| 1 IntroductionVascular aging is a major independent risk factor for cardiovascular diseases. Different pathophysiological stimuli are involved in its development and mediate extracellular matrix (ECM) remodeling leading to vascular stiffness. Cellular senescence is considered to play a causal role in the pathogenesis and development of vascular aging. Cellular senescence causes disorders of ECM metabolism, initiates vascular remodeling and finally results in a reduction in compliance and an increase in stiffness. The circulating level of testosterone decreases with age gradually. Testosterone deficiency is associated with a higher incidence of cardiovascular disease in men. Furthermore, it is associated with the markers of vascular aging such as increased carotid intima-media thickness and aortic calcification. The newest large observational cohort with extended follow-up shows that normalization of total testosterone level after testosterone replacement therapy (TRT) is associated with a significant reduction in all-cause mortality, myocardial infarction and stroke. However, the underlying relevance of testosterone delaying vascular aging as well as cellular senescence remains poorly understood.Gas6 is a member of the vitamin K-dependent protein family, and interacts with receptor tyrosine kinases of the TAM (Tyro3, Axl, Mer) family. Gas6 has the highest affinity for Axl among TAM receptors and is often called Gas6/Axl pathway. Numerous studies have identified that Gas6/Axl pathway plays a pivotal role in vascular biology and diseases such as vascular calcification, vascular remodeling and atherosclerosis. Moreover, the polymorphism of gas6 gene has been associated with stroke and acute coronary syndrome in humans. Clinical research shows that the plasma concentration of Gas6 decreases with age, and is associated with the testosterone level. Moreover, Son et al. finds that there are functional androgen-response elements in the Gas6 promoter, so androgen receptor pathway could regulate Gas6 transcription directly. More importantly, we demonstrate that Gas6/Axl plays an important role in regulating cell cycle arrest and delaying VSMCs senescence recently.Thus, we hypothesized that testosterone might delay VSMCs senescence and decrease collagen expression via Gas6/Axl pathway. Firstly, we established the induced senescence model by Ang ?, and determined the relationships among testosterone, Gas6 and Axl expression, VSMCs senescence and collagen expression. To further elucidate the role of Gas6 and Axl in testosterone delaying VSMCs senescence, we used specific inhibitors Axl-Fc and R428 to explore the function of Gas6 and Axl in testosterone delaying VSMCs senescence. Finally, we detected the effects of downstream signal molecules, Akt and FoxO1a, and used a specific inhibitor of Akt and FoxO1a gene silencing to confirm the role of Akt/FoxO1a in testosterone delaying VSMCs senescence.2 Objectives(1) We intended to explore the anti-senescence effect of testosterone on Ang ?-induced VSMCs senescence model;(2) Whether Gas6/Axl/Akt/FoxO pathway is involved in the process of testosterone delay VSMCs senescence.3 Methods3.1 Cell cultureVSMCs were extracted from the aortas of 8-10-week-old C57BL/6J wild-type mice. Briefly, mice were sacrificed, and aortas were isolated under sterile conditions. The aortas were minced, and tissue blocks were spread on the bottom of culture flasks without medium for about 2 h until the tissue blocks were attached. Then culture medium was added, and a large number of cells grew from blocks 3-5 day after. All cells were maintained in DMEM/F12 supplemented with 10% FBS in 5% CO2 and 95% humidified air at 37?.3.2 Establishment of cell senescence model To establish induced-senescence model, we treated VSMCs with Ang ? (10-6 mol/L) for 48 h.3.3 Identification of cell senescence modelWe evaluated the degree of cell senescence by SA-?-Gal Staining and Western blot analysis of p16INK4a and p21Cipl.3.4 Senescence-associated ?-galactosidase stainingRemoved the culture medium, and washed the cells one time with pre-cooled PBS. Then, cells were fixed with Fixative solution for 15 min at room temperature. After the P-Galactosidase Staining solution was added, the cells were incubated at 37? without CO2 overnight. Cells which had blue-green granules in their cytoplasm were regarded as positive staining.3.5 Western blot analysisProteins from VSMCs and MOVASs were extracted at the end of experience. The expression of Gas6, Axl, p16INK4a, p21Cipl,collagen ?, collagen ?, MMP-2, MMP-9, TIMP-1, TIMP-2, MT1-MMP, p-Akt, Akt, p-FoxO1a and FoxO1a were detected by western blot. The relative intensities of protein bands were analyzed by the ImageJ software.3.6 ELISAThe level of Gas6 in cell culture supernatants was measured by ELISA assay according to the manufacturer's instruction.3.7 Gelatin ZymographyThe enzymatic activity of MMPs in VSMCs was assayed by Gelatin Zymography. The relative activity of zymogrphy bands were analyzed by the Image J software.3.8 Transfection of siRNAA siRNA specific for FoxO1a was used to inhibit FoxO1a synthesis, and a randomly mixed sequences siRNA was used as negative control. The oligoribonucleotides were transfected into MOV AS grown in a six-well plate using LipofectamineTM 2000 transfection according to the manufacturer's protocol.3.9 Statistical analyses All data were presented as meanąSD. SPSS 20.0 was used for statistical analysis. Differences between two groups were analyzed by independent samples t-test and one-way ANOVA was used to compare results among groups. P<0.05 was considered significant.4 Results4.1 The selection of optimal concentration and incubation time of testosteroneTo explore the suitable testosterone concentration which could inhibit the expression of p16INK4a and p21Cip1 in VSMCs, cells were maintained in DMEM with 0 nM,3 nM, 30 nM and 300 nM testosterone for 24 h. The protein expression of p16INK4a and p21Cip1 significantly decreased after VSMCs were maintained in 30 nM testosterone and 300 nM, so 30 nM testosterone was used as a suitable concentration in subsequent experiments. Then VSMCs were maintained in DMEM with 30 nM testosterone for 0, 6,12,24,36 and 48 h. The protein expression of p16INK4a and p21Cip1 markedly reduced after VSMCs were exposed to testosterone for 24,36,48 h, therefore 24 h was used in the following experiments.4.2 Testosterone delays Ang ?-induced VSMCs senescenceAng ? greatly increased the expression of p16INK4a and p21Cip1 and SA-?-Gal staining rate compared with control group. With testosterone treatment, the increased expression of p16INK4a and p21Cip1 were significantly decreased, as the same of SA-?-Gal staining rate.4.3 The regulating effect of Ang ? on Gas6/Axl expressionAng ? (10-6 mol/L,48 h) decreased the Gas6 and Axl expression in VSMCs and reduced the Gas6 content in supernatant compared with control group.4.4 The regulating effect of testosterone on Gas6/Axl expressionOn the contrary, testosterone elevated the Gas6 and Axl protein expression in VSMCs and supernatant in a concentration-and time-dependent manner.4.5 Gas6 is involved in testosterone-mediated anti-senescence effectsAfter Axl-Fc treatment, the expression of p16INK4a and p21Cip1 as well as the staining rate of SA-?-Gal increased significantly compared with control group, regardless of whether testosterone was added. And Axl-Fc could reverse the downregulating effects of testosterone on p16INK4a and p21Cipl expression induced by Ang ?.4.6 Axl is involved in testosterone-mediated anti-senescence effectsWith R428 treatment, the p16INK4a and p21Cipl expression as well as the SA-?-Gal staining rate elevated markedly, in spite of whether testosterone was added. Likewise, R428 reversed inhibitory effects of testosterone on p16INK4a and p21Cipl expression induced by Ang ?.4.7 Testosterone reduces Ang ?-induced collagen expressionWhen stimulated by Ang ?, the protein expression of collagen ? and ? increased significantly compared with control group. With testosterone treatment, the elevated collagen ? and ? expression decreased significantly compared with Ang ?-treated group.4.8 Testosterone reduces Ang ?-induced collagen expression via Gas6When treated with Axl-Fc, the expression of collagen ? and collagen ? increased significantly compared with control group, regardless of whether testosterone was added. And Axl-Fc could reverse the downregulating effects of testosterone on collagen ? and collagen ?expression induced by Ang ?.4.9 Testosterone reduces Ang ?-induced collagen expression via AxlWith R428 treatment, the collagen ? and collagen ? expression elevated markedly, in spite of whether testosterone was added. Likewise, R428 reversed inhibitory effects of testosterone on collagen ? and collagen ? expression induced by angiotensin ?.4.10 The regulating effect of testosterone on MMP-2 expression and activityAng ? reduced the protein expression of TIMP-2 while elevated the protein expression of MMP-2 and MT1-MMP, as well as the MT1-MMP to TIMP-2 ratio compared with control group. In keeping with the elevated MMP-2 expression, the MMP-2 activity was increased in Ang ?-treated group as well. With testosterone treatment, the MMP-2 and MT1-MMP expression, the MT1-MMP to TIMP-2 ratio and the MMP-2 activity were markedly decreased while the expression of TIMP-2 was increased compared with Ang II-treated group.4.11 Testosterone regulates the expression and activity of MMP-2 through Gas6We used Axl-Fc to block the function of Gas6. The regulating effects of testosterone on MMP-2, TIMP-2 and MT1-MMP expression, as well as the MT1-MMP to TIMP-2 ratio were reversed by Axl-Fc. Simultaneously, the inhibitory effect of testosterone on the MMP-2 activity was reversed by Axl-Fc. And there was no difference in the expression of MMP-2, TIMP-2 and MT1-MMP, the MT1-MMP to TIMP-2 ratio and MMP-2 activity between the two Axl-Fc-treated groups.4.12 Testosterone regulates the expression and activity of MMP-2 through AxlR428 was used to inhibit Axl. With R428 treatment, the regulating effects of testosterone on MMP-2, TIMP-2 and MT1-MMP expression, as well as the MT1-MMP to TIMP-2 ratio were reversed. Similarly, the inhibiting effect of testosterone on the MMP-2 activity was reversed by R428 as well.4.13 The regulating effect of testosterone on Akt/FoxO1a phosphorylationThe phosphorylation of Akt and FoxO1a were decreased in Ang ?-treated group compared with the control group. Testosterone markedly restored the suppressed phosphorylation of Akt and FoxOla.4.14 Testosterone regulates Akt/FoxO1a phosphorylation through Gas6Axl-Fc reversed the enhancing effects of testosterone on the phosphorylation of Akt and FoxOla. And there was no difference in the phosphorylation of Akt and FoxOla between the two Axl-Fc-treated groups.4.15 Testosterone regulates Akt/FoxO1a phosphorylation through AxlThe enhancing effects of testosterone on the phosphorylation of Akt and FoxO1a were reversed by R428. And no difference was found in the phosphorylation of Akt and FoxO1a between the two R428-treated groups.4.16 The role of Akt in the regulating effects of testosterone on MMP-2 expression and activityThe regulating effects of testosterone on MMP-2, TIMP-2 and MT1-MMP expression, the MT1-MMP to TIMP-2 ratio and MMP-2 activity were reversed by LY294002 compared with angiotensin ?+testosterone group.4.17 The transfection efficacy of FoxO1a-siRNAThe transfection efficacy of FoxO1a-siRNA into MOVASs reached 70%. The expression of FoxO1a in the cells transfected with siRNA was significantly lower than in the cells transfected with the negative control-siRNA.4.18 The role of FoxOla in the regulating effects of testosterone on MMP-2 expression and activityAfter the expression of FoxO1a was inhibited, the regulating effects of testosterone on MMP-2, TIMP-2 and MT1-MMP expression, as well as the MT1-MMP to TIMP-2 ratio were reversed. Simultaneously, the increased MMP-2 activity induced by Ang ? was also decreased significantly after FoxOla was inhibited. And no difference was found in the MMP-2, TIMP-2 and MT1-MMP expression, MT1-MMP to TIMP-2 ratio and MMP-2 activity between Ang ?+FoxOla-siRNA group and Ang ?+ FoxOla-siRNA+testosterone group. In the presence of FoxOla-siRNA, testosterone cannot play an additional role in regulating MMP-2, TIMP-2 and MT1-MMP expression induced by Ang ?.4.19 Akt is involved in the anti-senescence effect of testosteroneThe PI3K inhibitor, LY294002 could reverse the downregulating effects of testosterone on the expression of pl6INK4a and p21Cipl induced by Ang ?. But the regulating effect of LY294002 on p16INK4a expression was much weaker.5 Conclusions(1) We have identified a novel molecular mechanism for testosterone in protecting against VSMCs senescence and collagen synthesis;(2) We have proved that testosterone alleviated VSMCs senescence and collagen synthesis mainly by modulating Gas6/Axl pathway;(3) We have found that Akt/FoxOla signaling pathway was involved in the protective effects of testosterone on VSMCs senescence and collagen synthesis.1 IntroductionVascular aging is a major independent risk factor and fertile ground for cardiovascular diseases. The aging vessels exhibit a series of characteristics which include intima-media thickening, luminal dilation, elastic membrane fragmentation, vascular stiffness and raised blood pressure. Testosterone, as the most important hormone of male, decreased gradually with age. Previous studies showed that testosterone deficiency is associated with a higher incidence of cardiovascular disease in men, and is associated with the markers of vascular aging such as increased carotid intima-media thickness and aortic calcification. Despite controversy over the benefits of testosterone replacement therapy (TRT), many studies demonstrate the benefits of TRT. TRT can improve the manifestation of vascular aging. However, the underlying mechanism of testosterone improve vascular aging is poorly understood. Cellular senescence is considered to play a causal role in the pathogenesis and development of vascular aging. Cellular senescence causes disorders of ECM metabolism, initiates vascular remodeling and finally results in a reduction in compliance and an increase in stiffness. In a prior study, we found that testosterone delayed Ang II-induced vascular smooth muscle cell senescence by promote the secretion of Gas6.Gas6 is a member of the vitamin K-dependent protein family, and interacts with receptor tyrosine kinases of the TAM (Tyro3, Axl, Mer) family. Gas6 has the highest affinity for Axl among TAM receptors and is often called Gas6/Axl pathway. Numerous studies have identified that Gas6/Axl pathway plays a pivotal role in vascular biology and diseases such as vascular calcification, vascular remodeling and atherosclerosis. Clinical research shows that the plasma concentration of Gas6 decreases with age, and is associated with the testosterone level. Moreover, Son et al. finds that there are functional androgen-response elements in the Gas6 promoter, so androgen receptor pathway could regulate Gas6 transcription directly. In our previous study, we further proof that testosterone could promote the expression of Gas6 in VSMCs. And we found that Gas6/Axl signaling pathway is involved in the anti-senescence effect of testosterone on Ang ?-induced VSMCs senescence.To further demonstrate the anti-aging effects of testosterone on vascular aging and the role of Axl in this process in vivo, we chose Axl-/- mice to established natural aging model and gave testosterone replacement therapy in this study. In addition, we pay a attention to the role of testosterone on vascular cell senescence, which is a source of vascular aging, and the ECM metabolism, which is the immediate cause of vascular stiffness. Meanwhile, we explore the downstream molecular mechanism of testosterone/Gas6/Axl signaling pathway.2 Objectives(1) We intended to explore the anti-senescence effect of physiological dose of testosterone on vascular stiffness.(2) Whether Gas6/Axl/Akt/FoxO pathway is involved in the improving effect of testosterone on vascular stiffness.3 Methods3.1 Animal model and drug treatment in vivoNinety wild type and ninety Axl-/- male mice on a B6129SF2/J background were obtained from Shandong Normal University (Jinan, China). Mice of each gene type were randomly divided into 3 groups:young group (n=30,3 month old), old group (n=30,18 month old), testosterone-treated group (n=30,18 month old). The testosterone-treated group receiving testosterone undecanoate (37.9 mg/Kg) at fifteenth month, once a month, to a total of three times, and sacrificed at 18 month. The old group receiving 0.05 ml corn oil the same as testosterone-treated group. At the end of the experiment, aorta and blood samples of the six groups were collected.3.2 Ultrasonography Ultrasonography was performed at 3,15 and 18 month. Intima-media thickness (IMT), diastolic diameter (Dd), systolic diameter (Ds) and maximal systolic velocity (Vmax) measurements of carotid artery and abdominal aorta were performed according to previously validated protocols in humans.3.3 Blood pressure measurementSystolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP) and heart rate was measured by a pulse based tail-cuff method with a photoelectric device at 3,15 and 18 month.3.4 Western blot analysisProteins from aorta were extracted at the end of experience. The protein expression of Gas6, Axl, p16INK4a, p21Cip1, collagen ?, collagen ?, MMP-2, MMP-9, TIMP-1, TIMP-2, MT1-MMP, Akt, p-Akt, FoxO1a, p-FoxO1a and ?-actin were detected by western blot. The relative intensities of protein bands were analyzed by the Image J software.3.5 Quantitative real-time PCRTotal mRNA from the aorta was prepared with Trizol Reagent and PrimeScriptTM Reverse Transcriptase according to the manufacturer's instructions. RT-PCR was used to determine the mRNA levels of Gas6, Axl and P-actin. Data analysis was performed with Bio-RadiQ5 (USA) and the 2-??CT method was used to assess the relative mRNA expression normalized in multiplex reactions using ?-actin as control.3.6 Telomere length measurementTotal DNA from aorta was extracted according to the manufacturer's instructions. Any sample was chose to establish standard curve. The Ct value of telomere and single copy gene were detected by RT-PCR. Data analysis was performed with Bio-RadiQ5 (USA) and the 2-??CT method was used to assess the relative T/S ratio, which was reflect relative telomere length.3.7 ELISAThe plasma level of Gas6 and testosterone was measured by ELISA assay according to the manufacturer's instruction.3.8 Histopathological analysis Morphological changes of aortic walls were observed in sections stained with HE staining, Sirius red staining, Masson staining and Verhoeff staining. The relative positive staining area for collagen and elastin were measured by an image analysis system.3.9 Immunohistochemical analysisTo indicate the expression and distribution of collagen ?, collagen ?, MMP-2 and MMP-9, immunohistochemical analysis were carried out. The color was developed with the DAB chromogen and counterstained with hematoxylin.3.10 ZymographyThe MMP-2 and MMP-9 activities of the aortic segment were assessed with zymography. The relative activity of zymogrphy bands were analyzed by the Image J software.3.11 Statistical analysesAll data were presented as mean ą SD. SPSS 20.0 was used for statistical analysis. Differences between two groups were analyzed by independent samples t-test and one-way ANOVA was used to compare results among groups. A P<0.05 was considered significant.4 Results4.1 The changes of testosterone and Gas6/Axl expression in aging vessel The serum testosterone level was significantly decreased in both WT and Axl-/- old groups compared with WT and Axl-/- young groups. With TU treatment, the serum testosterone level in WT and Axl-/- old+TU group reached the level of WT and Axl-/-young group. The serum Gas6 content and Gas6 mRNA and protein expression in aorta were markedly decreased in both WT and Axl-/- old groups. TU replacement therapy significantly increased the serum level and mRNA and protein expression of Gas6 in WT and Axl-/- old+TU groups compared with WT and Axl-/- young groups. The mRNA and protein expression of Axl were decreased in WT old group compared with WT young group. With TU treatment, the Axl mRNA and protein content elevated in WT old+TU group compared with WT old group. Both mRNA and protein expression of Axl were quite low in all Axl knockout mice.4.2 Axl is involved in the anti-senescence effect of testosterone on vascular agingTelomere length was significantly shortened in WT and Axl-/- old groups compared with WT and Axl-/- young groups. And telomere length in Axl-/- old group was shorter than WT old group. With TU treatment, the telomere length in WT old+TU group was significantly longer than WT old group. But telomere length in Axl-/- old+TU group was no longer than Axl-/- old group. The protein expression of p16INK4a and p21Cipl were increased in both WT and Axl-/- old groups compared with WT and Axl-/-young groups. And Axl-/- old group has the higher p16INIK4a and p21Cipl expression compared with all other groups. With TU treatment, the expression of p16INK4a and p21Cipl in WT old+TU group was significantly decreased compared with WT old group. But the expression of p16INK4a and p21Cipl in Axl-/- old+TU group was not significantly decreased compard with Axl-/- old group.4.3 The role of testosterone and Axl in structural changes of aging vesselThe intima-media thickness (IMT) was significantly increased in carotid artery and abdominal aorta in both WT and Axl-/- old groups compared with WT and Axl-/-young groups. The IMT of carotid artery and abdominal aorta in Axl-/- old group was not thicker than WT old group. With TU treatment, the IMT carotid arteryand abdominal aorta in WT old+TU group were decreased compared with WT old group The IMT carotid artery and abdominal aorta in Axl-/- old+TU group was not significantly thinner than Axl-/- old group. All of the carotid artery and abdominal aorta diastolic and systolic diameter in WT and Axl-/- old groups were significantly increased compared with WT and Axl-/- young groups. However, there were no significantly different in carotid artery and abdominal aorta diastolic and systolic diameter between WT and Axl-/- old+TU groups and WT and Axl-/- old groups. The distensibility coefficient of carotid artery and abdominal aorta in WT and Axl-/- old groups were smaller than the WT and Axl-/- young groups. And the coefficient distention of carotid artery and abdominal aorta in Axl-/- old group were smaller than the WT old group. With TU treatment, the coefficient distention of carotid artery and abdominal aorta in WT old+TU group were decreased compared with WT old group. But there were no significantly difference between Axl-/- old+TU group and Axl-/- old group in the coefficient distention of carotid artery and abdominal aorta.4.4 The role of testosterone treatment and Axl knockout in vascular stiffnessThe stiffness parameter (?) and pressure-strain elastic modulus (Ep) of carotid artery were significantly increased in WT and Axl-/- old group compared with WT and Axl-/-young group. The ? and Ep in Axl-/- old group were higher than in WT old group. With TU treatment, the ? and Ep of WT old+TU group were significantly decreased compared with WT old group. But there are not significantly difference between Axl-/-old+TU group and Axl-/- old group in ? and Ep. 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 group. The DC and CC of Axl-/- old group were lower than WT old group. With TU treatment, the DC and CC of WT old+TU group were significantly elevated compared with WT old group. But there are no significantly difference between Axl-/- old+TU group and Axl-/- old group in DC and CC.4.5 The role of testosterone treatment and Axl knockout in blood pressureBoth systolic and diastolic blood pressure (Ps and Pd) were increased in WT and Axl-/- old groups compared with WT and Axl-/- young groups. With TU treatment, the Ps and Pd of WT old+TU group were slightly decreased compared with WT old group. But there were no significantly difference between Axl-/- old+TU group and Axl-/- old group in Ps and Pd. The mean arterial blood pressure (MAP) of WT and Axl-/- old groups were increased significantly compared with WT and Axl-/- young groups. However, there were no significantly different in MAP between WT old+TU group and WT old group, Axl-/- old group and WT old group, and Axl-/- old+TU group and Axl-/- old group. The pulse pressure (PP) increased in WT and Axl-/- old group compared with WT and Axl-/- young group. With TU treatment, the PP in WT old+TU group markedly decreased compared with WT old group. But there are no significantly different in PP between Axl-/- old+TU group and Axl-/- old group.4.6 The role of testosterone treatment and Axl knockout in vascular fibrosisThe Masson trichrome staining, Picrosirius red staining and Verhoeff's Van Gieson staining results showed vascular fibrosis in the media. The collagen contents, collagen/elastin ratio and average aortic wall architecture score significantly increased in WT and Axl-/- old groups compared with WT and Axl-/- young groups. And the collagen content, collagen/elastin ratio and average aortic wall architecture score of Axl-/ old group was higher than WT old group. With TU treatment, the collagen content, collagen/elastin ratio and average aortic wall architecture score markedly decreased in WT old+TU group compared with WT old group. But there are no significantly difference in collagen content, collagen/elastin ratio and average aortic wall architecture score between Axl-/- old group and Ax-/- old+TU group. The elastin contents significantly decreased in WT and Axl-/- old groups compared with WT and Axl-/- young groups. And elastin contents of Axl-/- old group was slightly lower than WT old group. With TU treatment, elastin contents markedly increased in WT old+TU group compared with WT old group. But there are no significantly difference in elastin contents between Axl-/- old group and Axl-/- old+TU group.The immunohistochemical staining and western blot results showed that collagen ? and ? expression were significantly increased in WT and Axl-/- old groups compared with WT and Axl-/- young groups. The collagen ? and ? expression in Axl-/- old group was higher than WT old group. With TU treatment, the collagen ? and ? expression were significantly decreased in WT old+TU group compared with WT old group. But there were no significant difference in collagen ? and ? expression between the Axl-/- old+TU group and Axl-/- old group.4.7 The role of testosterone treatment and Axl knockout in MMP2/9 expression and activityThe 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. The MMP-2 and MMP-9 expression and acitivty in Axl-/- old group was higher than WT old group. With TU treatment, the MMP-2 and MMP-9 expression and activity were significantly decreased in WT old+TU group compared with WT old group. But there were no significant difference in the expression and activity of MMP-2 and MMP-9 between the Axl-/- old+TU group and Axl-/- old group.The MT1-MMP expression, MT1-MMP/TIMP-2 ratio and MMP-9/TIMP-1 ratio were significantly elevated, while TIMP-1 and TIMP-2 expression were decreased in WT and Axl-/- old groups compared with WT and Axl-/- young groups. The MT1-MMP expression, MT1-MMP/TIMP-2 ratio and MMP-9/TIMP-1 ratio in Axl-/- old group was higher than WT old group. With TU treatment, the MT1-MMP expression, MT1-MMP/TIMP-2 ratio and MMP-9/TIMP-1 ratio were significantly decreased in WT old+TU group compared with WT old group. But there were no significant difference in the MT1-MMP expression, MT1-MMP/TIMP-2 ratio and MMP-9/TIMP-1 ratio between the Axl-/- old+TU group and Axl-/- old group.4.8 The regulating effects of testosterone and Axl on Akt and FoxO1a phosphorylationThe phosphorylation levels of Akt and FoxO1a were significantly decreased in WT and Axl-/- old groups compared with WT and Axl-/- young groups. The phosphorylation levels of Akt and FoxO1a in Axl-/- old group was lower than WT old group. With TU treatment, the phosphorylation levels of Akt and FoxO1a were significantly increased in WT old+TU group compared with WT old group. But there are no significant difference in phosphorylation levels of Akt and FoxO1a between the Axl-/- old+TU group and Axl-/- old group.5 Conclusions(1) Testosterone can delay vascular aging and vascular stiffness;(2) The improving effect of testosterone on cell senescence plays a pivotal role in the anti-aging effect of testosterone on vascular aging;(3) Gas6/Axl played a pivotal role in testosterone alleviating vascular aging through modulating Akt/FoxOla pathway. |
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