Specimens were obtained from 12 patients who had undergone elective surgical repair of TAA (7 males and 5 females; average age, 65.8±6.9 years). Patients with degenerative aortic aneurysms recruited to the present study did not have connective tissue disorders, familial aortopathy or bicuspid aortic valve aortopathy, and aneurysms >6 cm in diameter were selected. All patients were admitted to the China Medical University Hospital (Liaoning, China) between January 2009 and December 2014. Patients with recent infection, active inflammatory disorder, malignancy, collagen disease or chronic renal failure were not included in the study. Normal aorta samples (used as controls) were obtained from heart transplantation donors (n=5). Controls were age-matched and asymptomatic, with no significant history of medication. The investigation conformed to the principles outlined in The Declaration of Helsinki. All protocols using human samples were approved by the Institutional Review Board of China Medical University Hospital and samples were obtained with written informed consent.

All animal experiments were conducted in strict accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (23). The protocol was approved by the Institutional Animal Care and Use Committee of China Medical University. A total of 30 male Wistar rats (weight, 200–250 g; age, 7–8 weeks; Experimental Animal Center of China Medical University, Liaoning, China) were used in the present study. The animals were housed in cages at room temperature (25±1°C) and 50% humidity, with a 12-h light/dark cycle and free access to chow and water. Rat TAA models were induced by periaortic application of CaCl₂ as previously described (19). Sham group animals (n=10) were treated with saline instead of CaCl₂. Following surgery, the CaCl₂-induced TAA rats were then randomly divided into the curcumin and vehicle groups. The curcumin group (n=10) was treated daily by oral gavage with 100 mg/kg curcumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) suspended in 1% carboxymethyl cellulose. The vehicle group (n=10) and sham group were treated daily by oral gavage with the same volume of 1% carboxymethyl cellulose only. The dose of curcumin was based on our previous experiment (19). Following 28 days, the rats were humanely sacrificed, and whole hearts and aortas were excised and analyzed.

The samples were either frozen in liquid nitrogen or fixed at 4°C in 4% phosphate-buffered formalin for 24 h. For each subject, one part of the aneurysm was used for histological examination and the other part was used for western blot analysis.

To accurately measure the aortic diameter, the aneurysm diameters were calculated using light microscopy (Olympus SZH Stereo Zoom microscope; Olympus Corporation, Tokyo, Japan) as previously described (19). Digital images of the descending thoracic aorta were obtained using a color charge-coupled device camera (7.2 Color Mosaic; Micro Video Instruments, Inc., Avon, MA, USA) linked to a computer with digital imaging software (SPOT Advanced version 3.5; Micro Video Instruments, Inc.).

Aortas were fixed at 4°C in 4% PBS-buffered paraformaldehyde for 24 h and then embedded in paraffin. For histological analysis, aortic tissue cross-sections (5 µm) from the human patients and rats were prepared. Sections were deparaffinized at room temperature with 2 applications of xylene for 10 min each. They were then rehydrated with 2 applications of absolute alcohol for 5 min each, and placed in distilled water. Hematoxylin and eosin (H&E) was applied to analyze general tissue morphology. Hematoxylin staining solution was added for 8 min at room temperature, followed by 1% acid alcohol for 30 sec, then tissues were counterstained with eosin-phloxine solution for 1 min at room temperature. Sections were then dehydrated with absolute alcohol, cleared with xylene and mounted. The change in elastic lamellar was observed by Orcein staining. The prepared sections placed in distilled water were stained in 1% Orcein for 1 h at room temperature, then dehydrated with 2 applications (5 min each) of absolute alcohol, cleared with 2 applications (10 min each) of xylene and mounted. For immunohistochemical analysis, aortic cross sections were deparaffinized twice in xylene (10 min each), rehydrated twice with absolute alcohol (5 min each) and placed in distilled water. Following heat-induced antigen retrieval in a microwave, the cross sections were blocked at room temperature with 3% hydrogen peroxide for 10 min. Diluted primary antibodies were applied to the sections and incubated overnight at 4°C. The primary antibodies, anti-VEGF (cat. no. bs-1313R; 1:150; BIOSS, Beijing, China), anti-CD3 (cat. no. MCA772; 1:200; T cell marker; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and anti-CD68 (cat. no. BA3638; 1:200; macrophage marker; Wuhan Boster Biological Technology, Ltd., Wuhan, China) were used to locate inflammatory cell infiltration in the aortic walls. Immune complexes were visualized using 3,3′-diaminobenzidine staining and slides were counterstained with hematoxylin. All sections were then dehydrated in graded alcohols and covered by coverslips with neutral balsam for light microscopy. For negative control experiments, the primary antibodies were omitted and the sections were processed as aforementioned. Non-specific staining was not observed. For all sections, the inflammatory cells were determined by counting the mean number of nuclei surrounded by positive immunostaining within ×400 high power fields (5–6 fields of view/section).

Frozen aortic tissues from patients, and aortic tissues and whole hearts from rats, were lysed in radioimmunoprecipitation buffer containing a protease inhibitor cocktail, 1 mM β-glycerolphosphate, 2 mM sodium vanadate, 2.5 mM sodium pyrophosphate, 1 mM EDTA and 1 mM EGTA. Total protein levels were measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins (40 µg) were then separated by 10% SDS-PAGE and were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBS containing 0.1% Tween-20 for 2 h at room temperature and were then incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Anti-VEGF (cat. no. bs-1313R; 1:500; BIOSS), anti-monocyte chemoattractant protein-1 (cat. no. bs-1101R; MCP-1; 1:1,000; BIOSS), anti-tumor necrosis factor (TNF)-α (cat. no. bs-2081Rl; 1:1,000; BIOSS), anti-vascular cell adhesion molecule-1 (cat. no. sc-13160; VCAM-1; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-intercellular adhesion molecule-1 (cat. no. sc-8439; ICAM-1; 1:1,000; Santa Cruz Biotechnology, Inc.) and rabbit anti-β-actin (cat. no. bs-0061R; 1:1,000; BIOSS). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat. no. bs-0295G; 1:5,000; BIOSS) and HRP-conjugated goat anti-mouse IgG (cat. no. bs-0296G; 1:5,000; BIOSS) for 1 h at room temperature. They were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) and ChemDoc XRS with Quantity One version 4.1 software (Bio-Rad Laboratories, Inc.). The results were normalized to those of β-actin. Blots were repeated at least three times for each condition. To quantify and compare the levels of proteins, the density of each band was measured by densitometry (Image Lab software version 4.1; Bio-Rad Laboratories, Inc.).

Terminal aortic size was expressed as a percentage increase from the baseline size in each rat. The mean percentage of change was then determined and compared to the control group. All values are expressed as the mean ± standard error of the mean. All statistical analyses were carried out using IBM SPSS Statistics (version 19; IBM Corp., Armonk, NY, USA) and experiments were repeated three times. One-way analysis of variance with the Dunnett's post hoc test were used to determine the significance of differences in multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

H&E and Orcein staining were performed in human TAA samples. H&E staining revealed that there was an increase in microvessel density in the medial and adventitia of the human TAA samples when compared with the control aortic samples. In addition, Orcein staining identified degradation in the wavy structure of elastin when compared with the control aortic samples (Fig. 1A and B).

It has recently been reported that VEGF has an important role in the pathogenesis of AAA (12). Therefore, the present study detected the expression levels of VEGF in TAA. Western blotting demonstrated that the expression of VEGF was significantly increased in TAA samples compared with the control aortic samples (Fig. 1C and D).

To examine the effects of curcumin on vasculogenesis and inflammation in TAA, a rat model of TAA was established, and rats were then treated with curcumin daily for 4 weeks. Aortic diameter was measured in the TAA animal model 28 days following the operation. The diameter of the thoracic aorta was larger in the vehicle group compared with in the sham-operated group. However, treatment with curcumin resulted in a decrease in thoracic aortic diameter compared with in the vehicle group (Fig. 2A and B). These results revealed that TAA may be prevented in CaCl₂-treated rats by curcumin treatment.

As observed in the human TAA samples, H&E staining revealed that the number of microvessels in the aortic wall was increased in the adventitia of the rat TAA samples compared with the control group. However, this effect was attenuated by treatment with curcumin (Fig. 2C).

In parallel with thoracic aortic dilatation with CaCl₂ treatment, vascular elastin integrity was altered, as determined by Orcein staining (Fig. 2D). The aortic wall of the sham group was intact without damage. However, in the TAA vehicle group, the medial elastic lamellae in some parts of the aorta lost its wave shape and developed fragmentations. Conversely, administration of curcumin inhibited elastin fragmentation and preserved the elastic laminar wave (Fig. 2D). These results suggested that curcumin treatment may effectively decrease the formation of microvessels and elastin degeneration in the rat TAA model.

To determine the effects of curcumin on the expression levels of VEGF in TAA, immunohistochemical staining and western blotting were performed. There was a low expression of VEGF in the normal aorta; however, in vehicle-treated rats the expression of VEGF in the aorta was significantly increased when compared with the sham group. In addition, curcumin treatment significantly reduced the expression of VEGF when compared with the vehicle group (Fig. 3A and B). To further determine whether curcumin had an inhibitory effect on other organs, the rat heart tissues were homogenized and total proteins extracted. Western blot analysis revealed that VEGF expression was not decreased in the curcumin-treated group, which indicated that curcumin treatment may not affect VEGF expression in other organs (Fig. 3C).

To evaluate inflammatory cell infiltration in the vascular wall, immunostaining of CD68 (macrophage marker) and CD3 (T cell marker) was conducted, and the number of positive cells was counted. The number of CD68⁺ (Fig. 4A and B) and CD3⁺ (Fig. 4C and D) cells was increased in the aortas of TAA rats compared with the sham rats, indicating that there may be increased infiltration of macrophages and T cells in TAA. However, treatment with curcumin inhibited macrophage and T cell infiltration in the aortas of TAA rats when compared with the vehicle group.

VCAM-1, ICAM-1 and TNF-α are markers of immune-inflammatory activation. Curcumin decreased inflammatory cell infiltration in the aorta; therefore, the associated inflammatory factors were assessed by western blotting (Fig. 5A). The protein expression levels of VCAM-1 (Fig. 5B), ICAM-1 (Fig. 5C), MCP-1 (Fig. 5D) and TNF-α (Fig. 5E) were significantly increased in the aortic walls of vehicle-treated rats compared with sham-operated rats. Conversely, curcumin significantly decreased the levels of inflammatory factors in the vehicle-treated rats, to a comparable level to the sham control group. These results indicated that curcumin effectively decreased inflammatory activation in the aorta in TAA.