The experimental study was conducted in accordance with the Guide for the Care and Usage of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA, 2011) and approved by the Committee for Animal Research of Shanghai Jiao Tong University (Shanghai, China).

Male Sprague-Dawley rats (n=70; 8-weeks-old; 250–300 g) were kept in a specific-pathogen-free facility by Ruijin hospital (Shanghai, China). A 12-h light/dark cycle was used and a temperature of 22°C was maintained in the facility. Rats had free access to food and water at all times. All rats were supplied by Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and were randomly allocated to TAA group (CaCl₂-treated; n=18), sham group (NaCl-treated; n=18), DMSO group (treated with CaCl₂ and DMSO; n=12) or rapamycin group (treated with CaCl₂ and rapamycin; n=12). The rat TAA model was established with CaCl₂, as previously described (12). Briefly, all animals were subjected to orotracheal intubation and mechanical ventilation. The chest cavity was opened, and the descending thoracic aortic segment was wrapped with a strip (sterilized medical cotton pad) presoaked in 0.5 M CaCl₂ solution or normal saline for 15 min. The experimental mortality for the treatment and control groups was ~10–20% due to anesthesia side effects or surgical trauma.

For the rapamycin treatment, 2 mg/kg rapamycin (AG Scientific, Inc., San Diego, CA, USA) in DMSO (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or DMSO alone (vehicle) was administered once intraperitoneally 1 day prior to the injury and continued for 2 weeks daily.

A ketamine/xylazine cocktail made with normal saline was used for deep anesthesia prior to performing terminal tissue collection. Specifically, 80 mg/kg ketamine (Zhejiang Jiuxu Pharmaceutical Co., Ltd., Jinhua, China) and 10 mg/kg xylazine (Selleck Chemicals, Houston, TX, USA) were administered as a single intraperitoneal injection. All rats were euthanized 2 days or 2 weeks following induction. The thorax was opened beneath the xiphoid process. The rats' right atrium was incised to provide an outlet for blood and perfusate. The cardiovascular system was perfused with normal saline from the left ventricle at a pressure of 100 mmHg for 10 min, and then the entire aorta was harvested. Only the part that was treated with the medical cotton pad (CaCl₂ or NaCl) was used in the following experiment. Aortic tissues were stored at −80°C until protein isolation was conducted for western blotting analysis. For histological and immunohistochemical studies, the cardiovascular system was subjected to perfusion with 4% formalin for 10 min, stored in 4% formalin solution at 4°C, and then embedded in paraffin. Sections of 5 µm thickness were cut.

Formalin-fixed, paraffin-embedded sections were deparaffinized at 56°C, washed with xylene and rehydrated using serial concentrations of ethanol. For immunohistochemistry staining, heat-mediated antigen retrieval (98°C for 6 min) was performed to enhance antigen exposure prior to detection. H₂O₂ (0.3%) was used to suppress endogenous peroxidase activity to reduce background staining at room temperature for 15 min. Normal goat serum (10%; Fuzhou Maixin Biotech, Co., Ltd., Fuzhou, China) was used prior to immunostaining and incubated with membranes at room temperature for 1 h to reduce non-specific staining. Hematoxylin and eosin (H&E) staining was performed for morphometric analysis. Sections were stained using a modified Verhoeff-Van Gieson (VVG) stain kit (Sigma-Aldrich; Merck KGaA) to evaluate the elastic fiber content following the manufacturer's instructions. Elastin breaks were quantified in triplicate by two independent investigators. The disruption of elastic fibers was graded on a scale of 1–4 (1, normal or disruption <25%; 2, disruption 25–49%; 3, disruption of 50–75%; 4, disruption >75% or total absence).

The antibodies and dilutions used in the present study are listed in Table I. All primary antibodies were diluted and incubated with slides at 4°C overnight. Biotin-conjugated antibodies (cat. no. BA-1300; 1:200; Vector Laboratories, Inc., Burlingame, CA, USA) were used as secondary antibody and were incubated with membranes at room temperature for 1–2 h. For immunohistochemistry, the positively stained area was determined using the threshold-based digital planimetry software (Image-Pro Plus 6.0; Media Cybernetics, Inc., Rockville, MD, USA), and expressed as the percentage of the positive area in the intima-media or adventitia.

SMCs were explanted from the descending thoracic aortas, as previously described (11). SMCs were subcultured in a complete SMC medium (Lonza Group, Basel, Switzerland) supplemented with 20% fetal bovine serum, antibodies, L-glutamine, HEPES, sodium pyruvate, insulin, recombinant human epidermal growth factor, and recombinant human fibroblast growth factor) in a 37°C, 5% CO2-humidified incubator. SMCs from passages 2–6 were used for experiments, and all experiments were confirmed in at least three different explants of SMCs.

SMCs were divided into different groups: group 1 was divided into subgroups treated with different concentrations of epidermal growth factor (EGF; 0, 1.25, 2.5 and 5.0 µg/ml). Group 2 was divided into four subgroups treated with either saline, rapamycin (20 nmol/l), EGF (2.5 µg/ml) or rapamycin (20 nmol/l) + EGF (2.5 µg/ml). Group 3 was divided into four subgroups treated with either saline, rapamycin (20 nmol/l), siTSC2 (small interfering RNA targeting TSC2), and rapamycin (20 nmol/l) + siTSC2.

The TSC2 siRNA and control nonspecific siRNA were synthesized by GeneChem Co., Ltd. (Shanghai, China). SMCs were transfected with TSC2 siRNA according to the manufacturer's instructions. In parallel, untreated cells and cells transfected with nonspecific siRNA were used as controls. The experiments were performed in triplicate for each experimental condition.

At each time point, the cells were harvested, washed in ice-cold phosphate-buffered saline (PBS), and lysed with radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA). The protein was extracted, and its concentration was determined using the Bio-Rad protein assay. Equal cell lysates (10 µg) were separated by 10% SDS-PAGE with Tris-HCl gel (Ready Gel; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P; EMD Millipore, Billerica, MA, USA). Following blocking in buffer (5% non-fat milk in T-PBS) for 3 h, the membranes were incubated overnight at 4°C with a primary antibody diluted in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA). Western blot analysis was performed using primary antibodies against α-tubulin (cat. no. T8203; 1:2,000; Sigma-Aldrich; Merck KGaA), mTOR (cat. no. 2972; 1:1,000), phospho-mTOR (cat. no. 5536; 1:500), S6 (cat. no. 2317; 1:1,000), phospho-S6 (cat. no. 5364; 1:500), p70S6k (cat. no. 9202; 1:1,000) and phospho-p70S6k (cat. no. 9206; 1:500; all from Cell Signaling Technology, Inc., Danvers, MA, USA), smooth muscle myosin heavy chain (cat. no. ab53219; 1:1,000), osteopontin (OPN; cat. no. ab8448; 1:1,000) and S100A4 (cat. no. ab41532; 1:1,000; all from Abcam, Cambridge, MA, USA). The membranes were probed with horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibody (cat. nos. 115-035-003 and 111-035-003, respectively; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The secondary antibody was incubated with membranes at room temperature for 1 h, at a dilution of 1:5,000. Signals were developed using an enhanced chemiluminescence Western Blotting Detection kit (GE Healthcare Life Sciences, Chalfont, UK). α-tubulin was used as a control.

The morphometric analysis was performed blinded for the statistician. The results were statistically analyzed using one-way analysis of variance, with the Dunnett's C post hoc test. A two-sided probability level of P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using the SPSS for Windows software (version, 13.0; SPSS, Inc., Chicago, IL, USA).

A CaCl₂-induced rat TAA model was previously established to explore the potential role of mTOR signaling pathway in the disease development (12). In the present study, male Sprague-Dawley rats (250–300 g) underwent periarterial exposure of thoracic aorta to either 0.5M CaCl₂ or normal saline (0.90% NaCl). Strikingly, immunohistochemical assessment of proinflammatory cytokines [i.e., tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β] by computerized planimetry in the aortic adventitia and aortic media revealed enhanced protein expression as early as 2 days following TAA induction (Fig. 1A and B). These results confirmed that the infiltration of inflammatory cells contributes to the pathogenesis of TAA.

Besides the enhanced expression of proinflammatory cytokines, immunoblot analyses revealed a significantly increased phosphorylation of mTOR (Ser2448) and S6K (Thr389) as early as 2 days following TAA induction (Fig. 1C). The activation of S6K was further confirmed by the increased phosphorylation of its substrate, S6, at both Ser240 and Ser244. Interestingly, mTOR signal enhancement was not detected 2 weeks post-induction, suggesting a role of the mTOR signaling pathway in the early development of TAA.

At 2 weeks post-induction, H&E staining was performed for measuring the aortic diameter in each group, presenting aneurysmal alteration in the CaCl₂-treated segments (Fig. 2A). In addition, more severely disrupted elastic fibers were observed in CaCl₂-treated segments than in CaCl₂-untreated segments by VVG staining (Fig. 2A).

SMCs contract in response to changes in pulse pressures through a cyclic interaction between thin and thick contractile filaments composed of SMC-specific isoforms of α-actin (α-SMA) and myosin heavy chain, respectively. Heterozygous mutations in α-SMA, encoded by the ACTA2 gene, predispose TAAs and acute aortic dissections (13). Therefore, the present study aimed to determine the expression of contractile proteins including α-SMA and calponin in aortic SMCs. Immunohistochemical staining revealed decreased expression of both proteins in the CaCl₂-treated segments. In contrast, an increased expression of SMC dedifferentiation markers S100A4 and osteopontin (OPN) was observed in the CaCl₂-treated segments (Fig. 2C).

MMPs are a family of zinc-dependent endopeptidases capable of degrading different extracellular matrix components including collagens and elastin. Under normal physiological conditions, the enzymatic activity of MMPs is tightly controlled by their TIMPs that always keep a balance in the MMP/TIMP system, but in vascular pathologies (e.g., aneurysm, vasculitis, atherosclerosis), the increased expression of MMPs has been often observed (14–16). At 2 weeks post-induction, MMP-2 expression was detected diffusely across the entire layers of CaCl₂-treated segments, which was significantly higher than the expression across the NaCl-treated segments (Fig. 2C). Similar changes were also observed for MMP-9 expression (Fig. 2C).

Pretreatment with rapamycin, a pharmacological inhibitor of the mTOR signaling pathway, reversed the augmentation of CaCl₂-treated segments (Fig. 2A) in both diameters and elastic fiber degradation. Fig. 3 demonstrated that, with rapamycin pretreatment (RAPA group), IOD (integral optical density) values of TNF-α, IL-6 and IL-1β significantly decreased (P<0.01) compared with those without treatment (TAA and DMSO groups). As presented in Fig. 3A, in the RAPA and sham groups, immunostaining-positive cells in the media layer of aorta were arranged in line with less cytoplasm coloring. In contrast, in the TAA and DMSO groups, a large number of immunostaining-positive cells, in a mass, presented deep cytoplasm coloring. Staining with a macrophage-specific antibody indicated that all aneurysm tissue sections contained CD68⁺ macrophages that were frequently present diffusely in the adventitia of the aortas from the CaCl₂-treated group at 2 weeks, but were rarely found in the control aortas (Fig. 3A, CD68 staining). IOD values of CD68 in the RAPA group significantly decreased (P<0.01) compared with those without treatment (TAA and DMSO groups). The results suggested that rapamycin pretreatment suppressed the hypersecretion of TNF-α, IL-6, IL-1β and CD68 and therefore protected the aorta against proinflammatory stress.

Immunohistochemical assays of α-actin and calponin contractile proteins revealed that the expression of both proteins progressively increased following treatment with rapamycin (Fig. 2C). In contrast, following treatment with rapamycin, a significant reduction in expression was observed for dedifferentiation markers S100A4 and OPN (Fig. 2C).

As described previously, in the CaCl₂-treated segments, MMP-2 significantly increased in the entire layers of aorta, while the NaCl-treated segments presented only mild staining (Fig. 2C). MMP-9 protein was also demonstrated at a peak level in the adventitia and elastic lamellae 2 weeks post-TAA induction. Following treatment with rapamycin, a significant reduction in MMP-2 and MMP-9 was observed (Fig. 2C).

To further explore the mechanism underlying the rapamycin-associated inhibition of TAA formation, rat aortic SMCs were isolated to conduct the in vitro cell culture assay. mTOR signaling was induced by treating cells with different concentrations of EGF (0, 1.25, 2.5 and 5.0 µg/ml). The EGF treatment of SMCs was indicated to activate mTOR signaling and led to an increased expression of dedifferentiation marker OPN (Fig. 4A). However, pretreating aortic SMCs with rapamycin inhibited EGF-induced mTOR activation, leading to decreased expression of OPN and S100A4 (Fig. 4B).

In another study, siRNA technology was used to downregulate TSC2 expression (11). TSC2 is a negative regulator of mTOR signaling (11). Therefore, when TSC2 was downregulated, the mTOR signaling pathway was activated. An increased expression of OPN and S100A4 was observed with TSC2 downregulation. However, when the cells were pretreated with rapamycin, the activation of the mTOR signaling pathway was inhibited, which is consistent with the fact that mTOR is a downstream mediator of TSC2. In the siTSC2 and rapamycin-double-treated cells, the expression levels of OPN and S100A4 were similar to the levels observed in rapamycin-treated cells (Fig. 4C).