| Background: Hypertension is an important risk factor for cardiovascular diseases,and cardiac damage is one of the important target organ damage related to blood pressure.Myocardial fibrosis is the main pathophysiological change of hypertensive heart remodeling.Due to the excessive deposition of myocardial extracellular matrix and the excessive proliferation of cardiac fibroblasts,it leads to the imbalance of collagen proportion and the disorder of collagen arrangement,resulting in the change of cardiac structure and functional damage.Phosphatase and tension homolog(PTEN)is a crucial regulator that controls many physiological processes,negatively regulates phospholnositide-3 kinase(PI3K)/protein kinase B(AKT),which involves in cell growth,cell polarization,cell migration,cell structure change,cell cycle and other cellular processes,but also involves in various cancers,neurodegenerative diseases,metabolic disorders and wound healing.The previous study showed that the PTEN signaling pathway decreased apoptosis of myocardial infarction and the change of macrophages phenotype.However,no studies have directly demonstrated that the PTEN signaling pathway improves hypertensioninduced myocardial fibrosis by changing the macrophage phenotype.Macrophages play important roles in initiation and progression of inflammatory and fibrotic responses.Macrophages can differentiate into “classical” inflammatory(M1)and “nonclassical” patrolling(M2)macrophages.M1 macrophages express tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin(IL)-1β and IL-6 and they induce a strong proinflammatory reaction and contribute to hypertension-induced cardiac fibrosis.M2 macrophages mainly secrete anti-inflammatory factors such as IL-10,IL-4 and IL-13 to achieve the anti-fibrosis effect.The previous study showed that the PTEN related pathway improved myocardial fibrosis by increasing the level of antiinflammatory M2 macrophages.However,whether effect of the PTEN signaling pathway on hypertension-induced cardiac fibrosis and macrophage polarization remains elusive.The hypothesis was that the PTEN pathway was related to hypertension-induced myocardial fibrosis by changes in macrophage phenotype.Aim: The purpose of this study was to explore the effect of macrophage phenotype on myocardial fibrosis in spontaneously hypertensive rats(SHRs),which may be related to the activation of the PTEN signaling pathway.Second,whether the PTEN inhibitor VO-OHpic improved myocardial fibrosis by increasing the level of M2 macrophage markers.Methods: In this study,both in vivo and in vitro were combined.1.In vivo experiments: Twenty SHRs were divided into two groups: the SHR group(no treatments,n = 10)and the VO-OHpic group(n = 10).Ten Wistar-Kyoto rats(WKY)were considered normal control group.PTEN inhibitor VO-OHpic(10μg/kg/day,subcutaneous injection)was administrated for 8 weeks.After the rats were sacrificed,heart weight(HW),tibia length(TL)and left ventricular weight(LVW)were measured.Then,HW/TL and LVW/TL were calculated to evaluate the gross weight of the heart.Cardiac function and structure were measured by echocardiogram.The levels of fibrotic markers(Col-Ⅰ,Col-Ⅲ and α-SMA)and M1/M2 macrophages(M1: CD80 and CD86;M2: CD163 and CD206)were evaluated by Masson’s trichrome staining,enzyme-linked immunosorbent assay(ELISA),and Western blot.In addition,the level of M1/M2 macrophages and the proliferation in hearts were measured by immunohistochemistry(IHC).2.In vitro experiments: Angiotensin Ⅱ(Ang II;0.1 μM,24 h)stimulated THP-1-induced macrophages.THP-1-induced macrophages were transfected with si RNA to knockdown AKT or treated with a PI3 K inhibitor(LY294002),in order to investigate the relationship between macrophage phenotype and the PI3K/AKT pathway.To assess the effect of VO-OHpic-induced macrophages on cardiac fibrosis in vitro,cardiac fibroblasts(CFs)alone and CFs cocultured with macrophages after different treatments.The levels of M1/M2 macrophages markers and fibrotic markers were evaluated by immunofluorescence(IF),Western blot,ELISA,and q RT-PCR.All data in vivo and in vitro were expressed as mean ± standard deviation,and multiple comparisons were made between groups using one-way ANOVA and Turkey test.Statistical analysis was performed using Graph Pad Prism 8.0 statistical software.Results: 1.In vivo experiments: The development of SHR induced myocardial fibrosis: HW/TL and LVW/TL were increased in the SHR group compared to the WKY group(P < 0.05).The results of the echocardiogram showed that fraction shortening(FS)and ejection fraction(EF)were decreased in the SHR group compared to the WKY group(P < 0.05).Whereas,left ventricular internal dimension-diastole(LVIDd),left ventricular internal dimension-systole(LVIDs)were higher in the SHR group than those in the WKY group(P < 0.05).Masson staining showed that SHR had an increased area of interstitial fibrosis compared to the WKY group(P < 0.05).Western blot and IHC showed that the SHR group had higher levels of CD80,CD86,Col-Ⅰ,Col-Ⅲ andα-SMA compared to those in the WKY group(P < 0.05).Besides,the SHR group had lower levels of CD206 and CD163 compared to those in the WKY group(P < 0.05).ELISA showed that the levels of TNF-α,Col-Ⅰ,Col-Ⅲ and α-SMA increased in the SHR group than those in the WKY group(P < 0.05).The protein level of PTEN increased in the SHR group compared to the WKY group(P < 0.05).Whereas the protein of PI3 K and AKT decreased in the SHR group compared to the WKY group(P< 0.05).The effect of PTEN inhibitor VO-OHpic on myocardial fibrosis: HW/TL and LVW/TL were decreased in the VO-OHpic group compared to the SHR group.The results of the echocardiogram showed that FS and EF were increased in the VO-OHpic group compared to the SHR group.Whereas,LVIDd and LVIDs were lower in the VOOHpic group than those in the SHR group(P < 0.05).Masson staining showed that the VO-OHpic group had a decreased area of interstitial fibrosis compared to the SHR group(P < 0.05).Western blot and IHC showed that the VO-OHpic group had lower levels of CD80,CD86,Col-Ⅰ,Col-Ⅲ and α-SMA compared to those in the SHR group(P < 0.05).Besides,the VO-OHpic group had higher levels of CD206 and CD163 compared to those in the SHR group(P < 0.05).ELISA showed that the levels of TNF-α,Col-Ⅰ,Col-Ⅲ and α-SMA decreased in the VO-OHpic group than those in the SHR group(P < 0.05).The protein level of PTEN decreased in the VO-OHpic group compared to the SHR group.Whereas the protein of PI3 K and AKT increased in the VO-OHpic group compared to the SHR group(P < 0.05).2.In vitro experiments: Western blot and IF showed that Ang Ⅱ-induced macrophages increased the levels of CD80 and CD86,and decreased the levels of CD163 and CD206 compared to the control group(P < 0.05).VO-OHpic and Ang Ⅱ-induced macrophages decreased the levels of CD80 and CD86,and increased the levels of CD163 and CD206 compared to the Ang Ⅱ group(P < 0.05).ELISA and q RT-PCR showed that VO-OHpic and Ang Ⅱ-induced macrophages decreased the levels of TNF-α and IFN-γ,and increased the levels of IL-10 compared to the Ang Ⅱ group(P < 0.05).To further verify the role pf PTEN signaling pathway in myocardial fibrosis,silencing of AKT expression and a PI3 K inhibitor(LY294002)was performed.After Ang Ⅱ stimulation,the levels of CD80,CD86,TNF-α and IFN-γ increased in VOOHpic-treated si AKT/LY294002 macrophages compared with VO-OHpic-treated macrophages(P < 0.05).The levels of CD163,CD206 and IL-10 decreased in VOOHpic-treated si AKT/LY294002 macrophages compared with VO-OHpic-treated macrophages(P < 0.05).Western blot of PTEN signaling pathway showed that the protein expression of PI3 K and AKT increased in VO-OHpic-treated macrophages compared with VOOHpic-treated si AKT/LY294002 macrophages and Ang-Ⅱ-treated macrophages(P <0.05).The protein expression of PTEN decreased in VO-OHpic-treated macrophages compared with VO-OHpic-treated si AKT/LY294002 macrophages and Ang-Ⅱ-treated macrophages(P < 0.05).Next,a Transwell coculture system was used to coculture CFs and macrophages following VO-OHpic and Ang Ⅱ stimulation.CFs incubated with VO-OHpic-and AngⅡ-induced macrophages decreased the levels of Col-Ⅰ,Col-Ⅲ,α-SMA,TGF-β and Smad 2/3(P < 0.05).However,CFs incubated with VO-OHpic-and Ang Ⅱ-induced si AKT-macrophages increased the above indicators(P < 0.05).Conclusion: PTEN signaling pathway may be involved in the formation of hypertension-induced myocardial fibrosis,relating to the change of macrophage phenotypes.The PTEN inhibitor VO-OHpic mediated a fibroprotective effect and increased M2 macrophage polarization via the PI3K/AKT/TGF-β/Samd2/3 pathway.Background: Based on the first part,we found that phosphatase and tension homolog(PTEN)/phospholnositide-3 kinase(PI3K)/protein kinase B(AKT)participated in the pathological process of hypertensive cardiac fibrosis.The previous study of our team showed that 60.4% of young and middle-aged women with hypertension had sexual dysfunction compared with 26% of normotensive women.Hypertension induces systemic vascular remodeling and arteriosclerosis,leading to the narrowed lumen of genital vessels,increased blood flow resistance,and finally decreased libido and sexual dysfunction.The female reproductive tract is rich in blood vessels and should be considered as one of hypertension-mediated organ damage.Our previous study found that the antihypertensive effect of felodipine combined with irbesartan was similar to that of felodipine combined with metoprolol,however,felodipine combined with irbesartan improved sexual function of female patients with hypertension,which is related to improved sex hormones levels and reduced oxidative stress levels.In further animal studies,we found that irbesartan had an improved effect on vaginal fibrosis and vaginal vascular remodeling in spontaneously hypertension rats(SHRs).Based on our team’s study of angiotensin Ⅱ receptor blockers,this study further explored the effect of angiotensin receptor-neprilysin inhibitor(ARNI)on female sexual dysfunction caused by hypertension.Sacubitril/valsartan(SAC/VAL)is a new class of cardiovascular drugs.The positive effect of SAC/VAL on hypertension and heart failure is superior to the effect of VAL,which may be beneficial for hypertension-induced female sexual dysfunction.NEP,the main target of SAC,is a membrane-bound circulating enzyme.NEP can recruit endogenous PTEN to the cell membrane,prolong the stability of PTEN protein and increase the activity of PTEN phosphatase.the part of SAC reduces cardiomyocyte death,cardiac fibrosis,and cardiac hypertrophy and improves the contractility of damaged cardiomyocytes by the level of PTEN.Previous studies found that PTEN played a pivotal role in maintaining stratified squamous epithelium and epithelial cell homeostasis.Cell proliferation and differentiation of stratified squamous epithelia must be tightly regulated and coordinated during homeostasis,and the vaginal epithelium is similar to other stratified squamous epithelia.The PTEN/PI3K/AKT pathway is one of the major signaling pathways that coordinate the activation,growth,and differentiation of follicles.However,the effect of the PTEN/PI3K/AKT signaling on clitoral and vaginal tissues in hypertension is unknown.Therefore,the significance of this part lies in whether SAC/VAL superior to VAL in hypertension-induced female sexual dysfunction,and the effect of the PTEN/PI3K/AKT signaling pathway may play an important role in SAC/VAL.Aim: We investigated the effect of SAC/VAL on the fibrosis of vaginal and clitoral tissues,estrus cyclicity and sexual behavior,including sexual receptivity(lordosis quotient and intensity),plus the number of proceptive and aggressive events.In addition,we evaluated the effect of SAC/VAL on the PTEN level of the clitoral and vaginal tissues in spontaneously hypertensive rats(SHRs).Methods: In this study,both in vivo and in vitro were combined.1.In vivo experiments: Thirty female SHRs were administered VAL(30 mg/kg/day,intragastric administration),SAC/VAL(60 mg/kg/day,intragastric administration)or saline.Ten normotensive female Wistar-Kyoto(WKY)rats were included in the control group.We assessed blood pressure,estrous cyclicity and sexual behavior in female rats.In addition,the thickness of epithelia and wall/lumen ratio in clitoral and vaginal tissues was evaluated by hematoxylin and eosin(H&E)and Masson’s trichrome staining.Western blot and enzyme-linked immunosorbent assay(ELISA)were used to assess the levels of fibrotic markers in vaginal and clitoral tissues.Furthermore,the protein levels of PTEN,PI3 K and AKT expression were measured by Western blot.2.In vitro experiments: Angiotensin Ⅱ(Ang Ⅱ)induced VK2/E6E7 cells,simulating the microenvironment of hypertension.VK2/E6E7 cells were stimulated with 0.1 μM Ang II for 24 h.After stimulation,VK2/E6E7 cells were treated with SAC/VAL or VAL for 24 h.SAC/VAL and VAL were dissolved in 100% dimethyl sulfoxide as 10 mmol stock solutions and stored at-80℃ before use.Then the compound stock solution was diluted in cell culture to a final concentration of 10 μmol/L.Data obtained in vivo were expressed as mean ± standard error.Data obtained in vitro were expressed as mean ± standard deviation.According to Gaussian distribution analysis,sexual behavior and sexual cycle data were analyzed using the Kruskal-Wallis variance.Other data were in line with normal distribution.Single factor analysis and Tukey analysis were selected for data processing.All data analysis was performed using Graph Pad 8.0 statistical software.Results: 1.In vivo experiments: The development of female sexual dysfunction in SHR: The systolic blood pressure(SBP)and diastolic blood pressure(DBP)were lower in the SHR group than those in the WKY group(P < 0.05).The SHR group exacerbated hypertension-induced sexual dysfunction,exhibited as a prolonged estrus phase,increased receptivity and proceptive events,and decreased aggressive events,compared to those of the WKY group(P < 0.05).H&E staining and Masson staining showed that the SHR group decreased the thickness of epithelia and increased wall/lumen ratio in genital tissues compared to those in the WKY group(P < 0.05).Besides,Western blot and ELISA showed that the SHR group had higher levels of Col-Ⅰ,Col-Ⅲ and α-SMA,and lower levels of estradiol and estrogen receptor α/β than the levels of the WKY group(P < 0.05).The SHR group increased PTEN expression and decreased PI3 K and AKT expression at the protein level compared to those in the WKY group(P < 0.05).The improvement of SAC/VAL on hypertension-induced female sexual dysfunction: SAC/VAL and VAL administered alone significantly lowered the systolic blood pressure(SBP)and diastolic blood pressure(DBP)in the SHRs compared to SHRs without treatment(P < 0.05).Additionally,SAC/VAL achieved significantly greater SBP and DBP reductions than VAL after 8 weeks of treatment(P < 0.05).SAC/VAL treatment improved hypertension-induced sexual dysfunction,exhibited as a prolonged estrus phase,increased receptivity and proceptive events,and decreased aggressive events,compared to those of VAL treatment(P < 0.05).In addition,SAC/VAL-treated SHRs had lower levels of fibrotic markers,and had higher levels of estradiol and estrogen receptor α/β than the levels of VAL-treated SHRs(P < 0.05).Moreover,SAC/VAL decreased PTEN expression and increased PI3 K and AKT expression at the protein level compared to those in VAL treatment alone(P < 0.05).2.In vitro experiments: Lower PI3 K and AKT expression and higher PTEN expression at the protein level were seen in Ang Ⅱ-induced VK2/E6E7 cells than in the control group(P < 0.05).However,Ang Ⅱ-induced VK2/E6E7 cells with SAC/VAL treatment had higher PI3 K and AKT expression and lower PTEN expression than Ang Ⅱ-induced VK2/E6E7 cells with VAL treatment or without any treatment(P < 0.05).The results indicated that the effect of SAC/VAL may be related to the PTEN/PI3K/AKT signaling pathway.Conclusions: SAC/VAL treatment is superior to VAL treatment in attenuating sexual dysfunction and fibrosis of vaginal and clitoral tissues,which may be related to the PTEN/PI3K/AKT pathway.Phosphatase and tension homolog(PTEN)is a dual phosphatase with both protein and lipid phosphatase activities.As the lipid phosphatase that dephosphorylates phosphatidylinositol-3,4,5-phosphate(PIP3),PTEN reduces the level of PIP3,a critical 2nd messenger.When cardiomyocytes are damaged,PIP3 activates and accumulates in microcysts and lipids in the cytoplasm of cardiomyocytes,improving the structure and function of damaged cardiomyocytes.PTEN is also sensitive to the cellular redox status by modification of cysteine residues in the active site,whereas certain agonists induce changes in the expression of PTEN by both transcriptional and translational mechanism.PTEN expression negatively regulates phosphoinostitide 3-kinases(PI3K)signaling pathway,which can improve the survival of cardiomyocytes,cardiac fibroblasts,vascular smooth muscle cells and endothelial cells.PTEN is involved in cardiovascular diseases,including cardiac hypertrophy,cardiac systolic dysfunction and heart failure.The diverse effects mediated by the PI3K/PTEN signaling in the heart clearly support an important biological and pathophysiological role for this signaling cascade.PI3 Ks are a family of evolutionarily conserved lipid kinases that mediate many cellular responses to physiological and pathophysiological stimuli.Class Ⅰ PI3 K can be activated by either receptor tyrosine kinase(RTK)/cytokine receptor activation(class ⅠA)or G-protein-coupled receptors(GPCR,class ⅠB).Once activated PI3 Ks generate Ptd Ins(3,4,5)P3 leading to the recruitment and activation of AKT,PDK1 and monomeric G-proteins,which then activate a range of downstream targets including glycogen synthase kinase-3β,mammalian target of rapamycin(m TOR),endothelial nitric oxide synthase and several anti-apoptotic effectors.Antagonistic actions of PTEN on PI3 K signaling are evolutionary-conserved and occur in various mammalian tissues including the heart.Overexpression of catalytically active PTEN decreases insulinstimulated phosphatidylinositols(Ptdlns)(3,4,5)P3 levels,whereas overexpression of wild-type PTEN causes a decrease in intracellular levels of Ptdlns(3,4,5)P3.In contrast,in embryonic stem cells lacking PTEN,Ptdlns(3,4,5)P3 levels are increased with greater insulin-like growth factor-1-induced accumulation of Ptdlns(3,4,5)P3,whereas expression of a catalytically inactive PTEN causes increased cellular Ptdlns(3,4,5)P3 levels.Class ⅠA(PI3Kα,β,and δ)and class ⅠB PI3 Ks mediate distinct phenotypes in the heart under negative control by the 3’-lipid phosphatase PTEN.In this review,we discuss the biochemistry and molecular biology of the PTEN/PI3K/AKT pathway and their critical role in cardiovascular diseases. |
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