Angiopoietin-2 attenuates angiotensin II-induced aortic aneurysm and atherosclerosis in apolipoprotein E-deficient mice

Abstract

Angiogenesis and inflammation are implicated in aortic aneurysm and atherosclerosis and regulated by angiopoietin-2 (Angpt2). The effect of Angpt2 administration on experimental aortic aneurysm and atherosclerosis was examined. Six-month-old male apolipoprotein E deficient (ApoE^-/-) mice were infused with angiotensin II (AngII) and administered subcutaneous human Fc-protein (control) or recombinant Angpt2 (rAngpt2) over 14 days. Administration of rAngpt2 significantly inhibited AngII-induced aortic dilatation and rupture of the suprarenal aorta (SRA), and development of atherosclerosis within the aortic arch. These effects were blood pressure and plasma lipoprotein independent and associated with Tie2 activation and down-regulation of monocyte chemotactic protein-1 (MCP-1) within the SRA. Plasma concentrations of MCP-1 and interleukin-6 were significantly lower in mice receiving rAngpt2. Immunostaining for the monocyte/macrophage marker MOMA-2 and the angiogenesis marker CD31 within the SRA were less in mice receiving rAngpt2 than controls. The percentage of inflammatory (Ly6C^hi) monocytes within the bone marrow was increased while that in peripheral blood was decreased by rAngpt2 administration. In conclusion, administration of rAngpt2 attenuated angiotensin II-induced aortic aneurysm and atherosclerosis in ApoE^-/- mice associated with reduced aortic inflammation and angiogenesis. Up-regulation of Angpt2 may have potential therapeutic value in patients with aortic aneurysm and atherosclerosis.

Figure 1. Effect of rAngpt2 on AngII-induced aortic dilatation and atherosclerosis in ApoE^−/−mice.

(A) Kaplan-Meier curves of survival free of aneurysm rupture in AngII-infused ApoE^−/− mice administered rAngpt2 (○; 4.0 mg/kg/48 hrs, s.c.) or control peptide (•); *P = 0.034 compared to controls using Log-rank (Mantel-Cox) test. (B) Regional aortic diameters for AngII-infused ApoE^−/− mice administered rAngpt2 (white; 4.0 mg/kg/48 hrs, s.c.; n = 24) or control peptide (grey; n = 23) determined by morphometry at sacrifice (day 14). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for maximum diameters (mm) and compared by Mann-Whitney U test. TA, thoracic aorta; SRA, suprarenal aorta; IRA, infrarenal aorta. (C) Sudan IV staining area within the aortic arch of mice receiving rAngpt2 (4.0 mg/kg/48 hrs, s.c.; n = 24) compared to controls (n = 19). Data expressed as mean ± SEM for positive staining area relative to total specimen area (%); *P = 0.017 compared by unpaired-t test. (D) Atherosclerotic plaque within the brachiocephalic artery (BCA) of mice receiving rAngpt2 (4.0 mg/kg/48 hrs, s.c.; n = 8) compared to controls (n = 7). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for plaque cross-sectional area relative to total luminal area (%); *P = 0.009 compared by Mann-Whitney U test.

Figure 2. Effect of rAngpt2 on Tie2 phosphorylation and inflammation markers within the suprarenal aorta.

(A) Representative western blots for Tie2/phosphorylated Tie2^Tyr992, nuclear (NFκB) p65/PCNA, and MCP-1. Tie2^Tyr992 phosphorylation (B), nuclear p65 (C), and MCP-1 (D) levels within the SRA of AngII-infused ApoE^−/− mice administered rAngpt2 (4.0 mg/kg/48 hrs, s.c.; n = 6) or controls (n = 6) after 14 days. Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for relative density units (RDU) per microgram (μg) protein, compared by Mann-Whitney U test.

Figure 3. Effect of rAngpt2 on monocyte/macrophage infiltration and markers of angiogenesis within the suprarenal aorta.

(A) Immunostaining for macrophages (arrow) and angiogenesis with MOMA-2 (upper panel) and CD31 (lower panel), respectively. Response to AngII after 14 days in SRA of ApoE^−/− mice administered rAngpt2 (4.0 mg/kg/48 hrs, s.c.; n = 6) or control (n = 6). L, lumen; cell nuclei stained blue; 200x magnification; scale bar, 50 μm; negative is isotype IgG control and represents background fluorescence. Data for MOMA-2 (B) and CD31 (C) expressed as median and interquartile range with maximum and minimum data points (whiskers) for positive staining area relative to total specimen area (%); *P < 0.01 compared by Mann-Whitney U test. (D) Concentration of VEGF within the SRA of mice receiving rAngpt2 (4.0 mg/kg/48 hrs, s.c.; n = 6) compared to controls (n = 6). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for picograms (pg) of VEGF per μg protein, compared by Mann-Whitney U test.

Figure 4. Effect of rAngpt2 on blood and tissue profile of Ly6C-positive inflammatory monocytes.

(A) Circulating concentration of MCP-1 and IL-6 in AngII-infused mice receiving rAngpt2 (4.0 mg/kg/48 hrs, s.c.; n = 6) or control peptide (n = 6) after 14 days. Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for picograms (pg) of protein per ml plasma; *P = 0.010 and ^†P = 0.013 compared by Mann-Whitney U test. Inflammatory (Ly6C^hi) monocytes within the circulation (B), bone marrow (C), and spleen (D) of AngII-infused mice receiving rAngpt2 (4.0 mg/kg/48 hrs, s.c.; n = 10) or control peptide (n = 7) after 14 days. Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for CD11b⁺Ly6G^-Ly6C^hi cells relative to total leukocyte population (%); *P = 0.019 (B,C) compared by Mann-Whitney U test.