| BackgroundHypertension is the most common risk factor for cardiovascular disease. The adult heart undergoes myocardial remodeling when subjected to longstanding hypertension. Hypertensive myocardial remodeling is an independent risk factor for lethal cardiovascular events,and is the key pathological manifestation during the transition of heart function from compensate to decompensate. Heart responds to such stresses by increasing cell size and extracellular matrix, reorganizing sarcomeres and activating a fetal cardiac gene program. Although these responses may initially maintain heart function, it induces cardiac hypertrophy and increases the risk of chamber dilation, heart failure and sudden death.Cardiac remodeling has been shown to be the independent risk factor of heart failure recently. The pathogenesis mechanism of cardiac remodeling is complex and still remains unclear. It is well-accepted that abnormal activation of neurohormonal factors, oxidative stress and cytokines are involved in cardiac remodeling. A complex set of signal transduction pathways might underlie hypertensive myocardial remodeling, and the study on the pathogenesis and control of cardiac remodeling has become a worldwide hotful topic.Study on the signal transduction pathway involved in cardiac hypertrophy and remodeling is one of the hot issues in the field of cardiovascular research. Left ventricular hypertrophy is prompted by increased protein synthesis and aggregation in left ventricle when the pressure overload is increased. We hypothesized that autophagy might play an important role during this process. Autophagy is a highly conservative cell process which is responsible for the removal or recycling life long proteins and organelles. It is also involved in the secondary utilization of nutrients including cellular proteins and organelles. This kind of behavior can provide energy for cells. However, autophagy may also be harmful in some cases due to its phagocytosis and cellular damage, its exact roles in cardiac hypertrophy and remodeling is still unclear although numerous studies have been performed to determine the role of autophagy in myocardial hypertrophy,The nuclear factor κB (NF-κB) family of Rel-related transcription factors, existing in almost all cells, is widely involved in functions such as immunity, inflammation, and stress response. In unstimulated cells, NF-κB, sequestered in the cytoplasm by binding to inhibitory κB(IκB) proteins, can be activated by various stimuli. Once cells are activated, the IκB proteins degrade. NF-κB translocates to the nucleus and binds to its DNA-binding sites, which enhances transcriptional activity of the respective genes involved in the control of different cellular responses. Recent studies have discovered that autophagy is required for the activation of NF-κB. NF-κB has been found to negatively regulate the autophagy in specific cell type in vitro, which indicates an intimate crosstalk between the two signaling pathways. In the present study, we investigated whether autophagy is involved in the physiopathology of myocardial hypertrophy in SHR and whether NF-κB signaling pathways modulate the autophagic capacity of hippocampal cells during myocardial hypertrophy by using pretreatment with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC).The most commonly used animal model for cardiac hypertrophy and remodeling study is induced by transverse aortic constriction (TAC). But this animal model has several limitations, such as surgery itself induced stress reaction which can affect the evaluation of autophagy. The similar limitations have also been seen in drug-induced cardiac hypertrophy and remodeling, and some transgenic animal models, which limit these animal models from autophagy study.Spontaneously hypertensive rat (SHR) is an important animal model for hypertension and cardiac hypertrophy and remodeling study without any surgery or drug interventions. However, there is no comprehensive study on cardiac autophagy and its association with the development of cardiac hypertrophy and remodeling in this important animal model.Therefore, this study was designed to explore the regulation of cardiac autophagy in SHR, the involved signaling pathways focusing on AMPK and LC3 related signal-transduction pathways and downstream factors, and their relationship with cardiac hypertrophy and remodeling in SHR. The results from this study would help us to understand the cellular and molecular mechanisms of hypertensive cardiac remodeling better, and provide novel theoretical evidences and strategy for prevention and treatment of hypertensive cardiac remodeling.Objective1.To determine the development of left ventricular hypertrophy in spontaneously hypertensive rats using the technique of nuclear magnetic resonance; to study the changes of myocardial fibrosis and cardiac function during the development of hypertension; to determine the activation of cardiac autophagy and its association with myocardial hypertrophy in spontaneously hypertensive rats.2. To investigate the time course of the regulation of cardiac autophagy, left ventricular hypertrophy and fibrosis, heart function, and their correlations in different periods of hypertension in spontaneously hypertensive rats.3.To explore the trend of cardiac autophagy changes and the possible signaling pathways involved in spontaneously hypertensive rats.4.To investigate the expression of LC3 and the related pathways at both mRNA and protein levels in the heart of spontaneously hypertensive rats.5.To evaluate the effects and the mechanism of the prevention and treatment of cardiac remodeling in cardiac autophagy.MethodsFourteen 4-months-old male SHRs were obtained from Beijing animal center. Forteen age-matched normotensive male WKY rats were purchased from animal center of Beijing as the control. In the experiments, rats were randomly divided into four groups:WKY group (WKY), SHR group (SHR), PDTC-treated WKY group (PDTC+WKY), and PDTC-treated SHR group (PDTC+SHR). Each group has 7 rats. All rats were housed with free access to food and water in a 22-25℃,55-58% relative humidity environment on a 12-h light/dark cycle. Every effort was made to minimize the number and suffering of animals used. Animals were given rodent diet and tap water ad libitum during the whole course of experiments. The hearts were immediately harvested and weighed when animals were sacrificed.The follwing parameters were measured during the study:(1) The rat body weight, heart rate and tail blood pressure; (2) Blood was collected. Plasm lipid was determined using routine standard method; (3) MRI was used to evaluate the functions of heart, LVEDd, IVSd and LVPWd, RWT and LVEF were calculated; (4) Histopathological study of heart tissue for the detection of remodeling, the ventricular tissue was stained with H-E and Masson’s trichrome staining; (5) The left ventricular mass index (LVMI) was used to estimate the degree of cardiac hypertrophy; (6) Hydroxyproline content assay, and CVF and PVCA were used to estimate the cardiac fibrosis; (7) Western-blot for the expression of AMPK, AKT, mTOR andS6K, NF-κBp65.Results1. The experimental animalsThree rats died in the entire experiment, one from WKY group and two from SHR group died.2. Comparison of SBP, BW, HR among groupsThere were no significant differences in terms of body weight, heart rate, tail systolic blood pressure at the beginning of the experiment. SHRs had higher SBP than the WKY rats (P<0.01). There were no significant differences in the BW/HR in WKY group. SBP of SHRs increased during the experiment, while it remained unchanged in WKYgroup.3. MR evaluationMR was performed respectively. Interventricular septum (IVS), left ventricular posterior wall (LVPW) and left ventricular diastolic diameter (LVDd) of SHRs increased significantly (P<0.01) compared with the SHR+P and those in WKY group, and these changes were attenuated by PDTC (P<0.01).4. HE stainingHE staining under optical microscopy showed that in WKY group, cell size of cardiomyocytes was smaller, uniform, and in regular organization; In SHR groups, cardiomyocytes size was greater, not uniform, cell form was irregular, and cell arrange was disorder; Cell wall was not clear and cell fusion was observed at intersection of cells.5. Left ventricular mass index analysisThe left ventricular mass index (LVMI) is the ratio of left ventricular weight (in milligrams) to body weight (in grams) (LVW/BW), LVMI of SHR increased significantly (P<0.01) compared with the WKY group, (P<0.01).6. Cardiac fibrosisAs revealed by Masson’s trichrome staining, marked deposition of collagen was detected in the cardiac interstitial and perivascular areas of the SHRs compared with the WKY rats, which was ameliorated by PDTC. Consistent with this finding, hydroxyproline content, CVF and PVCA were elevated in the left ventricles of SHRs compared with WKY rats (P<0.01), and PDTC significantly attenuated this elevation (P<0.05).7. Characterization of autophagic vacuoles after PDTC treatment by electron micrographAutophagic vacuoles were observed by transmission electron microscopy. The hippocampal tissue from WKY rats displayed normal structure and proper organelle distribution. No alteration of tissue integrity was observed in low magnification images. SHR rats showed autophagic vacuolization. A number of irregularities were seen sporadically in high-power electron microscopic images, including a large autophagosome containing other organelles, herniation of outer membranes of endoplasmic reticulum into adjacent lysosomalstructures. Other changes included prominent matrix granules and crystalline-like inclusion which was seen in SHR rats. The PDTC-treated rats also showed double-membrane autophagicvesicles in the cytoplasm, with loss of discernable organellar fragments, but the number was lower. Moreover, there were many differences among the sham group, the WKY group, and the PDTC-treated group in the performance of the rats’ hippocampus, indicating that intraperitoneal administration of PDTC has an impact on autophagic vacuolization.8. Protein expression by Western-blot assayCompared to WKY rats, SHRs showed a significant increase in LC3 expression in heart tissue (P<0.01). In SHR group, the expression of AMPK, mTOR, S6K significantly increased during the experimental course (P<0.01), while these gene expression in WKY rats remained unchanged. Consistent with these findings, western blot analysis showed that the NF-κBp65 protein level in SHR was much higher than that in WKY rats, WKY+PDTC, and SHR+PDTC rats(P<0.05;). There were no significant differences of NF-KBp65 protein levels between WKY rats and WKY+PDTC rats8. MDA measurementCompared with WKY and other groups, the level of MDA in SHRs significantly increased (P<0.05).9. Relationship among blood pressure, cardiac hypertrophy, fibrosis and gene expressions of MDA and LC3The LVMI, IVS, LVPW, LVDd, hydroxyproline content, CVF and PVCA were all positively related to the systolic blood pressure, and reduction of blood pressure by PDTC contributed to the attenuation of cardiac hypertrophy and fibrosis. The LVMI was also significantly correlated with the hydroxyproline content. Cardiac hypertrophy was positively correlated with the expression of LC3 and the collagen content in the heart.Conclusions1.Cardiac hypertrophy and fibrosis occur with the development of hypertension. This lays the foundation for further study on the mechanisms of hypertensive cardiac remodeling.2. Our study demonstrates that autophagy is induced during myocardial hypertrophy. The induction of autophagy was examined by a transmission electron microscopy, which revealed that autophagy vacuoles contained cytoplasmic organelles. This study also found the activation of autophagy in hypertensive heart by independent methods: immunhistostaining and western blot. The experimental data showed that the formation of autophagosomes increased in SHR, and the degradation of autophagosomes decreased in WKY and SHR+PDTC rats.3. There were significant positive correlations between autophagy and the parameters of cardiac remodeling, which suggests that autophagy was involved in the pathogenesis of cardiac hypertrophy and remodeling. The stimuli of pressure might be the factors inducing NF-κB activation. NF-κB is the most important signaling pathway activated in response to cell damage and environmental danger. We observed that NF-κB activity can be modulated by autophagy-related signaling. Autophagy following myocardial hypertrophy was found to be able to upgrade the IκB kinase (IKK), which is an upstream activator of NF-κB. Recent studies have demonstrated that autophagy is required for the activation of NF-κB and that TNF-α stimulates the lipidation of LC3. To analyze the role of the NF-κB signaling pathway in autophagy of the myocardial hypertrophy, PDTC, low-molecular weight substance that inhibits NF-κB-activation, was used as pretreatment. PDTC treatment resulted in a decrease of autophagy. As shown by immunoblot, PDTC treatment significantly reduced the shift of LC3-Ⅱ into LC3-Ⅰ compared to controls. The decreased expression in the SHR+PDTC group demonstrated that the administration of PDTC might help to inhibit complete autophagy.4. Our study indicate that a pathologic mechanism of autophagy that ultimately results in cardiac damage or an adaptive mechanism that is activated by oxidation stress. The results from pharmacological agents of PDTC that can inhibit autophagy suggests the adverse effects of autophagy on some certain stage during the development of myocardial hypertrophy and remodeling.Dissertation II; Regulation of autophagy by the nuclear factor kB signaling pathway in the aortic remodelingBackgroundIncreased vascular resistance resulting from enhanced vasoconstriction and impaired vasodilation is a hallmark of hypertension.Gnhanced vasocon striction is associated with the increase dreactivity to vasocon strictors.Thevasocon striction is exaggerated during hypertension period,but the reason is still unknown.NF-kB signaling pathway regulates 1:he expression of cytokines,adhesion molecules and apoptosis—related genes and therefore plays, an important rolein the rejection of foreign organs. In l;his study, we iinvestigated the effects of PDTC on the NF-kB signaling pathway and the autophagy.The mechanisms of vascular remodeling are complex involving a series of molecular and cellular changes,which contribe to the abnormality of vascular stmctuire,function and clinical phenotypes. These changesinclude:vascular hypertrophy,apoptosis,fibroblast proliferation,reexpression of fetal genes and proteins.Ajnong these changes,smooth muscle cell(SMG) proliferation is the key factor of vascular remodeling.Autophagy is an evolutionarily conserved catabolic process for subcellular degradation of prcrteins, organelles, and other cytoplasmic components. As the degradation of cellular organelles generates free fatty acids, amino acids, and nucleotides that can be reused to fuel mkochcmdrial ATP production and maintain prot:ein synthesis, autophagy is primarily considered as a cell survival mechanism,especially during nutrient deprivation. However, it has also been argued thatautophagy may act as an initiator of cell death. Excessive autophagic activity may directly destroy the major proportion of the cytosol and organelles,or alternatively, it may trigger apoptosis rather than plays the direct role in the process. For instance,stresses in certain organelles, such as mitochondria and endoplasmic reticulum(ER),were stimulators of autophagic response because autophagy could mediate the specific removal of dysfunctional or damaged cytoplasmic organelles. Several pathways that link the apoptotic and autophagic mechanisms also have been described at the molecular level. Thus the result of a common stimulation may be decided by the crosstalk bctween autophagy and apoptosis or as resuit of a cellular "decision",between these two responses.These exciting data together with the preliminary evidence showing the increased autophagy in human cardiac remodeling and heart failure, it is needed to further investigate the role of autophagy in the vascular rcmodeling and the subsequent cardiac remodeling and heart failure.Objective1To investigate the expression of autophagy and related molecular pathways in the aorta.2To investigate the involvement and the signal-transduction pathway of NF-kB in fibroblast proliferation.3To investigate whether autophagy is involved in the aortic disorder of physiopathology occurring with hypertension and whether NF-kB signaling pathways modulate the autophagic capacity in SMC during sepsis by using pretreatment with the NF-kB inhibitor pyrrolidinedithiocarbamate(PDTC).MethodsStudies were carried out in spontaneously hypertensive rats(SHR) and Wistar Kyoto(WKY) rats served as controls. SHR and WKY rats were maintained in the animal care facility with a 12 hour light and 12 hour dark cycle and were given free access to standard rodent chow and drinking water. The initial studies for the project were performed in 4 months old SHR and WKY rats. For pyrrolidinedithiocarbamate(PDTC)treatment,4 months old SHR and WKY rats were used and were divided into4 groups: 1) Untreated WKY rats(WKY);2) Untreated SHR(SHR); 3) SHR treated with PDTC SHR-PDTC); and 4)WKY treated wUh PDTC(WKY-PDTC).SHR and WKY were treated with PDTC in drinking water at a dose of 105 mg/kg/day. Age matched untreated SHR and WKY rats served as controls. The PDTC treatment was carried for a period of 16 weeks at the end of which l;he animals were used fbr blood pressure measurement and tissues were collected for biochemical studies.The following parameters were measured during the study:(1) The blood pressure;(2) Blood was collected and plasm lipid was determined using standard method;(3)Histopathological study of aortic tissue,the aortic tissue was stained with H-E and Masson’s trichrome staining for the remodeling determination;(5) The malondialdehyde(MDA) was used to estimate the degree of lipid peroxidation products in plasma;(6) Hydroxyproline Content assay, GVF and PVGA were used to estimate tihe aortic fibrosis;(7) Westem-blot for 1:he expressions of LC3, AKT,mTor,IKKB, NF-kB, and p65.Lipid peroxidation products in plasma of SHR and WKY rats were assessed by measuring thiobarbituric acid reactive substances namely malondialdehyde(MDA)using the standard mertiod.Frozen plasma was thawed on ice,diluted using 1.150/opotassium chloride and then boiled with 15% trichloroacetic acid, 0.375%thiobarbituri cacid and 0.25 N hydrochloric acid for 15 min. The reaction mixture was allowed to cool down and centriged at l000g for 10 min to remove the occulent precipitate. The color was read at 535 nm using a spectrophotometer. The MDA levels were calculated using l;he molar extinction co-efficient(1.56 X 105 M-1 cm-1).ResultsBlood pressureThe arterial pressure of SHR at 3-4 months of age was similar to 1:hat of WKY rats.The hypertension in SHr started to develop around 5-6 weeks of ae and blood pressure Consitently increased with age until it reached plateau at 13-14 weeks of age.Hence, 3-4 week old in SHRs represent an appropriate age for studying the factors contributing to the pathogenesis of hypertension.Characterization of autophagic vacuoles after PDTC treatment by electron microscopyAutophagic vacuoles were observed by transmissionelectron microscopy. As shown in the figures, the aortictissue from WKY rats and WKY+PDTC rats displayed nearly normal structure and proper organelle distribution. No alteration of tissue integrity was observed in low magnificationimages. Autophagic vacuolization was demonstrated in the aortic tissues of SHR rats. A number of irregularities were seen sporadically in high-power electron microscopicimages, including a large autophagosome containing other organelles, herniation of outermembranes of endoplasmic reticulum into adjacent lysosomalstructures. Other changes including prominentmatrix granules and crystalline-like inclusion were scenin elected examples of SHR rats. The PDTC-treated rats also showed double-membrane autophagicvesicles in the cytoplasm, with loss of discemableorganellar fragments,but the number with lower. As shown in the figures, there were many differences amonge control group,the WKY group,and the PDTC-treated group in the performanceof autophagy in the rats,aortic,which indicates thatintraperitoneal administration of PDTC has an impacton the autophagic vacuolization.Western blot for signaling transduction pathwayThe levels of the p65 subunit of NFkB were determined in nuclear and cytosolic fractions of aortas in SHR and WKY rats using western blotting. We found that the nuclear level of p65 in SHR was significantly higher as compared to WKY rats which indicated the increased NFkB activation in SHR vascular tissue. Furttiermore,pexpression was also significantly elevated in SHR as compared to SHR+PDTC rats.Thus, there appears to be an increase in total expression of NFkB p65 subunit in the SHR.SHR showed increased vascular oxidative stress associated with increased NFkB activation in response to the development of hypertension. To investigate the hypothesis that this early vascular oxidative stress contributes to hypertension, SHR were treated with an antioxidant pyrrolidinedithiocarbamate(PDTC), which has been well recognized to have NFkB inhibitory activity. The resuhs showed that the adultuntreated SHR had increased p65 subunit expression in the vascular homogenate compared with WKY rats,as determined by western blotting. In addition, PDTC treatment caused a marked reduction of the elevated p65 expression in SHR.The present study used immunofluorescence staining !;〇 del;ect the expression and localization of LC3 in the aorta tissues. The results showed that PDTC induced punctuate distribution of LC3 immunoreactivity, and t;he formation of autophagosomes was enhanced by PDTC. There are two fbrms of LC3, LC3-I and LC3-IL During the formation of autophagosomes,cytoplasmic fbrm of LC3-I is cleaved and liquefied to give rise to membranous form LC3-II. To determine if PDTC increases the production of LC3-II,Western blotting analysis was used to detect the profit! levels of LC3-I and LC3-II. The results showed that the levels of LC3,particularly LC3-II,was increased, leadingto an increased ratio of LC3-II/LC3-I after PDTC treatment. Beclin 1 is an autophagy regulator and plays an important role in tumorigenesis and autophagic activation Similar increases in Beclin1 proteins were also detected after PDTC treatment.Conclusion1.Aortic vascular remodeling is induced by hypertrophy of SMC,proliferation of cardiac fibroblasts and synthesis of collagen.2.NF-KB pathway and autophagy signal pathway were involved in aortic vascular remodeling.3.PDTC plays an important role in autophagy induced proliferation of aortic vascularremodeling. |
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