mTOR inhibition prevents angiotensin II–induced aortic rupture and pseudoaneurysm but promotes dissection in Apoe-deficient mice

Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II–induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II–induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.


Biomechanical Assessment
Tissue Preparation: Biaxial mechanical tests and data analysis were performed on excised segments of the suprarenal abdominal aorta from Apoe -/mice at 12 weeks of age treated for 7 days with saline, AngII, or AngII and rapamycin. The aortic segments were gently cleaned of excess perivascular tissue and branches were ligated with 9-0 nylon suture. The excised vessels were cannulated on custom-drawn glass pipets, secured with sutures at each end, and mounted on a biaxial testing device, submerged in Krebs-Ringer's solution at 37 °C and oxygenated with 95% O2 / 5% CO2.
Active Mechanical Contraction Testing: The viability of the active tone in the aorta was assessed through two initial contractions to 100 mM KCl at two different combinations of pressures and vessel lengths followed by washouts with normal Krebs-Ringer solution. The vessels were then set to 90 mmHg and in vivo value of axial stretch and contracted with 100 mM KCl for 15 minutes followed by 10 minutes of relaxation by washout. This was repeated for 1 μM phenylephrine and, before washing out, 10 μM acetylcholine was added to assess endothelial function.
Passive Mechanical Biaxial Testing: After completing the active testing, the testing chamber was drained and refilled with Hanks' buffered salt solution and maintained at room temperature to minimize smooth muscle contractility. Vessels were mechanically preconditioned, by cyclic pressurization between 10 to 140 mmHg at the estimated in vivo value of axial stretch, to minimize viscoelastic contributions to the mechanical behavior. The aortic segments were then subjected to a series of seven biaxial protocols consisting of cyclic pressurization from 10 to 140 mmHg while the vessel was held fixed at three different axial stretches (95, 100, and 105% of the in vivo value), and cyclic axial stretching at four fixed pressures (10, 60, 100, and 140 mmHg).
Data Analysis of Active and Passive Mechanical Properties: The contractile properties were assessed by calculating changes in inner radius and mean circumferential stress between relaxed and contracted states. The passive pressure-diameter and axial force-length data were fit with a validated four-fiber family constitutive model via a nonlinear regression of a data set from all seven testing protocols. Specifically, we used a Holzapfel-type nonlinear stored energy function W,

Flow Cytometry
Aortic tissue was minced and incubated in 0.5 ml DMEM with 10% FBS, 1.5 mg/ml collagenase A, and 0.5 mg/ml elastase for 60 min at 37°C while the tissue fragments were gently

Quantitative RT-PCR
Frozen aortas without hematomas were crushed and the tissue fragments were immersed in RLT lysis buffer (Qiagen) and vigorously vortexed. Total RNA was isolated using a RNeasy Mini Kit and DNase Digestion Set (Qiagen) according to the manufacturer's protocol. Reverse transcriptions were performed using an iScript cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCR was performed using a real-time PCR detection system (CFX96, Bio-Rad) by mixing equal amount of cDNAs, Taqman gene master mix, and primers from Applied Biosystems for Col1a1 Tnc (Mm00495662_m1), Ccn2 (Mm01192933_g1), and Gapdh (Mm99999915_g1). RNA-free ddH2O was used as negative controls for qPCR instead of cDNA samples. All reactions were in a 12.5 μl volume, in duplicate. PCR amplification consisted of 10 min of an initial denaturation step at 95 °C followed by 40 cycles of PCR at 95 °C for 15 s, and 60 °C for 1 min. We confirmed stable expression of a second housekeeping gene, Hprt1 (Hs02800695_m1).

Cell Culture
Murine SMCs were derived by explant outgrowth from minced fragments of thoracic aortas from C57BL/6J mice. Human SMCs were derived by explant outgrowth from medial fragments of non-diseased ascending aortas of 3 organ donors whose hearts were not used for clinical transplantation. The cells were cultured in DMEM with 10% FBS and used between passage 1-3 for mouse SMC and passage 4-5 for human SMC.

Stress calculations
Dilated circumferential stress (kPa) 155 ± 7 128 ± 11 75 ± 5 Change in circumferential stress (%) 24.4 ± 6.7 13.5 ± 4.7 16.6 ± 5.9  *Suprarenal abdominal aortas of 12-week-old, male Apoe -/mice (n = 145) were comprehensively characterized after various pharmacological interventions of saline, AngII, or AngII plus rapamycin (Rapa). The aortic segments were of limited size and each specimen was analyzed using a single technique. Microscopy included histology, histomorphometry, immunohistochemistry, immunofluorescence, and confocal procedures. Aortas with hemorrhagic lesions were analyzed by microscopy but excluded from other assays to avoid the confounding influence of extravasated blood. Additional cohorts of 12-week-old, male mice (n = 6) used to derive SMC cultures and 12week-old, female mice (n = 6) and 3 week-old, male mice (n = 6) used for preliminary signaling studies are not included. Colorimetric assay for number of murine aortic SMCs adherent to fibronectin-coated plates after 1 h following cell pretreatment with (A) CTGF and/or thrombospondin-1 (TSP1), (B) CTGF and/or tenascin-C (TNC), and (C) thrombospondin-1 and/or tenascin-C at 12.5 and 25 μg/mL for 45 min (n = 3-7, pooled from 2 experiments); OD405 readings normalized to untreated controls. Individual data shown, bars represent mean ± SEM, ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with Tukey's multiple comparisons test. Additive interactions are defined as the effects from combined matricellular proteins at a particular dose (12.5 μg/mL, each) as equal to that of both individual matricellular proteins at twice that dose (25 μg/mL), and synergistic interactions are defined as the effects from combined matricellular proteins at a particular dose (12.5 μg/mL, each) as greater than that of both individual matricellular proteins at twice that dose (25 μg/mL). No positive interactions among the matricellular proteins were identified by these definitions.