Testosterone ameliorates vascular aging via the Gas6/Axl signaling pathway

TU treatment up-regulated testosterone and Gas6/Axl expression in aging mice

Serum testosterone level was significantly decreased in both WT (P<0.001) and Axl^-/- (P<0.001) old groups compared with WT and Axl^-/- young groups (Figure 1A). With testosterone undecanoate (TU) treatment, the serum testosterone level in WT (P<0.001) and Axl^-/- (P<0.01) old+TU groups was increased compared with WT and Axl^-/- old groups (Figure 1A).

Figure 1. TU treatment up-regulated testosterone and Gas6/Axl expression in aging mice. (A) The serum level of testosterone. (B) The serum level of Gas6. (C) Relative mRNA expression of Gas6 and Axl. (D) Relative protein content of Gas6 and Axl. Data are mean ± SD; ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. WT young group; ^†P < 0.05 and ^‡P < 0.001 vs. WT old group; ^&P < 0.05 and ^&&&P < 0.001 vs. Axl^-/- young group; ^#P < 0.05 and ^##P < 0.01 vs. Axl^-/- old group; A-B: n = 8 for each group, C-D: n = 3 for each group.

The serum Gas6 content was markedly decreased in WT and Axl^-/- old groups (P<0.05) compared with WT and Axl^-/- young groups (Figure 1B). TU replacement therapy significantly increased the serum Gas6 level in WT and Axl^-/- old+TU groups compared with WT and Axl^-/- old groups (P<0.05) (Figure 1B). Similarly, the mRNA and protein expression of Gas6 in aorta were markedly decreased in WT and Axl^-/- old groups (P<0.05~P<0.01) compared with WT and Axl^-/- young groups (Figure 1C, 1D). With TU replacement therapy, the mRNA and protein expression of Gas6 in aorta were increased in WT and Axl^-/- old+TU groups compared with WT and Axl^-/- old groups (P<0.05) (Figure 1C, 1D).

The mRNA and protein expression of Axl were decreased in WT old group compared with WT young group (P<0.05~P<0.01) (Figure 1C, 1D). With TU treatment, the Axl mRNA and protein expression levels were elevated in WT old+TU group compared with WT old group (P<0.05) (Figure 1C, 1D). Both mRNA and protein expression of Axl were quite low in all Axl knockout mice (Figure 1C, 1D).

Gas6/Axl was involved in the anti-senescence effect of testosterone on vascular cell senescence

Telomere length was significantly shortened in WT (P<0.01) and Axl^-/- (P<0.01) old groups compared to WT and Axl^-/- young groups (Figure 2A), and telomere length in Axl^-/- old group was shorter than WT old group (P<0.05) (Figure 2A). With TU treatment, the telomere length in WT old+TU group was significantly longer than WT old group (P<0.05) (Figure 2A). Telomere length in Axl^-/- old+TU group was not longer than Axl^-/- old group (Figure 2A). The protein expression of p16^INK4a and p21^Cip1 were increased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.01~P<0.001) (Figure 2B). The Axl^-/- old group had the highest p16^INK4a (P<0.05) and p21^Cip1 (P<0.05) expression compared with all other groups (Figure 2B). With TU treatment, the expression of p16^INK4a (P<0.05) and p21^Cip1 (P<0.05) in WT old+TU group was significantly decreased compared with WT old group (Figure 2B). But the expression of p16^INK4a and p21^Cip1 in Axl^-/- old+TU group were not significantly decreased compared with Axl^-/- old group (Figure 2B). The changing trend of SA-β-Gal staining rate was the same as that of p16^INK4a and p21^Cip1 (Figure 2C, 2D).

Figure 2. Gas6/Axl was involved in the anti-senescence effect of testosterone on vascular cell senescence. (A) The relative telomere length was detected by real-time PCR. (B) Representative Western blot and analysis of p16^INK4a and p21^Cip1. (C): Representative b-galactosidase staining with the descending aorta from the mouse (scale bar: 20μm). (D): Analysis of the relative dyeing rate. Data are mean ± SD; ^**P < 0.01 and ^***P < 0.001 vs. WT young group; ^†P < 0.05 and ^††P < 0.01 vs. WT old group; ^&&P < 0.01 and ^&&&P < 0.001 vs. Axl^-/- young group; A-B: n = 3 for each group, C-D: n = 5 for each group.

The role of testosterone and Gas6/Axl in structural changes of aging vessel

Coinciding with prior studies, the intima-media thickness (IMT) was significantly increased in carotid artery and abdominal artery in both WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.001) (Figure 3A, 3B, 3E). The IMT of carotid artery and abdominal aorta in Axl^-/- old group was not significantly thicker than WT old group (Figure 3A, 3B, 3E). With TU treatment, the IMT of carotid artery (P<0.05) and abdominal aorta in WT old+TU group decreased compared to WT old group (Figure 3A, 3B, 3E). The IMT of carotid artery and abdominal aorta in Axl^-/- old+TU group were not significantly thinner than Axl^-/- old group (Figure 3A, 3B, 3E).

Figure 3. The role of testosterone and Gas6/Axl in structural changes of aging vessel. (A) Carotid artery (CA) and abdominal aorta (AA) of ultrasonography. (B) Intima-media thickness (IMT) of carotid artery. (C) Systolic diameter (Ds) and diastolic diameter (Dd) of carotid artery. (D) Coefficient distention (CD) of carotid artery. (E) IMT of abdominal aorta. (F) Ds and Dd of abdominal aorta. (G) CD of abdominal aorta. Data are mean ± SD; ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. WT young group; ^†P < 0.05 vs. WT old group; ^&P < 0.05, ^&&P < 0.01 and ^&&&P < 0.001 vs. Axl^-/- young group; ^#P<0.05 vs. Axl^-/- old group; n = 8 for each group.

Both diastolic diameter (Ds) and systolic diameter (Dd) of the carotid artery and abdominal aorta in WT and Axl^-/- old groups were significantly increased compared with WT and Axl^-/- young groups (P<0.05~P<0.001) (Figure 3A, 3C, 3F). However, there was no significant difference of Ds and Dd between Axl^-/- old group and WT old group with or without TU treatment (Figure 3A, 3C, 3F). The coefficient distention (CD) of carotid artery and abdominal aorta in WT and Axl^-/- old groups were smaller than the WT and Axl^-/- young groups (P<0.05~P<0.01) (Figure 3A, 3D, 3G). The CD of carotid artery (P<0.05) and abdominal aorta (P<0.05) in Axl^-/- old group were smaller than the WT old group (Figure 3A, 3D, 3G). With TU treatment, the CD of carotid artery (P<0.05) and abdominal aorta (P<0.05) in WT old+TU group were decreased compared with WT old group (Figure 3A, 3D, 3G). There was no significant difference between Axl^-/- old+TU group and Axl^-/- old group (Figure 3A, 3D, 3G).

The role of testosterone and Gas6/Axl in vascular stiffness of aging vessel

The stiffness parameter (β) and pressure-strain elastic modulus (Ep) of carotid artery were significantly increased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.01~P<0.001) (Figure 4A, 4B). The β (P<0.05) and Ep (P<0.05) in Ax l^-/- old group were higher than in WT old group (Figure 4A, 4B). With TU treatment, the β (P<0.05) and Ep (P<0.05) of WT old+TU group were significantly decreased compared with WT old group (Figure 4A, 4B). There was no significant difference between Axl^-/- old+TU group and Axl^-/- old group (Figure 4A, 4B). The distensibility coefficient (DC) and compliance coefficient (CC) of carotid artery were significantly decreased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.05, P<0.001) (Figure 4C, 4D). The DC (P<0.05) and CC of Axl^-/- old group were lower than WT old group (Figure 4C, 4D). With TU treatment, the DC (P<0.05) and CC (P<0.05) of WT old+TU group were significantly elevated compared with WT old group (Figure 4C, 4D), but no statistical difference was found compared with Axl^-/- old+TU group and Axl^-/- old group (Figure 4C, 4D).

Figure 4. The role of testosterone and Gas6/Axl in vascular stiffness and blood pressure of aging vessel. (A) Stiffness parameter (β). (B) Pressure-strain elastic modulus (Ep). (C) Distensibility coefficient (DC). (D) Compliance coefficient (CC). (E) Systolic pressure (SBP) and diastolic blood pressure (DSP). (F) Mean arterial pressure (MAP). (G) Pulse pressure (PP). Data are mean ± SD; ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. WT young group; ^†P < 0.05 and ^††P < 0.01 vs. WT old group; ^&P < 0.05, ^&&P < 0.01 and ^&&&P < 0.001 vs. Axl^-/- young group; #P<0.05 vs. Axl^-/- old group; n = 8 for each group.

The role of testosterone and Gas6/Axl in blood pressure

Both systolic pressure (SBP) and diastolic blood pressure (DBP) were increased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.05~P<0.001) (Figure 4E). The SBP and DBP of Axl^-/- old group were slightly higher than WT old group (Figure 4E). With TU treatment, the SBP and DBP of WT old+TU group were slightly decreased compared with WT old group (Figure 4E). However, no significant difference was found between Axl^-/- old+TU group and Axl^-/- old group in SBP and DBP (Figure 4E). The mean blood pressure (MBP) of WT (P<0.01) and Axl^-/- (P<0.05) old groups were increased significantly compared with WT and Axl^-/- young groups (Figure 4F). However, there was no significant difference in MBP between WT old+TU group and WT old group, Axl^-/- old group and WT old group, Axl^-/- old+TU group and Axl^-/- old group (Figure 4F).

The pulse pressure (PP) increased in WT (P<0.05) and Axl^-/- (P<0.05) old groups compared with WT and Axl^-/- young groups (Figure 4G). PP in Axl^-/- group was higher than WT old group (P<0.05) (Figure 4G). With TU treatment, the PP in WT old+TU group markedly decreased compared with WT old group (P<0.05) (Figure 4G), but no statistical difference was found compared with Axl^-/- old+TU group and Axl^-/- old group (Figure 4G).

Testosterone ameliorated vascular fibrosis in aging-related arterial stiffness

The Masson trichrome staining, Picrosirius red staining and Verhoeff’s Van Gieson staining results showed vascular fibrosis in the media. The collagen contents, collagen-to-elastin ratio, average aortic wall architecture score significantly increased, and elastin content decreased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.01~P<0.001) (Figure 5A_2~4, 5B–5E). And collagen-to-elastin ratio (P<0.05) of Axl^-/- old group were higher than WT old group (Figure 5A_2~4, 5B, 5D, 5E). With TU treatment, the collagen content (P<0.01), collagen-to-elastin ratio (P<0.01) and average aortic wall architecture score (P<0.05) markedly decreased, and elastin content (P<0.05) increased in WT old+TU group compared to WT old group (Figure 5A_2~4, 5B, 5D, 5E). But there was no significantly difference in collagen content, collagen-to-elastin ratio, average aortic wall architecture score and elastin content between Axl^-/- old group and Axl^-/- old+TU group (Figure 5A_2~4, 5B, 5D, 5E).

Figure 5. Testosterone ameliorated vascular fibrosis in aging-related arterial stiffness. (A1-4) Representative H&E staining, Masson trichrome staining, Picrosirius red staining and Verhoeff Van Gieson (scale bar: 20μm). (B–E) Quantitative analyses of collagen content, elastin content, collagen content/elastin content and average aortic wall architecture score. (F) Immunohistochemical staining for collagen I and III (brown staining considered positive staining; scale bar: 20μm). (G) Representative Western blots of collagen I and III. (H) Western blot analyses of collagen I and III. Data are mean ± SD; ^*P < 0.05 and ^**P < 0.01 vs. WT young group; ^†P < 0.05 and ^††P < 0.01 vs. WT old group; ^&P < 0.05, ^&&P < 0.01 and ^&&&P < 0.001 vs. Axl^-/- young group; ^#P<0.05 vs. Axl^-/- old group; A-F: n = 5 for each group, G-H: n = 3 for each group.

The immunohistochemical staining and western blot results showed that collagen I and III expression were significantly increased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.05~P<0.01) (Figure 5F, 5G, 5H). The collagen I and III expression in Axl^-/- old group were higher than WT old group (Figure 5F, 5G, 5H), and were significantly decreased in WT old+TU group compared to WT old group (P<0.05) (Figure 5F, 5G, 5H). However, between the Axl^-/- old+TU group and Axl^-/- old group, no statistical difference was found in collagen I and III (Figure 5F, 5G, 5H).

The protective effects of testosterone on vascular aging was attributed to inhibition of MMPs expression and activity

Immunohistochemical staining, Western blot and Zymography results showed that MMP-2 and MMP-9 expression and activity were significantly increased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.05, P<0.01) (Figure 6A–6C, 6F, 6G). The MMP-2 and MMP-9 expression and activity in Axl^-/- old group were higher than WT old group (P<0.05) (Figure 6A–6C, 6F, 6G), and were significantly decreased in WT old+TU group compared with WT old group (P<0.05) (Figure 6A–6C, 6F, 6G). But there was no significant difference between the Axl^-/- old group and Axl^-/- old+TU group (Figure 6A–6C, 6F, 6G).

Figure 6. The protective effects of testosterone on vascular aging were attributed to inhibition of MMPs expression and activity through promotion of Akt/FoxO1a phosphorylation. (A) Immunohistochemical staining for MMP-2 and MMP-9 (brown staining considered positive staining; scale bar: 20μm). (B) Representative Western blot of MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP. (C–E) Western blot analyses of MMP-2, MMP-9, TIMP-1, TIMP-2, MT1-MMP, MT1-MMP/TIMP-2 and MMP-9/TIMP-1. (F) Representative Gelatin Zymography of MMP-2 and MMP-9 activity. (G) Gelatin Zymogrphy analyses of MMP-2 and MMP-9 activity. (H) Representative Western blot of p-Akt, t-Akt, p-FoxO1a and t-FoxO1a. (I) Western blot analyses of p-Akt, t-Akt, p-FoxO1a and t-FoxO1a. Data are mean ± SD; ^*P < 0.05 and ^**P < 0.01 vs. WT young group; ^†P < 0.05 and ^††P < 0.01 vs. WT old group; ^&P < 0.05, ^&&P < 0.01 and ^&&&P < 0.001 vs. Axl^-/- young group; ^#P<0.05 vs. Axl^-/- old group; A: n = 5 for each group, B-H: n = 3 for each group.

The MT1-MMP expression, MT1-MMP to TIMP-2 ratio and MMP-9 to TIMP-1 ratio were significantly elevated, while TIMP-1 and TIMP-2 expressions were decreased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.05~P<0.001) (Figure 6B–6E). The MT1-MMP expression, MT1-MMP to TIMP-2 ratio (P<0.05) and MMP-9 to TIMP-1 ratio (P<0.05) in Axl^-/- old group were higher than WT old group (Figure 6B–6E), and were significantly decreased in WT old+TU group compared to WT old group (P<0.05~P<0.01) (Figure 6B–6E). There was not significantly different between the Axl^-/- old+TU group and Axl^-/- old group (Figure 6B–6E).

Elevated phosphorylation of Akt/FoxO1a was implicated in the protective effects of testosterone on vascular aging

The phosphorylation levels of Akt and FoxO1a were significantly decreased in WT and Axl^-/- old groups compared with WT and Axl^-/- young groups (P<0.05~P<0.01) (Figure 6H, 6I). The phosphorylation levels of Akt (P<0.05) and FoxO1a (P<0.05) in Axl^-/- old group were lower than WT old group (Figure 6H, 6I). With TU treatment, the phosphorylation levels of Akt (P<0.05) and FoxO1a (P<0.05) were significantly increased in WT old+TU group compared with WT old group (Figure 6H, 6I). But there was no significant difference in phosphorylation levels of Akt and FoxO1a between the Axl^-/- old+TU group and Axl^-/- old group (Figure 6H, 6I).

Animal model and drug treatment in vivo

Ninety Axl^-/- male mice were offered by Professor Pan J. from Shandong Normal University (Jinan, China). Ninety wild-type male mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China), all mouse lines were on a B6129SF2/J genetic background. The animals were housed at 22°C with 12-h light-dark cycles and maintained on a normal chow diet and water. Mice of two gene types were then randomized into three groups: the young group (n = 30, 3 months old), the aging group (n = 30, 18 months old) and the testosterone undecanoate treatment group (TU, n = 30, 18 months old). Mice in TU group were given testosterone undecanoate (37.9 mg/Kg) by subcutaneous injection on the back at fifteen-month-old, once a month, a total of three times. Old group received solvent reagent (corn oil) by the same method. At the end of the experiment, the mice were sacrificed. All animals procedures were performed corresponding to the institutional guidelines of Qilu Hospital of Shandong University and were approved by Shandong University Institutional Animal Care and Use Committee.

Ultrasonography

Ultrasonography was performed at 3, 15 and 18 month. All animals were anesthetized and laid supine on a heated table, and warmed ultrasound transmission gel was placed on the chest. The two-dimensional imaging was performed by using the Real-TimeMicro Visualization Scanhead (RMV 704) with a central frequency of 40 MHz at the mechanical transducer (Vevo 770; VisualSonics, Toronto, ON, Canada). Intima-media thickness (IMT), diastolic diameter (Dd) and systolic diameter (Ds) measurements were performed according to previously validated protocols in humans. Two-dimensional ultrasonography was used to calculate arterial stiffness index (β), Peterson’s elastic modulus (Ep), distensibility coefficient (DC) and compliance coefficient (CC). All measurements reported represented the means of three consecutive cardiac cycles and were performed by the same investigator.

β, Ep, DC and CC were estimated automatically by the following formulae:

β = ln {(Ps / Pd) / [(Ds – Dd) / Dd]}

Ep = (ΔP / ΔD) × Dd = [(Ps – Pd) / (Ds – Dd)] × Dd (10⁶dyn / cm²)

DC = (2ΔD / D) / ΔP

CC = (πD × ΔD) / 2ΔP

Blood pressure measurement

Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate were measured by a pulse based tail-cuff method with a photoelectric device (Natsume, Tokyo, Japan) at 3, 15 and 18 month. Blood pressure and heart rate were reported as a mean of 3 consecutive measurements.

Tissue preparation

The aorta was rapidly removed and washed in a phosphate-buffered solution (PBS). The upper part of the aorta (approximate 5 mm) was fixed in 4% neutral formaldehyde for 48 h. The tissues were dehydrated in ethanol, embedded in paraffin, and cross sectioned (4-μm thickness) for histology staining. The remaining portions of the aortas were immediately frozen in liquid nitrogen and stored at -80 °C for molecular studies.

Western blotting analysis

The aortas were snap-frozen in liquid nitrogen, pulverized, and resuspended in ice-cold lysis buffer (Beyotime). Lysates were placed on ice for 30 min to solubilize, and supernatant was collected by centrifugation (15,000g) for 15 min at 4°C. Protein concentrations were determined with the Bicinchoninic Acid Assay (BCA) method, and then the supernatants were analyzed by SDS-polyacrylamide gel electrophoresis. About 30 μg of total protein per well was loaded and then electrophoresed at 80 V for 30 minutes and 110V for 60 minutes. The gels were then transferred at 200mA for 120minutes onto a PVDF membrane. Membranes were blocked with 5% non-fat milk in TBS with 0.1% Tween-20 for 1.5 hours. After blocking, membranes were incubated at 4°C for overnight with the specific primary antibodies in antibody diluent (P0023A, Beyotime). Gas6 (Goat, Santa Cruz Biotechology, USA), Axl (Rabbit, Cell Signaling Technology, USA), p16^INK4a (Mouse, Santa Cruz Biotecholog, USA), p21^Cip1 (Rabbit, Santa Cruz Biotecholog, USA), collagen I (Rabbit, AbcamAbcam, USA), collage III (Rabbit, Abcam, USA), MMP-2 (Rabbit, Abcam, USA), MMP-9 (Rabbit, Abcam, USA), TIMP-1 (Rabbit, Santa Cruz Biotecholog, USA), TIMP-2 (Rabbit, Santa Cruz Biotecholog, USA), MT1-MMP (Rabbit, Santa Cruz Biotecholog, USA), Akt (Rabbit, Cell Signaling Technology, USA), p-Akt (Rabbit, Cell Signaling Technology, USA), FoxO1a (Rabbit, Cell Signaling Technology, USA), p-FoxO1a (Rabbit, Cell Signaling Technology, USA) and β-actin (Mouse, Zhongshan Biotech, China), followed by anti-IgG horseradish peroxidase-conjugated secondary antibody. The membrane bands were visualized by use of chemiluminescence (Millipore) and quantified by densitometry with application of the Image J software (U.S. National Institutes of Health, Bethesda, USA).

Quantitative real-time RT-PCR

Total RNA was extracted using TRIzol reagent (Takara), then quantified by spectrophotometry and reverse-transcribed with the Reverse Transcriptase System (TAKARA) and oligo (dT) primers. RT-PCR was performed using the following primers: β-actin, forward 5’-CAT GTA CGT TGC TAT CCA GGC-3’and reverse 5’-CTC CTT AAT GTC ACG CAC GAT-3’; Gas6, forward 5’-GCT TCT GCT GCT CCT GCT G-3’and reverse 5’-ATA GTC CGT CTC GGG GTC GT-3’; Axl, forward 5’-TGA TAA CAC CCA GAC CCA GG-3’ and reverse 5’-TGA CTC CCT TGG CAT TGT GG-3. The mRNA levels for Gas6 and Axl were determined by a real-time PCR thermocycler (CFX Real-Time PCR cycler; Bio-Rad) with SYBR green as fluorescence dye. All values obtained were normalized to mouse β-actin.

Telomere length measurement

The genomic DNA of the descending aorta was extracted and the CT values of telomere and single copy gene were measured by RT-PCR. The data was collected by Bio-Rad iQ5 system. Relative ratio of telomere/single copy gene = 2^-ΔΔCT. The primer sequences were as follows: Tel 1 5’-GGT TTT TGA GGG TGA GGG TGA GGG TGA GGG TGA GGG T-3’, Tel 2 5’-TCC CGA CTA TCC CTA TCC CTA TCC CTA TCC CTA TCC CTA-3’; HBG 1 5’-GCT TCT GAC ACA ACT GTG TTC ACT AGC-3’, HBG 2 5’-CAC CAA CTT CAT CCA CGT TCA CC-3’.

Senescence-associated β-galactosidase staining

After the β-galactosidase staining solution was added, the sections of aortic walls were incubated at 37 °C without CO2 overnight. Cells which had blue-green granules in their cytoplasm were regarded as positive staining. For each group, six fields of the sections were selected randomly to calculate the positive staining rate using Image-Pro Plus 6.0 software.

ELISA

The serum level of Gas6 (R&D Systems, USA) and testosterone (Abcam, USA) was measured by ELISA assay according to the manufacturer’s instruction.

Histopathological analysis

Morphological changes of aortic walls were observed in sections stained with Hematoxylin-eosin staining. The collagen deposition of aortic walls was evaluated with Sirius red staining and Masson staining. Elastin content was measured by Verhoeff staining. The relative positive staining area for collagen and elastin were measured by an image analysis system (Image-Pro Plus, Version 6.0; Media Cybernetics, Houston, TX).

Immunohistochemical analysis

Sections were conducted antigen retrieval with a microwave-based method. Primary antibodies for collagen I (Rabbit, Abcam, USA), collagen III (Rabbit, Abcam, China), MMP-2 (Rabbit, Abcam, USA), MMP-9 (Rabbit, Abcam, USA) were applied and incubated overnight in PBS at 4°C. After three washes in PBS, (10min/wash), sections were incubated with the appropriate secondary antibody for 30 minutes at 37°C. The stained sections were developed with diaminobenzidine and counterstained with hematoxylin. The results were viewed under a confocal FV 1000 SPD laser scanning microscope (Olympus, Japan) and evaluated using Image-Pro Plus software.

Zymography

MMP activities of the aortic segment were assessed with zymography. Briefly, samples were minced and homogenized in ice-cold PBS, then tissues were centrifuged to collect proteins. Protein content was measured by a Bio-Rad protein assay, and SDS-polyacrylamide gel electrophoresis zymography was performed. One part of homogenate containing 30 μg of protein was loaded on a 10% SDS-polyacrylamide gel containing 0.1% gelatin.

Statistical analyses

All data was presented as mean ± SD. SPSS 20.0 (SPSS, Chicago, IL) was used for statistical analysis. Results were compared by one-way ANOVA, followed by Tukey-Kramer post hoc test and independent samples t test. P<0.05 was considered statistically significant.