Locally applied leptin induces regional aortic wall degeneration preceding aneurysm formation in apolipoprotein E-deficient mice

Abstract

Objective: Leptin promotes atherosclerosis and vessel wall remodeling. As abdominal aortic aneurysm (AAA) formation involves tissue remodeling, we hypothesized that local leptin synthesis initiates and promotes this process.

Methods and results: Human surgical AAA walls were analyzed for antigen and mRNA levels of leptin and leptin receptor, as well as mRNA for matrix metalloproteinases (MMP)-9 and MMP-12. Leptin and leptin receptor antigen were evident in all AAAs, and leptin, MMP-9, and MMP-12 mRNA was increased relative to age-matched nondilated controls. To simulate in vivo local leptin synthesis, ApoE(-/-) mice were subjected to a paravisceral periaortic application of low-dose leptin. Leptin-treated aortas exhibited decreased transforming growth factor-β and increased MMP-9 mRNA levels 5 days after surgery, and leptin receptor mRNA was upregulated by day 28. Serial ultrasonography demonstrated accelerated regional aortic diameter growth after 28 days, correlating with local medial degeneration, increased MMP-9, MMP-12, and periadventitial macrophage clustering. Furthermore, the combination of local periaortic leptin and systemic angiotensin II administration augmented medial MMP-9 synthesis and aortic aneurysm size.

Conclusions: Leptin is locally synthesized in human AAA wall. Paravisceral aortic leptin in ApoE(-/-) mice induces local medial degeneration and augments angiotensin II-induced AAA, thus suggesting novel mechanistic links between leptin and AAA formation.

Figures

Figure 1

Comprehensive analyses of human AAA wall tissue. Representative Movat’s pentachrome staining – Low (A) and high (a) power images, respectively; (The black square in “A” depicts the analyzed area). The images show severe dissolution of elastin fibers and severe attenuation of the medial wall with collagen replacement. The irregular luminal surface shows areas of organizing thrombi. The tissue fragment in the upper left represents a proteoglycan-rich plaque disassociated from the parent aorta consisting mostly of SMCs and macrophages and organizing thrombus (black arrow). Low and high power magnifications (B and b), respectively, of an adjacent histologic section to “A” immunostained against smooth muscle α-actin highlights the attenuated media. Representative immunohistochemical staining smooth muscle α-actin (C), and the macrophage marker CD68 (D) from the area represented by the black box in “A”. Immunohistochemical staining against leptin (E & e) and leptin receptor (ObR) (F & f). Scale bar(s) = 2 mm for A and B, 200 μm for C, D, E, and F and 100 μm for e and f; G, mRNA expression of Leptin and ObR; H, MMP-9 and MMP-12 mRNA. Bar graphs also include the baseline control (non-dilated moderately atherosclerotic aorta) and retroperitoneal fat tissue. (i – intima; m – media; a – adventitia)

Figure 2

Aortic reference locations as depicted in 3 regions along the mouse abdominal aorta. A,B, The locations relate to the visceral vessels. Red arrow shows blood flow direction; yellow squares depict 3 reference locations, juxta-renal, para-visceral, and supra-celiac. C, Percentage of change in maximal diameter (compared to day 0) at three aortic reference locations of the leptin-treated and control mouse models. *P<0.05 and **P<0.01, vs. control at the same time point.

Figure 2

Aortic reference locations as depicted in 3 regions along the mouse abdominal aorta. A,B, The locations relate to the visceral vessels. Red arrow shows blood flow direction; yellow squares depict 3 reference locations, juxta-renal, para-visceral, and supra-celiac. C, Percentage of change in maximal diameter (compared to day 0) at three aortic reference locations of the leptin-treated and control mouse models. *P<0.05 and **P<0.01, vs. control at the same time point.

Figure 3

mRNA analysis for 23 selected leptin-modulated genes in leptin- and placebo-treated mouse aortas on POD-5 (A), and POD-28 (B). *P<0.05 and **P<0.01.

Figure 3

mRNA analysis for 23 selected leptin-modulated genes in leptin- and placebo-treated mouse aortas on POD-5 (A), and POD-28 (B). *P<0.05 and **P<0.01.

Figure 4

Representative staining for elastin and smooth muscle α-actin in leptin- and control-treated mouse abdominal aortas, and in distant descending aortas 45 days after deployment of peri-aortic leptin. Note the loose appearance and fragmentation of elastic lamellas (blue arrow), and the depletion of smooth muscle α-actin in leptin-treated abdominal aorta (n=3). Scale bars=100 μm.

Figure 5

Representative immunohistochemical staining for MMP-9, MMP-12, and MAC-3 antigens, and in situ zymography for MMP-9 in leptin- and placebo-treated mouse abdominal aortas 45 days after addition of the peri-aortic application. Quantification analysis for each antigen immunoreactivity signal is shown in the panel on the right. Note the increased antigen expression for MMP-9, MMP-12 and adventitial macrophages (Mac-3) in leptin-treated abdominal aorta (n=3). Also note the excessive fluorescence (arrow) -indicative of MMP-9 elastolytic activity in a representative leptin treated aorta slide untreated by EDTA, while EDTA treated parallel slide exhibits reduced fluorescence within the same area. Scale bars=100 μm.

Figure 6

Outcome of Ang II infusion with and without peri-aortic leptin deployment. A, representative findings in subjects that succumbed from aortic arch aneurysm rupture during the follow up period. Left to right, a post mortem view of the dilated aortic arch, a corresponding ultrasound view of the arch in the same mouse 1 day prior to death, and the hemothorax after aortic rupture. B, Representative serial sections of Masson stained transverse sections of the supra-renal aorta in Ang II leptin treated, upper row, vs Ang II placebo treated, in the lower row. Note the larger aneurysm size in Ang II plus leptin vs placebo controls. Blood flow direction is depicted by the red arrow. C, Maximal diameter, and maximal diameter change (%) relative to day 0 (P =0.06). D, Immunohistochemistry for MMP-9 in Ang II leptin, and Ang II placebo treated mice on POD-28. MMP-9 signal quantification revealed significantly higher antigen in leptin treated mice (P =0.003) (n=15). Scale bar = 500 μm.

Figure 6

Outcome of Ang II infusion with and without peri-aortic leptin deployment. A, representative findings in subjects that succumbed from aortic arch aneurysm rupture during the follow up period. Left to right, a post mortem view of the dilated aortic arch, a corresponding ultrasound view of the arch in the same mouse 1 day prior to death, and the hemothorax after aortic rupture. B, Representative serial sections of Masson stained transverse sections of the supra-renal aorta in Ang II leptin treated, upper row, vs Ang II placebo treated, in the lower row. Note the larger aneurysm size in Ang II plus leptin vs placebo controls. Blood flow direction is depicted by the red arrow. C, Maximal diameter, and maximal diameter change (%) relative to day 0 (P =0.06). D, Immunohistochemistry for MMP-9 in Ang II leptin, and Ang II placebo treated mice on POD-28. MMP-9 signal quantification revealed significantly higher antigen in leptin treated mice (P =0.003) (n=15). Scale bar = 500 μm.