg/d of the AT1R blocker Losartan. Systolic blood pressure (SBP) was measured at baseline and weekly via a tail cuff. The aortic diameter (AoD) was measured at baseline and terminal surgery at 21 days by digital microscopy. Aortic tissue was harvested for immunoblotting (phosphorylated extracellular signal-regulated kinase-1 and -2 [pERK1/2] to ERK1/2 ratio) and expressed as the fold-change from the BPN/3 control mice. Aortic vascular smooth muscle cells (VSMCs) underwent stretch with or without Losartan (1 μM) treatment to assess the mechanical stimulation of ERK1/2 activity. Statistical analysis of the blood pressure, AoD, and VSMC ERK1/2 activity was performed using analysis of variance. However, the data distribution was determined to be log-normal (Shapiro-Wilk test) for ERK1/2 activity. Therefore, it was logarithmically transformed before analysis of variance. Results At baseline, the SBP was elevated in the BPH/2 mice relative to the BPN/3 mice ( P < .05). Losartan treatment significantly reduced the SBP in both mouse strains ( P < .05). AAA induction did not affect the SBP. At 21 days after induction, the percentage of increase in the AoD from baseline was significantly greater in the BPH/2 mice than in the BPN/3 mice (101.28% ± 4.19% vs 75.59% ± 1.67% above baseline; P < .05). Losartan treatment significantly attenuated AAA growth in both BPH/2 and BPN/3 mice (33.88% ± 2.97% and 43.96% ± 3.05% above baseline, respectively; P < .05). ERK1/2 activity was increased approximately fivefold in the BPH/2 control mice relative to the BPN/3 control mice ( P < .05). In the BPH/2 and BPN/3 mice with AAA, ERK1/2 activity was significantly increased relative to the respective baseline control ( P < .05) and effectively reduced by concomitant Losartan therapy ( P < .05). Biaxial stretch of the VSMCs in the absence of angiotensin II demonstrated increased ERK1/2 activation ( P < .05 vs static control), which was significantly inhibited by Losartan. Conclusions In BPH/2 mice with spontaneous neurogenic, low-renin HTN, AAA growth was amplified compared with the normotensive control a...
Introduction Elevated interleukin-6 (IL-6) plasma levels have been associated with abdominal aortic aneurysm (AAA), but whether this cytokine plays a causative role in the degenerative remodeling or represents an effect from the inflammatory cascades initiated by infiltrating leukocytes remained unclear. This project aims to demonstrate that within the aortic wall, signaling from IL-6 through the STAT3 transcription factor is necessary for infiltration of proteolytically-active macrophages and development of small AAA. Methods Following measurement of baseline infrarenal aortic diameter (AoD, digital microscopy), C57Bl/6 and IL-6 knockout (IL-6KO) mice underwent AAA induction by application of peri-adventitial CaCl2 (0.5 M) +/− implantation of an osmotic mini-pump delivering IL-6 (4.36 µg/kg/day over 21 days). At the terminal procedure, AoDs were measured by digital microscopy and aortas harvested for immunoblot (pSTAT3/STAT3), matrix metalloproteinase (MMP) quantification, or flow cytometric analysis of macrophage content. Plasma was collected for cytokine analysis. Results IL-6 infusion significantly increased the plasma IL-6 levels in C57Bl/6 and IL-6KO animals. The C57Bl/6 + CaCl2 group developed AAA (AoD >50% above baseline) but IL-6KO + CaCl2 did not. In the IL-6KO + IL-6+CaCl2 group, AAA developed to match that of C57Bl/6 + CaCl2 mice. STAT3 activity was significantly increased in animals with advanced stages of dilation (>40% from baseline), compared to those with ectasia (≤25%). Although cytokine profiles did not support T-cells or neutrophils as being active contributors in this stage of aortic remodeling, changes in the profile of elaborated MMPs suggested macrophage activity with a trend toward alternatively activated pathways. Flow cytometry confirmed significantly increased macrophage abundance specifically in animals with upregulated STAT3 activity and advanced aortic dilation. Conclusion In this murine model of AAA, progressive dilation to development of true AAA was only accomplished when IL-6 signaling upregulated STAT3 activity to effect accumulation of proteolytically-active macrophages. This pathway warrants further investigation to identify potential therapeutic avenues to abrogate growth of small AAA.
Introduction: Serum biomarkers for abdominal aortic aneurysms (AAA) have been identified, but the prognostic relevance has remained unclear. Mechanical activation of aortic VSMCs can elicit production of interleukin-6 (IL-6), a cytokine consistently elevated in AAA. Expecting that stressed native aortic cells may drive degenerative remodeling, we hypothesize that biomarkers focused on matrix proteolysis may be elaborated directly from the aortic wall and assessed as an indicator of aortic strain. Methods: AAA was induced in C57Bl/6 mice with peri-adventitial CaCl 2 (n=5). Terminal procedure at 21 days quantified aortic diameter (AoD) by digital microscopy, obtained blood sampling, and collected aortic tissue. Aortic ultrasound (Vevo3100 with VevoVasc Software) was conducted on Days 0 and 21 to assess AoD and aortic strain parameters. Tissue and serum were analyzed to quantify IL-6 and the proteolytic matrix markers: matrix metalloproteinase-9 (MMP-9), Cathepsin S (CtsS), Cystatin C (CysC), and osteoprotegerin (OPG). Aortic VSMCs underwent mechanical stimulation with 12% Stretch and subsequent QPCR for expression of proteolytic matrix markers (n=4). Groups were analyzed by T-test and ANOVA. Results: At 21 days, all mice in AAA group developed true aneurysms by digital microscopy (>50% dilation compared to internal baseline), and this correlated to measurements from transabdominal ultrasound. Aortic strain assessment indicated increased stiffness parameters in AAA (elevated Pulse Wave Velocity, decreased Distensibility, and decreased Global Radial Strain). The AAA group also had significantly elevated levels of IL-6, MMP-9, and OPG (p<0.05 versus Control for each), with a trend toward increased CtsS and CysC. Alternatively, Stretch VSMCs demonstrated upregulated expression of IL-6, CysC, and OPG (p<0.05 vs Static), while the proteases MMP-9 and CtsS remained unchanged. Conclusion: Mechanical activation of aortic VSMCs can stimulate expression of biomarker proteins associated with AAA, but protease production may rely on the inflammatory infiltrate to promote degenerative matrix remodeling. Further investigation into the source of common AAA biomarkers will provide insight for effective integration into patient care.
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