Cardiomyocyte autophagy is regulated by angiotensin II type 1 and

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自噬的发展趋势

REVIEWEmerging strategies to effectively target autophagy in cancerVW Rebecca and RK AmaravadiINTRODUCTIONThe impact autophagy has on human health and disease are far and wide,with reports demonstrating important functions in bacterial 1and viral infections,2suppression of in ﬂammation,3adaptive immune responses 4and immunosurveillance,5neurodegeneration,6heart disease 7and cancer.8Aberrant auto-phagic activity is an emerging hallmark of cancer,9serving a critical function in the pathogenesis,survival and response to therapy in a growing number of cancers.In general,autophagy provides the means by which cells mitigate metabolic and therapeutic stresses,remove waste and manage toxic byproducts of anabolism and catabolism,such as reactive oxygen species.10The role autophagy serves speci ﬁcally in cancer has been controversial,with some reports indicating autophagy suppresses tumor development,whereas other reports providing evidence that autophagy promotes the growth of established tumors.11The overarching question is whether or not autophagy can be effectively modulated to impair cancer initiation or progression.Recent advances in the fundamental understanding of the context-dependent consequences of autophagy defects in the setting of activated oncogenes will likely pave the way for new strategies to either induce or impair autophagy therapeutically.Meanwhile,the ﬁrst deliberate attempt to modulate autophagy therapeutically has been accomplished through the publication of the ﬁrst seven clinical trials involving hydroxychloroquine (HCQ)in cancer patients.12–18Lessons learned from these clinical trials have raised new questions that can be answered in the laboratory.Finally,a deeper understanding of how autophagy is regulated at the genetic,epigenetic and posttranslational level,and how autop-hagy can regulate itself and be regulated by drugs,extracellular components and metabolites,may point to new therapeutic targets that can directly or indirectly modulate autophagy.Here we discuss the latest developments in the ﬁeld ’s understanding of autophagy in cancer and novel strategies to effectively modulate autophagic activity.AUTOPHAGY FORM AND FUNCTIONThe dissection of the autophagy pathway was ﬁrst described in yeast 19where it clearly serves as an intracellular,self-preservation mechanism providing internal nutrients to cells in times of stress.20Although autophagy is evolutionarily conserved across organisms,its role in multicellular organisms is more nuanced than it is in yeast.Recent evidence indicates autophagic ﬂux is not only dependent on the expression of the canonical autophagy machinery,but through genetic,epigenetic,metabolic,post-translational and extracellular regulation of this machinery.This complex regulation of autophagy may enable its multiple roles in cancer.Autophagic ﬂux occurs at a basal rate in all eukaryotic cells to maintain equilibrium through the recycling of nonessential components within the cell.8Under challenging conditions such as nutrient deprivation,21hypoxia 22or targeted therapy,23autophagic ﬂux can be increased via multiple stimuli to elicit homeostatic regulation over critical metabolic building blocks including amino acids,nucleic acids and monosaccharides necessary for cell survival (Figure 1).Multiple forms of autophagy exist in mammalian cells,each with well-characterized mechan-isms that differ in the way material destined for degradation is sequestered and transported to the lysosome (micro,chaperone mediated and macroautophagy).24Macroautophagy represents the most multifunctional and best-described form of autophagy,comprising a complex,tightly regulated process where double-membrane autophagic vesicles (termed autophagosomes)are generated.Autophagosomes function by sequestering damaged or misfolded proteins,engul ﬁng mitochondria (termed mito-phagy)and internalizing endoplasmic reticulum (ER;amongst other cytoplasmic components)through the aid of cargo adaptor proteins before ultimately fusing to the lysosome for degradation and recycling of internal contents to sustain cellular viability.25,26Autophagy can be characterized as canonical or non-canonical,depending upon the molecular machinery involved in the biogenesis of autophagosomes.Canonical autophagy is regulated by a number of autophagy-related (ATG)proteins and non-ATG proteins (such as class III phosphatidylinositol 3-kinase (PI3KIII),p150and (activating molecule in Beclin-1-regulated autophagy)Ambra1)that choreograph the initiation,elongation,maturation and fusion stages of the pathway.27Non-canonical autophagy is not as well understood,where autophagosomes can be created independently of Atg5or Atg7.28Recently,ferritin clusters have been reported to accumulate at the site of autophagosomeThe Department of Medicine and Abramson Cancer Center;University of Pennsylvania School of Medicine,Philadelphia,PA USA.Correspondence:Dr RK Amaravadi,The Department of Medicine and Abramson Cancer Center,University of Pennsylvania School of Medicine,16Penn Tower,3400Spruce Street,Philadelphia,PA 19063,USA.E-mail:ravi.amaravadi@Received 31December 2014;revised 18February 2015;accepted 18February 2015;published online 20April 2015Oncogene (2016)35,1–11©2016Macmillan Publishers Limited All rights reserved 0950-9232//oncformation along with p62in cells lacking Atg5,possibly shedding insight regarding non-canonical autophagosome biogenesis dynamics.29The classical and perhaps best-characterized environ-mental-mediated regulation of canonical autophagy occurs via the growth factor/receptor tyrosine kinase/phosphoinositide 3-kinase (PI3K)/protein kinase B (also known as AKT)/mechanistic target of rapamycin complex 1(mTORC1)signaling axis,which directly controls autophagic activity through the phosphorylation and inhibition of Unc-51-like kinase 1(ULK1),part of the ﬁrst protein complex involved in autophagic vesicle formation.30Under conditions in which growth factors and nutrients such as amino acids are rich in the extracellular space,the PI3K/AKT/mTORC1pathway is highly active and mTORC1inhibits ULK1through the phosphorylation at its serine-757residue.31However,when growth factors become limited,mTORC1becomes inactive and can no longer repress the complex consisting of ULK1,focal adhesion kinase family-interacting protein 200kDa,ATG13and ATG101,which favors the initiation of autophagy (the ﬁrst phase of autophagy).32AMP-activated protein kinase,in response to either glucose starvation or amino-acid deprivation,can also regulate ULK1activity via ﬁne-tuning of the phosphorylation status of ULK1.33Once activated,ULK1forms a complex with Beclin-1via assistance from TRIM5α,acting as a protein platform,leading to the phosphorylation and activation of Beclin-1.34Once active,Beclin-1activates the class III PI3K vacuolar sorting protein 34(Vps34),a component necessary both for endocytic sorting and in the ability of cells to respond to ﬂuctuations in nutrients such as amino acids and insulin.Vps34activity has also been demon-strated to not be inhibited by the TORC1inhibitor rapamycin,suggesting that Vps34can also function upstream of mTOR,serving as a vehicle for mTOR to monitor the levels of a wider net of critical nutrients for cell survival.35Following Vps34activation,autophagy cytoplasmic machinery is recruited onto the phospho-lipid membranes derived from various sources including theendoplasmic reticulum,36plasma membrane,37mitochondria 38and Golgi apparatus.39The second phase of autophagy (nuclea-tion)marks the beginning of autophagosome formation with the nucleation of membranes by Beclin-Vps34and either ATG14L,Rubicon,Ambra,among other proteins.The third phase (elonga-tion and maturation)allows for the maturation of autophago-somes and requires a ubiquitin ligase-like ATG5-ATG12-ATG16L complex (formed with the aid of ATG7and ATG10).40ATG4can also contribute to the elongation phase,and has recently been implicated as a biomarker and potential therapeutic target for chronic myeloid leukemia stem/progenitor cells (Figure 1).41The ubiquitin-like protein LC3/Atg8is subsequently conjugated to the lipid phosphatidylethanolamine on the surface of autophagosome membranes.Once integrated in the lipid bilayer,LC3interacts with adaptor proteins (autophagy receptors)such as p62,Nbr1,TRIM5αand NIX,which recruit cargo from the cytoplasm and promote autophagosome closure.34,42Proteomic network analysis in cells undergoing autophagy reveal high connectivity between LC3/Atg8and upstream autophagy components such as ULK1,Vps34and ATG2A,suggesting that LC3/Atg8may serve a more signi ﬁcant role in regulating autophagosome formation than was previously appreciated.43Once autophagosomes have engulfed cargo and closed,they are ultimately traf ﬁcked and fused to lysosomes forming autophagolysosomes.This fusion allows for the pH-dependent degradation of cytosolic cargo via hydrolases located within the acidic environment of the autophagolysosome.44Lysosomal permeases such as spinster permit the release of degradation products ranging from sugars,amino acids and nucleic acids into the cytosol for reuse by the cell 45(Figure 1).Our growing understanding of how autophagy is regulated has shed light on the potential novel druggable components for autophagy inhibition,which will be discussed later (see Figure 1andbelow).Figure 1.Autophagy regulators and points of intervention.(a )Autophagy occurs through a multistep process that includes four control points:initiation,nucleation,maturation,and lysosomal fusion and degradation of autophagosome contents.Successful autophagy results in the recycling of nutrients into the cytoplasm.(b –e )Autophagy is regulated on multiple levels with four major classes of regulation including posttranslational,transcriptional,epigenetic and metabolic regulation.Potential druggable targets are depicted (red star)with a promise to better modulate autophagy than strategies currently being implored.Autophagy inhibitor therapy VW Rebecca and RK Amaravadi2Oncogene (2016)1–11©2016Macmillan Publishers LimitedMOUSE MODELS ADDRESS THE ROLE OF AUTOPHAGY IN TUMOR INITIATION AND MAINTENANCEA major breakthrough in understanding the role of autophagy in tumorigenesis was made when spontaneous lung and liver tumors were found to arise in Beclin-1+/−mice.46Monoallelic deletion of the human homolog of Beclin-1(BECN1)was initially reported to occur in 40–75%of cases of human sporadic ovarian,breast and prostate cancer.47Taken together these results established BECN1as the ﬁrst autophagy-associated tumor suppressor gene.47,48However,the proximity of BECN1to the ovarian and breast tumor suppressor gene BRCA1on chromosome 17q21has decreased the certainty of Beclin-1’s role as a bona ﬁde tumor suppressor gene.A recent report demonstrated Beclin-1allele loss to be a rare event,assessed in human prostate,breast and ovarian tumor sequencing data from The Cancer Genome Atlas and other databases,except in the setting of loss of neighboring gene BRCA1.49Further,a larger panel of cancers was analyzed with no evidence for BECN1mutation or loss,leaving the function of BECN1as a tumor suppressor in human cancer unclear.Adding more complexity to the role Beclin-1serves in malignancy is a report showing Beclin-1to share regulation with p53at the level of proteasomal degradation in an ubiquitin-dependent manner;therefore sug-gesting that the spontaneous malignancy in Beclin-1+/−experimental systems may be due to lower p53levels.50Along a similar vein,Beclin-1and the antiapoptotic Bcl-2family member myeloid cell leukemia (Mcl-1)protein are both stabilized by binding to the deubiquitinase USP9X (ubiquitin-speci ﬁc peptidase 9X-linked),and negatively modulate the expression of each other through competitive displacement of USP9X.51Beclin-1expres-sion levels were discovered to decrease in patient-derived melanoma tissues as Mcl-1levels increased in a signi ﬁcant interdependent manner,independent of autophagy.51Though Beclin-1has recently been demonstrated to have a role in the response of lung cancer to epidermal growth factor receptor inhibition,52further experimental validation is needed to deter-mine the practical consequences of BECN1heterozygosity in human tumors and to delineate whether the observations involving Beclin-1are indeed dependent on the role autophagy serves in each of these experimental systems,or rather due to the confounding implications BRCA1,p53and Mcl-1each provide on cancer cell viability and disease progression.Beyond Beclin-1,mouse models with mosaic deletion of Atg5and liver-speci ﬁc deletion of Atg7also resulted in a greater incidence of spontaneous liver adenomas;however,the tumors were benign suggesting autophagy may be necessary for the progression beyond the benign state.53Deletion of Fip200also prevented the development of breast cancer.54,55Numerous mouse models have demonstrated autophagy to serve a critical capacity in disease progression in established oncogene-driven tumors,where inhibition of autophagy results in a reduction intumor volume in established tumors.In a mouse xenograft model utilizing immortalized baby mouse kidney epithelial cell lines engineered to express constitutive activity of RAS (H-ras V12)while also possessing defects in apoptotic machinery (Bax /Bak -de ﬁ-cient),autophagy was found to support survival of cancer cells undergoing metabolic stress and was localized to the poorly vascularized,hypoxic cores of tumors.56Further,cell lines engineered with constitutive activity of AKT (myr -AKT)along with apoptotic defects displayed high levels of necrosis,mechanisti-cally due to the coordinate inhibition of apoptosis (via Bax /Bak de ﬁciency)and autophagy (inhibited by AKT activity).Although these data were critical,what were sorely needed were genetically engineered mouse models of oncogene-driven cancers with and without defects in autophagy genes.These models have emerged recently (Table 1)and reveal a theme where the majority of mice with defects in key autophagy machinery display accelerated the development of benign tumors,however,autophagy appears to be essential for the progression of benign tumors to a more malignant state.Once a tumor is established,autophagy has been clearly demonstrated to also have a role in promoting the survival of existing tumor cells within the tumor microenvironment.57Two models of spontaneous Kras -driven lung cancer,one with tumor cell deletion of Atg758and one with tumor cell deletion of Atg5,59explored the importance of autophagy in the context of Ras oncogenes (Table 1).In the Kras G12D /Atg7ﬂ/ﬂmodel,the deletion of Atg7resulted in a signi ﬁcant reduction in tumor burden and an increase in tumor lipid accumulation;however,no difference in the overall survival could be noted due to an increase in death by in ﬂammation in mice with Atg7-de ﬁcient tumors.58In the Kras G12D /Atg5ﬂ/ﬂmodel,the deletion of Atg5resulted in increased tumor initiation;however,tumor cells exhibited decreased mitochondrial bioenergetics,and the deletion of Atg5also enhanced survival of mice.59Each of these mouse models revealed autophagy to be necessary for cancer cell proliferation and progression of lung tumors from adenomas to carcinomas.These ﬁndings strengthen the concept that Ras -driven cancers rely on autophagy for sustained metabolism and growth.A mouse model with Cre-activatable BRAF (Braf V600E )driven lung cancer,with and without the conditional knockout of Atg7was generated to determine the role of autophagy in BRAF -driven lung cancers.Autophagy was required for the growth of established Braf V600E -driven lung cancers via the preservation of mitochondrial function and the supply of metabolic substrates critical for sustained tumorigenesis.60Atg7-de ﬁcient mice experienced increased early tumorigenesis in an oxidative stress-dependent manner compared with mice with intact Atg7;however,as in the Kras -driven lung cancer model,Atg7deletion converted Braf V600E -driven adenomas to tumors that had the histological appearance of benign oncocytomas rather than carcinomas.60Autophagy inhibitor therapy VW Rebecca and RK Amaravadi3©2016Macmillan Publishers Limited Oncogene (2016)1–11In mouse models of pancreatic cancer,autophagy wasdiscovered to be vital and essential for tumorigenic growth of pancreatic cancers de novo.61Pancreatic ductal adenocarcinoma(PDAC)cell lines and primary tumor possess constitutivelyactivated autophagy(as seen by GFP-LC3puncta and cleavedLC3-A IHC(LC3-II))and a unique dependence upon autophagy. Importantly,the genetic(suppression of ATG5expression byshRNAs)or chemical inhibition(chloroquine)of autophagy leadsto robust tumor regression and prolonged survival in pancreaticcancer xenografts and genetic mouse models.61KRAS mutations are one of the known drivers in PDAC,and a recent reportleveraging an inducible mouse model of mutated Kras(Kras G12D)in a p53Lox/WT background shed further light on the roleautophagy serves in pancreatic cancer.In a temporal and pancreas-speciﬁc manner,the authors ablated KRAS activity,which resulted in pancreatic tumor regression within2–3weeksfollowed by relapse a few months thereafter.The cancer cells surviving KRAS ablation were studied with transcriptome analysisand gene set enrichment analysis revealing a signiﬁcant enrich-ment of genes involved in lysosomal activity,mitochondrialelectron transport chain and autophagy,among other cellular processes.62Although the genetically engineered mouse models describedabove were incredibly useful in shedding light on the effects ofautophagy defects on the tumorigenesis of oncogene-driven cancer,they did not effectively model the therapeutic ablation ofautophagy.With cancer therapy,drugs will typically impact thepathway throughout the body and are often administered only after the tumor becomes apparent(stage IV)or in a high-risk(stage III)setting.Although tumor xenografts address this to somedegree,those models are artiﬁcial because mice lack immunesystems and the tumor is typically grown out of context in the ﬂanks of the mice.To address all of these concerns,a genetically engineered mouse model of an inducible Kras-driven lung cancerwas generated where Atg7could be systemically deleted in aconditional manner.When systemic Atg7deletion was engaged in adult mice,mice initially were asymptomatic,but eventually died of neurodegeneration at roughly3months.63However,when Atg7 was systemically ablated in mice before the induction of Kras-driven lung cancer,the rate of lung nodules appeared to increase, but the nodules failed to progress to cancer before the mice succumbed to the effects of systemic Atg7depletion.When Atg7 was systemically deleted in mice after Kras-driven tumors were allowed to form,massive tumor regression and apoptosis was observed before the toxicity of Atg7depletion on normal tissue was evident.These observations are valuable as they reveal that chronic autophagy inhibition may yield toxicities,supporting the exploration of optimal treatment regimens that minimize expo-sure to autophagy inhibitors while still maximizing the antitumor beneﬁt conferred from autophagy inhibition.In general,mouse models show that autophagy is critical in the transition from premalignant to malignant,however,autophagy promotes growth of established tumors.These recent results partially reconcile the dichotomy of autophagy in tumorigenesis, and support a role for the inhibition of autophagy as a therapeutic strategy in certain advanced cancers.There was an exception reported,where a model of pancreas-speciﬁc Kras-mutant,Trp53−/−tumors was treated with autophagy inhibition with either genetic ablation of Atg5or Atg7,or chemically with HCQ,resulting in the promotion of tumorigenesis(Table1).64From both the strategies,autophagy inhibition was found to accelerate the formation of PDAC in mice due to enhanced glucose uptake and enrichment of anabolic pathways.65A wrinkle in this model is its use of an embryonic pancreas-speciﬁc homozygous deletion of Trp53in the context of Kras mutation,which results in advanced cancers in early development.In nature,p53is most frequently found as missense mutations in Kras-mutant pancreatic cancers.66 The heterozygous expression of mutant Trp53in the context of oncogenic Kras is postulated to give rise to precancerous lesions called pancreatic intraepithelial neoplasias,with the subsequent loss of heterozygosity of the wild-type TP53allele driving the progression from pancreatic intraepithelial neoplasias to PDAC.65 Thus,the model64utilizing homozygous deletion of Trp53did not fully recapitulate the step-wise progression of pancreas cancer as is found in humans.To address this important issue,a pancreas-speciﬁc Kras-mutant Trp53+/−mouse model was generated that experiences loss of heterozygosity of the wild-type Trp53allele during PDAC progression,therefore mirroring the step-wise development of human pancreas cancer.67Within this model with Trp53(loss of heterozygosity),autophagy inhibition via ablation of Atg5or with HCQ was found to increase the overall survival in a mouse preclinical trial leveraging cohorts of genetically characterized,patient-derived xenografts.Trp53status was not found to correlate with the response in tumor cell lines or patient-derived xenograft models,and although autophagy inhibition in the pancreas lead to an increase in tumor initiation, few of these premalignant lesions could develop into invasive tumors and the mice treated with autophagy inhibition lived longer overall.67Theseﬁndings are of the upmost importance,as conclusions drawn from the Trp53model that did not recapitulate human pancreas cancer development64lead to premature recommendations that patients with Trp53mutations should not receive treatment with HCQ.68Due to the high proﬁle of the Journal in which this opinion piece was published,it is possible that patients who may have beneﬁted from clinical trials utilizing HCQ may have been directed to other therapies by their physicians.Insight from these studies will also help design therapy regimens,where exposure to autophagy inhibitors will be strategically timed to allow for optimal therapeutic beneﬁt in the absence of potential hazards from the chronic inhibition of autophagy.It appears that in most cases autophagy defects lead to accelerated tumor initiation,but impaired tumor maintenance. It is for these reasons why much effort in developing therapeutics targeting autophagy is focused on advanced cancers where concerns about developing secondary benign tumors will be less problematic if the advanced cancer that is putting the patient’s life immediately at risk can be halted or regressed.A deeper understanding of how autophagy is regulated on multiple levels could unravel the switch that turns autophagy from a tumor suppressor to a tumor promoter.CANCER THERAPY CAN PRODUCE AUTOPHAGIC/ IMMUNOGENIC CELL DEATH:THE ARGUMENT TO INDUCE AUTOPHAGYObservations that therapy-induced autophagy can have a role in tumor cell cytotoxicity have been reported;however,they commonly depend upon pre-existing defective apoptotic machin-ery in order for the autophagic cell death to manifest.Bcl-2 homology3mimetics such as gossypol have been demonstrated to elicit autophagic cell death in apoptosis-deﬁcient malignant glioma and prostate cancer,by way of disrupting physical interactions between Bcl-2family members and Beclin-1.69 Autophagic cell death refers to cell death that is accompanied by extensive cytoplasmic vacuolization,often correlated to increased autophagicﬂux.70The use of the term autophagic cell death is controversial,as since its conception the phrase is commonly misused to suggest that autophagy actively contri-butes to cell death.Although autophagy frequently occurs concurrently with regulated cell death,autophagy is directly responsible for the death of tumor cells in only a few cases.71To date,there have been no deliberate attempts to induce autophagy speciﬁcally in a cancer model.Autophagy appears to be responsible for the death of some cancer cells with defective apoptotic machinery,such as inhibited caspase-8,in an ATG7andAutophagy inhibitor therapyVW Rebecca and RK Amaravadi4Oncogene(2016)1–11©2016Macmillan Publishers LimitedBeclin-1-dependent manner in vitro.72Another study reported re-expression of(ARHI)aplasia Ras homolog I in human ovarian cancer cell lines resulted in autophagic cell death in vitro.73 However,in vivo autophagy enabled these cells to remain dormant in the context of ARHI re-expression,with chloroquine treatment markedly reducing the regrowth of xenografts.Similar results were also observed in vitro when cells were cultured with factors found in vivo such as IGF-I,M-CSF and IL-8,suggesting autophagy serves a protective role when experimental conditions recapitulate those found within the tumor microenvironment.A recent consensus statement on cell death nomenclature warned about the fact that regulated cell death mechanisms frequently interact with each other and it may be that in many cases persistent autophagy can activate other forms of cell death that are actually responsible for the death that ensues.71There may exist multiple checkpoints that limit autophagic cell death from occurring in vivo,such as growth factor availability and functional apoptotic machinery.Interestingly,autophagy has also been reported to serve a role in the recruitment of immune system effectors.Chemotherapy in autophagy-competent cancers recruited dendritic cells and T lymphocytes to the tumor bed in an ATP-dependent fashion.74 Inhibiting autophagy suppressed the release of ATP and attenuated the recruitment of immune cells.Similar results were observed in melanoma where chemotherapy75or radiotherapy76 each led to an increase in mannose-6-phosphate receptor on the tumor cell surface,making tumor cells more susceptible to lysis by cytotoxic T cells,in an autophagy-dependent manner.The implications theseﬁndings hold in regard to the clinical utilization of autophagy inhibitors moving forward remain to be determined.A potential combination of an immune checkpoint inhibitor,such as anti-PD-1antibody,77with an autophagy inhibitor can be envisioned to ensure potential secondary effects on the immune response to cancer cells do not blunt the antitumor effect of autophagy inhibition.CANCER THERAPY CAN PRODUCE CYTOPROTECTIVE AUTOPHAGY:THE ARGUMENT TO INHIBIT AUTOPHAGY Autophagy was convincingly shown as a key survival mechanism in apoptosis-defective transformed cells subjected to growth factor withdrawal.Cells that survived growth factor withdrawal or other modes of starvation could be killed when autophagy was inhibited with either3-methyladenine or CQ,and the autophagic phenotype was reversible once growth factors were replenished.21 Utilizing a Myc-induced model of lymphoma,the role of autophagy in the survival of tumor cells in vivo was demonstrated where treatment with either CQ or ATG5shRNAs enhanced the ability of alkylating drug therapy to induce tumor cell death.78 Since then,a multitude of papers have been published demonstrating utility in combining autophagy inhibitors with cancer therapy.11In addition to autophagy serving a critical role in tumorigenesis,many cancer drugs have been reported to induce autophagy that can be cytoprotective.Traditional cytotoxic chemotherapeutics and targeted therapies induce autophagy through a number of signaling pathways including the DNA damage response,mTOR and AMP-activated protein kinase signaling,the ER stress response and others.11Inhibition of autophagy with chloroquine in preclinical models improves the response of tumor cells to alkylating agents,suggesting that autophagy promotes survival.79Another report observed cyto-protective autophagy to serve a critical resistance mechanism to BRAF inhibition in BRAF-mutant melanoma.23Thisﬁnding was of particular interest,as the role autophagy has in resistance to targeted therapies that target PI3K/AKT/mTOR signaling have been well studied;80,81however,the function of autophagy in the context of MAPK pathway inhibition has not been well characterized.Mechanistically,BRAF inhibition leads to a physical interaction between mutant BRAF and GRP78,a master regulatorof ER stress activity,which results in the downstream activation ofthe ER stress pathway effector PERK.PERK activation results in an induction of cytoprotective autophagy.BRAF inhibitor-induced autophagy was observed at a high rate in tumors obtained at thetime of progression on BRAF inhibitor therapy.23Targeting autophagy with HCQ concurrently with BRAF inhibitor therapy resulted in signiﬁcant tumor regression in mouse xenografts studies.Thisﬁnding was reproduced in in vitro and in vivo studiesin pediatric gliomas that harbor BRAFV600E mutations,and the addition of HCQ to a BRAF inhibitor overcame the resistance to BRAF inhibition in a patient with pediatric glioma.82Many other examples exist supporting the concept of combining chemo-therapy or targeted therapy with a chloroquine derivative,providing rationale for launching cancer clinical trials involving HCQ. CLINICAL TRIALS OF HCQ,THE FIRST AUTOPHAGY INHIBITORThe seminal discoveries of these recent mouse models and preclinical investigations dovetail nicely with the publishing of theﬁrst set of HCQ clinical trials in patients with advanced cancers (Table2).Six phase I/II trials were performed in human patients diagnosed with glioblastoma multiforme,16relapsed/refractory myeloma17and melanoma in addition to other advanced tumors.13–15One additional clinical trial was published whereinpet dogs diagnosed with spontaneously occurring lymphoma were also treated with HCQ-based combination therapies.12Eachtrial involved a combination therapy that had preclinical studies to justify clinical translation.78,83–86The majorﬁnding from these trials is that,based on electron microscopy-based pharmacody-namic assays,autophagy can be modulated therapeutically with chloroquine derivatives.Remarkably,across all of the trials o10%of patients had severe non-hematological toxicity.Speciﬁcally, there was no evidence of extensive metabolic toxicity,liver injuryor neurologic impairment in these trials despite some evidencethat chronic modulation of autophagy was achieved in patients,as seen by the accumulation of autophagic vesicles in peripheral blood mononuclear cells and tumor cells.When combined with radiation therapy and concurrent and adjuvant temozolomide, HCQ produced dose-limiting myelosuppression at doses above600mg HCQ.At these doses only a subset of patients had evidence of autophagy modulation detectable in their peripheral blood mononuclear cells,which may be one reason there was no signiﬁcant improvement in the overall survival compared with the historical controls of temozolomide and radiation alone.16 Signiﬁcant therapy-associated increases in AVs and LC3-II were observed in peripheral blood mononuclear cells in a concentration-dependent manner,demonstrating HCQ could modulate autophagy in bined treatment with the proteasome inhibitor bortezomib and HCQ resulted in a greater perturbation of tumor cell autophagy compared with peripheral blood mononuclear cell autophagy,arguing that HCQ may selectively accumulate in tumor cells.17Similar results were observed in the phase I trial of vorinostat and HCQ13and in the canine lymphoma trial using doxorubicin with HCQ.12Although these phase I studies were not powered to determine efﬁcacy, response rates in unselected patient populations were generally low.However,there were a number of striking responses and prolonged stable disease observed in patients with melanoma, renal cell carcinoma,colon cancer and myeloma,that suggest thata speciﬁc subset of cancers may be susceptible to regimens containing chloroquine-based autophagy inhibitors.Critical to the future success of autophagy-oriented clinical trials are biomarkersthat may aid in patient selection.Current biomarkers to assess autophagy modulation in clinical trials consist of monitoring the accumulation of autophagic vesicles in peripheral blood mono-nuclear cells and tumor cells by electron microscopy,as well as checking for changes in LC3lipidation by western blotting and Autophagy inhibitor therapyVW Rebecca and RK Amaravadi5©2016Macmillan Publishers Limited Oncogene(2016)1–11。

糖尿病肾脏疾病足细胞自噬改变研究进展

中国糖尿病杂志2020 年12 月第28 卷第 12 期Chin J Diabetes,December 2020,Vol. 28，N〇. 12•947••文献综述•糖尿病肾脏疾病足细胞自噬改变研究进展曾思皓何笑云左熠龚建萍黎玲刘展宏周素娴【提要】D K D是D M常见微血管病变，是终末期肾病的主要原因，已成为全球严重的公共卫生问题。

自噬是细胞应对各种压力的自我保护，去除蛋白质聚集物和受损细胞器以维持细胞内平衡。

作为重要的应激反应机制，自噬参与多种疾病的发病机制并调控信号通路。

足细胞自噬参与D K D的发生发展，可作为新的治疗靶点。

本文就影响D K D足细胞自噬的相关物质进行综述。

【关键词】糖尿病肾脏疾病；自噬；信号通路doi:10. 3969/j.issn. 1006-6187. 2020.12. 013P ro g ress in th e stu d y o f a u to p h a g y ch a n g e s in d ia b etic n ep h ro p a th y p o d o cy te ZENG Sihao, HE Xiaoyun,ZUO Yi, et al. Department o f Endocrinology, Affiliated Hospital of Guilin Medical University^ Guilin541001，ChinaCorresponding author: ZHOU Siurian，Email :zoe一doctor@ 163. com【S u m m a r y】Diabetic kidney disease(DKD)is a common microangiopathy of diabetes mellitus，whichis the main cause of end-stage renal disease,and has become a serious global public health problem.Autophagy is the self-protection of cells to deal with various stresses.It can remove protein aggregates and damagedorganelles to maintain the intracellular balance.As an important stress response mechanism,autophagy is involved in the pathogenesis of various diseases and the regulation of signal pathways.Podocyte autophagy isinvolved in the development of DKD,which can be used as a new therapeutic target.This article reviews therelated substances that affect the autophagy of DKD podocytes.【K ey w o r d s】Diabetic kidney disease;Autophagy;Signaling pathway2018年全球D M患者为4. 25亿，预计2045年将增至6.29亿⑴。

乳腺梭形细胞化生癌1例并文献复习

126Chin J Lab Diagn，January，2021，Vol 25，No. 1Pyroptosis in Diabetic Cardiomyopathy [J ]. Cell Physiol Bio-c h e m,2018,50(4)：1230.[34]Li P，Ruan X，Yang L, et al. A liver-enriched long non-codingR N A，IncL S T R，regulates systemic lipid metabolism in mice[J].C e l l M e ta b，2015,21(3):455.[35]Zhang M,Z h a n g L, Hu J ,et al. MST1 coordinately regulates au-tophagy and apoptosis in diabetic cardiomyopathy in miceCJ].Diabetologia, 2016,59(11) ： 2435.[36]Feng Y,X u W,Z h a n g W,e t al. LncRNA DCRF regulates cardio-myocyte autophagy by targeting miR-551b-5p in diabetic cardio- m y o p a th y[J].T h e r a n o s tic s，2019,9(15):4558.[37]Zhuo C,Jiang R,L in X,e t al. LncRNA H19 inhibits autophagyby epigenetically silencing of DIRAS3 in diabetic cardiomyopa- th y[J]. O n co targ et，2017,8(l):1429.[38] Zeng Z, Pan Y, Wu W, et al. Myocardial hypertrophy is improved with berberine treatm ent via long non-coding R N A MI-AT-mediated au to p h ag y[J]. J Pharm Pharm acol,2019»71( 12)：1822.[39] Zhao L，Li W，Zhao H. Inhibition of long non-coding RN ATU G1 protects against diabetic cardiomyopathy induced diastolic dyvsfunction by regulating miR-499-5p [ J ]. Am J TranslR e s,2020,12(3)：718.C4〇]L u o X.H e S,H u Y,e t al. Spl-induced LncRNA CTBP1-AS2 isa novel regulator in cardiomyocyte hypertrophy by interactingwith FU S to stabilize T L R4[J J.Cardiovasc P a th o l, 2019,42：21.[41]Z h ao J,L iu B.L i C. Knockdown of long noncoding R N A GASSprotects human cardiomyocyte-like AC16 cells against high glucose-induced inflammation by inhibiting miR-2 l-5p-mediatedThR^l/NF'-KB signaling[J]. Naunyn Schmiedebergs Arch P h a rm aco l,2019, 10. 1007/s00210-019-01795-z [42] Zhang M,G u H»C hen J, et al. Involvement of long noncodingRN A MALAT1 in the pathogenesis of diabetic cardiomyopathy [J]. Int J Cardiol,2016,202： 753.[43]Gao L，Wang X，Guo S，et al. LncRNA H O T A IR functions as acompeting endogenous RN A to upregulate SIRT1 by sponging miR-34a in diabetic cardiomyopathy [J],J Cell Physiol* 2019,234(4)：4944.[44]Yu M，Shan X，Liu Y，et al. RNA-Seq analysis and functionalcharacterization revealed IncRNA NONRATT007560. 2 regulated cardiomyocytes oxidative stress and apoptosis induced byhigh g lu c o s e[J].J Cell Biochem，2019，120(10): 18278.[45]Gong W，Zhu G，Li J，et al. LncRNA MALAT1 promotes theapoptosis and oxidative stress of human lens epithelial cells viap38MAPK pathway in diabetic cataract [J].Diabetes Res Clin P r a c t,2018,144：314.[46]Li X,W a n g H, Yao B,e t al. IncRNA H19/miR-675 axis regulates cardiomyocyte apoptosis by targeting VDAC1 in diabetic cardiomyopathy[J]. Sci R e p,2016,6：36340.[47]Gao Y, Wu F,Z h o u J ,et al. T he H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells[J].Nucleic Acids R e s,2014,42(22): 13799.[48]Liu SX,Zheng F,X ie K L,e t al. Exercise Reduces Insulin Resist +ance in Type 2 Diabetes Mellitus via Mediating the IncRNA M A LA T l/M icroR N A-382-3p/R esistin Axis[J]. Mol Ther N ucleic Acids. 2019.18 ： 34.[49] Liu S，Zheng F，Cai Y, et al. Effect of Long-Term ExerciseTraining on IncRNAs Expression in the Vascular Injury of Insulin Resistance[J]. J Cardiovasc Transl R e s,2018,11(6) ：459.(收稿日期：2019_11 —12)文章编号：1〇〇7 —4287(2021)01 —0126 —03乳腺梭形细胞化生癌1例并文献复习杨大涛、邢华〃，惠亚丹2,张祥恺2,张平波2(吉林大学中日联谊医院1.乳腺外科；2.内分泌代谢科，吉林长春130033)乳腺化生性癌（M B C)于是一组具有明显异质性的罕见的乳腺侵袭性肿瘤，占所有乳腺恶性肿瘤的〇. 2%-1%[1]。

自噬介导的蛋白质质控通路

自噬介导的蛋白质质控通路英文回答：Autophagy is a cellular process that plays a crucial role in maintaining cellular homeostasis and proteinquality control. It is a highly conserved process that involves the degradation and recycling of cellular components, including proteins, organelles, and even pathogens. The autophagy pathway is regulated by a complex network of proteins and signaling pathways.One important protein quality control pathway that is regulated by autophagy is the clearance of misfolded or aggregated proteins. When proteins are misfolded or aggregated, they can form toxic aggregates that can disrupt cellular function and contribute to the development of various diseases, including neurodegenerative diseases like Alzheimer's and Parkinson's. Autophagy helps to remove these toxic protein aggregates by sequestering them into autophagosomes, which are double-membrane vesicles thatengulf cellular components for degradation. These autophagosomes then fuse with lysosomes, where the enclosed proteins are degraded by lysosomal enzymes.Another protein quality control pathway that is regulated by autophagy is the selective degradation of damaged or dysfunctional organelles. For example, autophagy plays a crucial role in the clearance of damaged mitochondria through a process called mitophagy. Mitochondria are the powerhouses of the cell, but they can also generate reactive oxygen species (ROS) that can damage cellular components. When mitochondria are damaged, they can be selectively targeted for degradation by autophagy to prevent the accumulation of dysfunctional mitochondria and the release of ROS.In addition to protein quality control, autophagy also plays a role in cellular stress responses. For example, during nutrient deprivation, autophagy is activated to provide the cell with nutrients by degrading and recycling cellular components. This allows the cell to adapt to the stress and survive under nutrient-limited conditions.Autophagy can also be induced in response to other stresses, such as oxidative stress and hypoxia.中文回答：自噬是一种维持细胞稳态和蛋白质质量控制的重要细胞过程。

细胞自噬与疾病的研究进展

细胞自噬与疾病的研究进展焦寒伟;王红均;赵宇;伍莉;帅学宏;陈吉轩;甘玲;罗献梅;黄庆洲【摘要】Autophagy is a biological process in which cells transport the damaged organelles, waste cytoplasm and denatured or senescent proteins to lysosome, and then undergo enzymatic hydrolysis. Autophagyis a highly conserved process in eukaryotes, and autophagy is a cell self-protective mechanism which maintains the intracellular homeostasis. Autophagy plays an important role in the programmed cell death (PCD), cancer, cardiovascular disease, liver disease, neurodegenic diseases, cancer and tumor, autoimmune diseases and other disases. It was suggested that the autophagy played an important role in the process of immuno related diseases, thus clarifying that cell autophagy may be a new target and breakthrough in the treatment of immune related disease, and pave the way for the treatment of immune related diseases.%细胞自噬 (Autophagy) 是细胞将受损的细胞器、废弃细胞质和变性或衰老的蛋白质转运至溶酶体(Lysosome), 并进行酶解的一个生物学过程.在真核生物中, 细胞自噬是一个高度保守的过程, 而自噬是细胞的一种自我保护机制, 维持了细胞内稳态.自噬在细胞程序性死亡(Programmed cell death, PCD) 、心血管疾病、肝脏疾病、神经性疾病、癌症与肿瘤以及自身免疫性疾病等多种疾病过程扮演了重要的角色.该文综述了细胞自噬与多种疾病的研究进展, 提示了细胞自噬在免疫相关性疾病过程中发挥着重要的调控作用, 从而阐明细胞自噬可能作为疾病治疗的新靶点和突破口, 为治疗免疫相关性疾病做铺垫.【期刊名称】《安徽农业科学》【年(卷),期】2019(047)002【总页数】5页(P10-14)【关键词】细胞自噬;溶酶体;真核生物;疾病【作者】焦寒伟;王红均;赵宇;伍莉;帅学宏;陈吉轩;甘玲;罗献梅;黄庆洲【作者单位】西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌402460;西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌 402460;西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌 402460;西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌 402460;西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌 402460;西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌 402460;西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌 402460;西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌 402460;西南大学动物科学学院, 重庆市兽医科学工程研究中心, 重庆荣昌 402460【正文语种】中文【中图分类】Q291细胞自噬(Autophagy or autophagocytosis)是真核生物中细胞将自身胞浆蛋白或损伤细胞器包裹形成囊泡，并在溶酶体降解回收再利用的代谢过程。

激活小鼠的自噬方法

激活小鼠的自噬方法英文回答：Autophagy is a cellular process that involves the degradation and recycling of cellular components. It plays a crucial role in maintaining cellular homeostasis and has been implicated in various physiological and pathological conditions. Activating autophagy in mice can be achieved through several methods.One common method to activate autophagy in mice is through dietary restriction or caloric restriction. This involves reducing the amount of food intake or restricting the intake of specific nutrients. Caloric restriction has been shown to induce autophagy in various tissues,including the liver, muscle, and brain. For example, studies have shown that reducing calorie intake by 30-40% can significantly increase autophagy levels in the liver of mice.Another method to activate autophagy in mice is through pharmacological interventions. Several compounds have been identified that can induce autophagy. One such compound is rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR) pathway. mTOR is a key regulator of autophagy, and inhibiting its activity can lead to autophagy activation. Rapamycin has been widely used to induce autophagy in various animal models, including mice. Other compounds, such as resveratrol and spermidine, have also been shown to induce autophagy in mice.In addition to dietary restriction and pharmacological interventions, exercise has also been shown to activate autophagy in mice. Exercise-induced autophagy has been observed in various tissues, including skeletal muscle and the heart. For example, studies have shown that endurance exercise can increase autophagy levels in skeletal muscle of mice. The exact mechanisms by which exercise induces autophagy are still not fully understood, but it is believed to involve the activation of AMP-activated protein kinase (AMPK) and the inhibition of mTOR signaling.中文回答：自噬是一种细胞过程，涉及细胞成分的降解和再利用。

可卡因诱导神经细胞自噬的研究进展

可卡因诱导神经细胞自噬的研究进展沈宝玉，杨根梦，李媛媛，刘柳，黄俭，曾晓锋，李利华（昆明医科大学法医学院，云南昆明650500）［摘要］可卡因（Cocaine ）是一种强效的中枢兴奋剂，因其对中枢神经系统（central nervous system ，CNS ）的兴奋作用而导致滥用，可卡因的长期摄入可引起中枢神经损害，具有很强的神经毒性和药物依赖性。

此外，可卡因滥用者常因共用针具和无保护措施的性行为等高风险活动而感染人免疫缺陷病毒（human immunodeficiency virus ，HIV ）。

自噬（Autophagy ）是高度保守的分解代谢调控途径，可维持细胞能量稳态和调节细胞生长，是细胞死亡或存活的重要仲裁者，近年来受到了广泛关注。

对可卡因诱导神经细胞自噬和相关神经毒性的研究进行综述，为进一步研究可卡因与自噬提供参考。

［关键词］可卡因；自噬；人免疫缺陷病毒；艾滋病；神经毒性［中图分类号］R89［文献标志码］A ［文章编号］2095－610X （2020）01－0163－05Progress of Cocaine-induced Autophagy in CNSSHEN Bao-yu ，YANG Gen-meng ，LI Yuan-yuan ，LIU Liu ，HUANG Jian ，ZENG Xiao-feng ，LI Li-hua（School of Forensic Medicine ，Kunming Medical University ，Kunming Yunnan 650500，China ）［Abstract ］Cocaine is consumed by drug users as a psychostimulant.However ，chronic cocaine intaking cancause damage to the central nervous system （CNS ）and induce addiction.In addition ，cocaine users are infected with human immunodeficiency virus （HIV ）through high-risk activities such as needle sharing and unprotected sex.Autophagy is a highly conserved catabolism regulation pathway ，which can maintain cell energy homeostasis and regulate cell growth.Furthermore ，autophagy as an important arbiter of cell death or survival has attracted wide attention in recent years.We reviewed the studies on cocaine-induced autophagy and focused on CNS.［Key words ］Cocaine ；Autophagy ；HIV ；AIDS ；NeurotoxicityJournal of Kunming Medical UniversityCN 53-1221R［收稿日期］2019－10－22［基金项目］国家自然科学基金资助项目（81560303，81660310，81560302）；云南省教育厅科学研究基金资助项目（2019Y0343，2019Y345）［作者简介］沈宝玉（1995~），男，云南宣威人，在读硕士研究生，主要从事毒品神经毒性及依赖机制的研究工作。

在一个心脏骤停的猪的实验模型中凋亡是复苏后心功能障碍的一种机制

Brief ReportApoptosis is involved in the mechanism of postresuscitation myocardial dysfunction in a porcine model of cardiac arrestWei Gu MD,Chun Sheng Li MD⁎,Wen Peng Yin MD,Zhi Jun Guo MD,Xiao Min Hou MD,Da Zhang MMDepartment of Emergency Medicine,Beijing Chaoyang Hospital,Capital Medical University,Beijing,China Received16February2012;revised20April2012;accepted23April2012AbstractBackground:Postresuscitation myocardial dysfunction contributes to the low survival rate aftersuccessful resuscitation,but its mechanism remains poorly understood.This study investigated whethercaspase3–mediated apoptosis is activated in the heart after postresuscitation myocardial dysfunction.Methods:After pigs were subjected to8minutes of electrically induced cardiac arrest(CA),standardcardiopulmonary resuscitation was performed.Animals in the post–return of spontaneous circulation(ROSC)group were randomly assigned to be killed(n=6per group)at12and24hours after ROSC,and myocardial specimens were analyzed with electron microscopy,Western blotting,quantitative real-time polymerase chain reaction,and terminal deoxynucleotidyl transferase–mediated dUTP nick endlabeling assay.Results:Myocardial function was significantly impaired after ROSC.Expression of Bcl-2,Bax,andcaspase3protein was markedly increased in the post-ROSC group compared with the sham-operatedgroup(P b.05)at12and24hours after ROSC,whereas Bcl-2/Bax was significantly reduced in thepost-ROSC group compared with the sham-operated group(P b.05).The messenger RNA levels ofcaspase3were significantly elevated at12and24hours after ROSC,and increases in caspase3activityindicated activation of the mitochondrial apoptotic pathway.Typical apoptotic nuclei were observed incardiomyocytes24hours after ROSC.More apoptotic cells were observed in animals that hadundergone CA compared with sham-operated animals(P b.05).Conclusion:Caspase3–mediated apoptosis may be one of the main pathologic mechanisms ofpostresuscitation myocardial injury in a porcine model of CA.©2012Elsevier Inc.All rights reserved.1.IntroductionCardiac arrest(CA)is second only to all cancer deaths combined as a cause of mortality and is responsible for approximately275000deaths per year in the United States [1].In China,the incidence of sudden cardiac death per year is418.4/million[2].Despite major efforts to improve outcomes from sudden CA,the survival rate remains low [3].Many patients initially resuscitated from CA die before discharge from the hospital[4]because of so-called postresuscitation syndrome[5].Although postresuscitation myocardial dysfunction is a critical issue and has been reported in45%to60%of⁎Corresponding author.E-mail address:lcscyyy@(C.S.Li)./locate/ajem0735-6757/$–see front matter©2012Elsevier Inc.All rights reserved./10.1016/j.ajem.2012.04.031American Journal of Emergency Medicine(2012)30,2039–2045successfully resuscitated patients[5],the mechanisms responsible for postresuscitation myocardial dysfunction are not well understood.Whether apoptosis is involved in the mechanism of myocardial dysfunction after resuscita-tion remains controversial[6].Once apoptosis occurs,the loss of myocytes cannot be reversed,and the reduction in the number of contractile cells ultimately compromises the“pump”function of the heart[7].However,recent laboratory and clinical evidence indicates that the transient myocardial dysfunction after CA/cardiopulmo-nary resuscitation(CPR)is responsive to therapy and is fully reversible[5].Therefore,in this study,we established a swine model of CA.We examined whether caspase3–induced cardiomyo-cyte apoptosis is activated in the heart after resuscitation and further hypothesized that after CA/CPR,apoptosis is one of the main mechanisms involved in postresuscitation myocar-dial dysfunction.2.Methods2.1.Animal preparationTwenty-two inbred Wuzhishan miniature pigs(12-14 months of age,30±2kg)were used in this study.After premedication with0.5mg/kg intramuscular midazolam, anesthesia was induced by injection of propofol(1.0mg/kg) and maintained in a surgical plane of anesthesia with intravenous infusion of pentobarbital(8mg/kg per hour)[8]. Animals were mechanically ventilated with a volume-controlled ventilator(Servo900C;Siemens,Munich, Germany)with a tidal volume of15mL/kg and fraction of inspired oxygen of0.21.A Swan-Ganz catheter(7F; Edwards Lifesciences,Irvine,CA)was advanced from the right femoral vein andﬂow directed into the pulmonary artery for measurement of mean aortic pressure(MAP)and cardiac output(CO).All hemodynamic parameters were monitored with a patient monitoring system(M1165; Hewlett-Packard,Palo Alto,CA).2.2.Experimental protocolAfter surgery,the temporary pacemaker conductor was inserted into the right ventricle through the right sheathing canal and connected to an electrical stimu-lator(GY-600A;Kaifeng Huanan Equipment Co,Ltd, Henan,China)programmed in the S1S2mode(300/200 milliseconds),40V,8:1proportion,and10-millisecond step length to provide a continuous electrical stimulus until ventricularﬁbrillation(VF)[9].Ventricular fibrillation was deﬁned as an electrocardiogram show-ing waveforms corresponding to VF and a rapid decline in MAP toward zero.Ventilation was stopped while inducing VF.After8minutes of VF,manual CPR was carried out at a frequency of100compressions per minute with mechanical ventilation at a fraction of inspired oxygen of100%and a compression-to-ventilation ratio of30:2.The quality of chest compressions was controlled by a HeartStart MRx Monitor/Deﬁbrillator with Q-CPR(Philips Medical Systems,Best,Holland).Return of spontaneous circu-lation(ROSC)was deﬁned as10consecutive minutes of maintenance of systolic blood pressure at50mm Hg.If spontaneous circulation was not restored within 30minutes,we regarded the animal as dead.The same procedure without CA initiation was achieved in the sham operation group,including induction of anesthe-sia,mechanical respiration,and monitoring of physio-logic parameters.After successful resuscitation,animals in the post-ROSC group were randomly assigned to be killed(n=6per group) at12and24hours after ROSC.2.3.MeasurementsHemodynamic parameters,including CO and MAP,were measured continuously,and we recorded the values at baseline;30minutes;and2,4,and6hours after ROSC.Some myocardial specimens were preserved in4% paraformaldehyde to observe pathologic changes for trans-mission electron microscopy and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling(TUNEL), whereas others were stored at−80°C for quantitative real-time polymerase chain reaction(PCR)and Western blotting.2.4.Western blot analysis of Bcl-2,Bax,and caspase3A100-mg frozen heart sample was homogenized in2mL of ice-cold buffer.The tissues were homogenized and then centrifuged at12000rpm for10minutes at4°C.A total of 100μG of protein was loaded onto10%sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel in each sample.Western blotting was performed with the mem-branes blocked for2hours with5%nonfat milk and then incubated with the primary antibodies(diluted overnight at 4°C):Bax,1:500(sc-70407;Santa Cruz Biotechnology, Santa Cruz,CA);Bcl-2,1:200(MAB4625;EMD Millipore, Jaffrey Cheshire,NH);caspase3,1:500(4051;Abcam Biotechnology,Canbridge,United Kingdom);and GAPDH, 1:250(Santa Cruz Biotechnology).Blots were blocked and incubated at4°C overnight with the speciﬁc primary antibody.The immunoreactive bands were visualized on ﬁlm and scanned.2.5.Quantitative real-time PCR assay for caspase3Total RNA was extracted from50to100mg of tissue according to the protocol described for the BioEasy SYBR2040W.Gu et al.Green I Real-Time PCR Kit Manual(Bo Ri Technology Co, Ltd,Hang zhou,Zhejiang,China).Preincubation was performed at95°C for2minutes,followed by ampliﬁcation in45cycles at95°C for20seconds;59°C(caspase3);72°C for30seconds;and,ﬁnally,during slow heating up,72°C for 10minutes.After the ampliﬁcation,melting curve analysis with a temperature gradient from65°C to95°C was recorded every0.5°C(hold for5seconds).The obligation quantity (OD)of the target genes was compared with that of GAPDH. The primer sequences of the expected PCR products were as follows:for caspase3,sense5-CATGGCCTGTCAGAAAA-TAC-3and antisense5-TAACCCGAGTAAAATGTGC-3; and for GAPDH,sense5-GACCCAGAATACCAAGTG-CAGATGTA-3and antisense5-CTGTTTCAGGAT-TTAAGGTTGGAGATT-3.2.6.TUNEL stainingThe TUNEL staining was carried out strictly according to the manufacturer's instructions(Roche Molecular Biochem-icals,Mannheim,Germany)to identify the apoptotic cells in parafﬁn sections.For each specimen,cells with positive nuclei staining from5microscopicﬁelds(original magniﬁ-cation×400)were counted.The total number of cardiomyo-cytes was also counted using light microscopy at a magniﬁcation of×400.Data were expressed as the mean number of TUNEL-positive cells among the total cardio-myocytes per microscopicﬁeld.2.7.Statistical analysesThe experimental data were analyzed by SPSS17.0 (SPSS,Chicago,IL).The results are expressed as mean±SD,and the Student t test was used for comparisons between2groups.Differences at different time points within groups were compared with repeated-measures analysis of variance(ANOVA).A2-tail value of P b.05was considered signiﬁcant.3.ResultsOf the16animals,4died in the post-ROSC groups during CPR;12survived.3.1.Hemodynamic statusBaseline hemodynamic measurements between the2 groups are shown in Fig.1.Neither MAP nor CO differed signiﬁcantly between the2groups(P N.05).After successful resuscitation,MAP was signiﬁcantly reduced at4and6 hours in the post-ROSC group compared with the sham-operated group.Cardiac output decreased signiﬁcantly Fig.1A,Mean aortic pressure.B,Cardiac output.The values are reported as mean(SD).The square represents the post-ROSC group;the circle represents the sham-operated group.⁎P N.05,#P b .05,and▲P b.01vs sham(1-way repeated-measures ANOVA).relative to baseline values at all time points after ROSC between the2groups(Fig.1).3.2.Ultrastructural changes in cardiomyocytesUnder transmission electron microscopy,nuclear degen-eration was typical with aberrant and crimped morphology at 24hours after cardiac resuscitation.Fine granular chromatin emerged in the normal nuclei(Fig.2A).Twelve hours after ROSC,partial nuclear chromatin condensation and periph-eral margins with a deep color were noted(Fig.2B).Twenty-four hours after ROSC,intracellular damage became more severe.The nucleus appeared aberrant and crimpled with condensed chromatin(Fig.2C).Micrographs of cardiomyo-cytes at24hours post-ROSC showed irreversible cell damage to apoptosis(Fig.2D).2041Apoptosis postresuscitation myocardial dysfunction3.3.Elevated Bcl-2/Bax and caspase 3apoptosis-related protein markersAs shown in Fig.3,Western blot analysis showed that the expressions of Bcl-2,Bax,and caspase 3progressively increased in the 12-hour post-ROSC group compared with the sham-operated group (P b .05).In particular,expressions of Bax and caspase 3were signi ﬁcantly up-regulated at 24hours after ROSC,whereas Bcl-2did not signi ﬁcantly increase but decreased at 24hours after ROSC.Furthermore,expression of Bcl-2/Bax was markedly reduced in the 12-and 24-hour post-ROSC groups compared with the sham-operated group (P b .05).3.4.Increased messenger RNA level of caspase 3The messenger RNA expression of the caspase 3gene in the 12-and 24-hours post-ROSC groups signi ﬁcantly increased compared with that in the sham-operated group (P b .01),but there was no difference between the 12-and 24-hour post-ROSC groups (P N .5)(Fig.4).3.5.TUNEL assay of cardiomyocyte apoptosisThe percentage of TUNEL-positive cardiomyocytes was signi ﬁcantly higher in the 24-hour post-ROSC group compared with that in the sham-operated group (P b .05).The brown nuclei in Fig.5are TUNEL positive stained.4.DiscussionCirculatory failure and myocardial dysfunction resulting from CA largely contribute to morbidity and mortality after initially successful CPR [10].In the present study,pigs that underwent CA and resuscitation all presented with severe postresuscitation myocardial dysfunction manifested by low MAP and decreased CO (Fig.1).Ultrastructural analysis within 24hours after ROSC showed swelling and disruption of the sarcoplasmic reticula,mitochondria,and sarcomeres in the cytoplasm and typical apoptotic alterations in the nuclei (Fig.2),which indicate myocardialdamage.Fig.2Cytoplasmic ultrastructure of the myocardium under an electron microscope.2042W.Gu et al.Although some experimental evidence has supported to some extent that apoptosis was not the main mechanism of postresuscitation myocardial dysfunction [11,12],these experiments differ from ours in several aspects.First,the experimental animals were different.Swine,whose heart structure is quite similar to that of humans,were used in the present study,whereas rodents,whose heart structure has certain differences from that of humans,were used in their study.Second,the de ﬁbrillation time was different.Electric de ﬁbrillation was performed after VF had been sustained for 8minutes in the present study but was performed after 4minutes in their study.Apoptosis may not be initiated if the ﬁbrillation time is too short.Third,the differences in our ﬁndings may be attributed to the longer observation interval in the present study.Apoptosis indices of the myocardium were detected 12and 24hours after VF in the present study and 4hours after VF in their previous study;4hours may be too short to detect the occurrence of apoptosis.The animals whose myocardial function recovered to normal and had no apoptosis were all alive in the previous study.However,it is not clear whether a similar mechanism develops in subjects who do not survive and succumb to severe myocardial dysfunction.Our results showed that,at 12and 24hours after ROSC,the expression of Bcl-2and Bax began to increase,Bcl-2/Bax began to decrease,and caspase 3activity simultaneously increased.The most principal pathways for apoptosis initiation is termed the Bcl-2/Bax –controlled pathway,which comprise antiapoptotic members such as Bcl-2and proapoptotic members such as Bax [13].When Bax is overexpressed in cells,death signal is enhanced.When Bcl-2is overexpressed,it heterodimerizes with Bax,and death is repressed.Thus,the ratio of Bcl-2to Bax serves as a rheostat to determine the susceptibility to apoptosis.Caspase 3is one of the executioner caspases and is responsible forapoptoticFig.3A,Western blots of expressions of Bax,Bcl-2,and active caspase 3proteins of myocardial tissue 12and 24hours after ROSC.B,Quanti ﬁcation of Bax,Bcl-2,and active caspase 3protein levels.C,Expressions of Bcl-2/Bax proteins of myocardial tissue 12and 24hours after ROSC.The values represent mean ±SE (n =6).▲P b .05and #P b .01vs sham (1-way repeated-measuresANOVA).Fig.4Messenger RNA expressions of caspase 3:quantitative data for caspase 3.Results are expressed as mean ±SEM.⁎P b .01and ▲P b .05vs sham.2043Apoptosis postresuscitation myocardial dysfunctioncell death,leading to internucleosomal DNA fragmentation [14].Besides promoting cell death,caspase 3activation can cleave structural and regulatory sarcomeric proteins,leading to contractile dysfunction [15].Thus,the possibility remains that caspase 3activation still plays a pathogenic role in postresuscitation myocardial dysfunction by altering con-tractile function.Cardiac arrest results in whole-body ischemia/reperfusion and represents the most severe shock state,during which delivery of oxygen and metabolic substrates is abruptly halted and metabolites are no longer removed.Cardiopulmonary resuscitation only partially reverses this process,achieving CO and systemic oxygen delivery that are much less than normal [5].Furthermore,the myocardium tissue destruction progresses even after circulation has been successfully restored.Cardiac myocytes are terminally differentiated,and once apoptotic or dead,they cannot be replaced.Consequently,the loss of contractile myocytes reduces the capability of the myocardium to sustain its pump function.In the present study,24hours post-ROSC,signi ﬁcant myocar-dial damage and apoptosis emerged,accompanied by increased protein expression of Bcl-2,Bax,and active caspase 3.These ﬁndings indicate that the initiation of the Bcl-2/Bax –controlled and caspase 3–mediated apoptoticsignal may thus contribute,at least,in part,to cardiomyocyte apoptosis after resuscitation.5.ConclusionCaspase 3–mediated and Bcl-2/Bax –controlled apoptotic pathway may be one of the main pathologic mechanisms of postresuscitation myocardial injury in a porcine model of CA.AcknowledgmentsWe sincerely thank Profs Ping Wei and Yilin Sun,who provided technical help for the analysis of myocardial specimens using electron microscopy and TUNEL assay.References[1]Curfman GD.Hypothermia to protect the brain.N Engl J Med2002;346:546.[2]Wang Yuan-Long,Zhong Jing-Quan,Tao Wen,et al.Initialdefibrillation versus initial chest compression in a 4-minuteventricularFig.5Changes in the number of TUNEL-positive myocytes in the experimental pigs 24hours after ROSC and in the sham-operated pigs.A,Representative TUNEL-stained sections from the sham-operated and 24-hour post-ROSC groups.The brown nuclei indicate TUNEL-positive nuclei.B,Percentages of cardiomyocytes with TUNEL-positive nuclei.Results are expressed as mean ±SEM.⁎P b .01vs sham;n =6per group.2044W.Gu et al.fibrillation canine model of cardiac arrest.Crit Care Med2009;37(7): 2250-2.[3]Nichol G,Thomas E,Callaway CW,et al.Regional variation in out-of-hospital cardiac arrest incidence and outcome.JAMA2008;300:1423-31.[4]Louise M,Malena L,Angela B,et al.Survival in out-of-hospitalcardiac arrest before and after use of advanced postresuscitation care:a survey focusing on incidence,patient characteristics,survival,and estimated cerebral function after postresuscitation care.Am J Emerg Med2010;28:543-51.[5]Neumar RW,Nolan JP,Adrie C,et al.Post-cardiac arrest syndrome:epidemiology,pathophysiology,treatment,and prognostication A consensus statement from the International Liaison Committee on Resuscitation(American Heart Association,Australian and New Zealand Council on Resuscitation,European Resuscitation Council, Heart and Stroke Foundation of Canada,InterAmerican Heart Foundation,Resuscitation Council of Asia,and the Resuscitation Council of Southern Africa);the American Heart Association Emergency Cardiovascular Care Committee;the Council on Cardio-vascular Surgery and Anesthesia;the Council on Cardiopulmonary, Perioperative,and Critical Care;the Council on Clinical Cardiology;and the Stroke Council.Circulation2008;118:2452-83.[6]Kern KB,Rhee KH,Raya TE,et al.Global myocardial stunningfollowing successful resuscitation from cardiac arrest.Circulation 1994;90(Suppl.I):I-5.[7]Abbate A,Biondi-Zoccai GG,Bussani R,et al.Increased myocardialapoptosis in patients with unfavorable left ventricular remodeling andearly symptomatic post-infarction heart failure.J Am Coll Cardiol 2003;41:753-60.[8]Wu JY,Li CS,Liu ZX,et al.A comparison of two types of chestcompressions in a porcine model of cardiac arrest.Am J Emerg Med 2009;27:823-9.[9]Han Yi,Li Chun-sheng,Zhi-yu Su,et al.Effects of norepinephrine onkidney in a Swine model of cardiopulmonary resuscitation.Am J Emerg Med2011;29:731-7.[10]Oddo M,Ribordy V,Feihl F,et al.Early predictors of outcome incomatose survivors of ventricular fibrillation and nonventricular fibrillation cardiac arrest treated with hypothermia:a prospective study.Crit Care Med2008;36:2296-301.[11]Radhakrishnan J,Ayoub IM,Gazmuri RJ.Activation of caspase-3may not contribute to postresuscitation myocardial dysfunction.Am J Physiol Heart Circ Physiol2009;296:H1164-74.[12]Song Fengqing,Shan Yi,Tang Wanchun,et al.Apoptosis is notinvolved in the mechanism of myocardial dysfunction after resusci-tation in a rat model of cardiac arrest and cardiopulmonary resuscitation.Crit Care Med2010;38(5):1329-34.[13]Borner C.The Bcl-2protein family:sensors and checkpoints for life-or-death decisions.Mol Immunol2003;39:615-47.[14]Arends MJ,Morris RG,Wyllie AH.Apoptosis.The role of theendonuclease.Am J Pathol1990;136:593-608.[15]Moretti A,Weig HJ,Ott T,et al.Essential myosin light chain as atarget for caspase-3in failing myocardium.Proc Natl Acad Sci U S A 2002;99:11860-5.2045Apoptosis postresuscitation myocardial dysfunction。

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Autophagy 1215Autophagy 5:8, 1215-1216; November 16, 2009; © 2009 Landes BioscienceAutophAgic puNctumAutophAgic puNctumPunctum to: Porrello ER, D’Amore A, Curl CL, Allen AM, Harrap SB, Thomas WG, Delbridge LM. Angiotensin II type 2 receptor antago-nizes angiotensin II type 1 receptor-mediated cardiomyocyte autophagy. Hypertension 2009; 53:1032-40; PMID: 19433781; DOI: 10.1161/HYPERTENSIONAHA.108.128488.Key words: autophagy, cardiac hyper-trophy, angiotensin II, AT 1 receptor, AT 2 receptorAbbreviations: AngII, angiotensin II; AT 1, AngII receptor type 1; AT 2, AngII receptor type 2; HHR, hypertrophic heart rat; NHR, normal heart rat;PLZF, promyelocytic zinc finger protein; PtdIns3K, phosphatidylinositol 3-kinase; RAS, renin-angiotensin system Submitted: 09/20/09Accepted: 09/22/09Previously published online:/journals/autophagy/article/10153Correspondence to: Lea M.D. Delbridge; Email: lmd@.auAutophagic activity increases in the heart in response to a variety of stresses including hypertension, ischemia and neonatal starvation. Constitutive autophagy plays an important role in the maintenance of cellular homeostasis in the heart, whereas unrestrained autophagic activity accentuates the maladaptive car-diac remodeling response to stress (e.g. hypertension) and may contribute to the pathogenesis of heart failure. A detailed understanding of the molecular mecha-nisms governing autophagy induction and autophagosome maturation is evolv-ing, but little is currently known about the extra- and intracellular cues that trig-ger autophagic induction in the heart. The renin-angiotensin system (RAS) is implicated in the pathogenesis of a num-ber of cardiovascular conditions includ-ing hypertension, cardiac hypertrophy, myocardial infarction and heart failure. We now provide the first link between angiotensin II (AngII) and autophagy regulation in the heart. We demonstrate that AngII increases autophagosome formation via the AngII type I (AT 1) receptor and that this response is consti-tutively antagonized by co-expression of the AngII type 2 (AT 2) receptor in neo-natal cardiomyocytes.The RAS is one of the most extensively studied and best characterized systems in biology. Numerous studies have examined the effects of the RAS on protein synthesis and turnover, cellular hypertrophy, pro-liferation, and apoptosis in many organ systems including the heart, vasculature, kidneys and brain. To date, most of the classical biological actions attributed toCardiomyocyte autophagy is regulated by angiotensin II type 1 and type 2 receptorsEnzo R. Porrello and Lea M.D. Delbridge*Department of Physiology; University of Melbourne; Victoria, AustraliaAngII, including cellular hypertrophy, proliferation and apoptosis, have been attributed to the AT 1 receptor subtype. AT 1 receptor signaling mechanisms are well characterized and involve coupling to the G αq/11 family of G proteins. In con-trast, the signaling pathways and biologi-cal functions associated with AT 2 receptor activation are ambiguous. Some studies suggest that the AT 2 receptor may act as an antagonist of AT 1-mediated signaling, but there are also reports of synergistic inter-play between these two receptor subtypes. Interestingly, autophagic activity is com-monly increased in the heart under condi-tions where the RAS is upregulated (e.g., hypertension, chronic ischemia, heart failure), suggesting a potential mechanis-tic link between AngII and autophagic induction.We have previously used AT 1 and AT 2 receptor-expressing adenoviruses in a neo-natal rat cardiomyocyte culture model system under in vitro conditions which allow quantitative modulation of recep-tor expression levels, to reveal synergistic interplay between AngII receptor sub-types in cardiomyocyte growth regula-tion. Both AT 1 and AT 2 receptors exert pro-hypertrophic effects in neonatal myo-cytes when overexpressed, but only AT 1-mediated effects are AngII-dependent, suggesting that the AT 2 receptor is consti-tutively active. In the present study, we use the same in vitro conditions to show that AngII also exerts a pro-autophagic effect mediated via the AT 1 receptor subtype. A novel anti-autophagic role for the AT 2 receptor subtype was identified, involving a constitutive, nonligand regulated mech-anism. Apoptotic activity was not affected1216 Autophagy Volume 5 issue 8in cardiomyocytes expressing either the AT 1 or AT 2 receptor, distinguishing the autophagy and apoptosis induction path-ways. We further tested the pathophysi-ological significance of AT 1 receptor stimulation on cardiomyocyte autophagy in a genetic model which exhibits cardiac growth abnormality, the Hypertrophic Heart Rat (HHR). Neonatal HHR cardi-omyocytes were more susceptible to AT 1-induced autophagy in vitro. However, this increased AT 1-mediated autophagic induction in the HHR was completely suppressed by co-expression of the AT 2 receptor. Collectively, these findings indi-cate that the AT 1 and AT 2 receptor sub-types reciprocally regulate cardiomyocyte autophagy.While the signaling pathways respon-sible for the anti-autophagic effects asso-ciated with AT 2 receptor activation have not yet been elucidated, our studies are consistent with a phosphatidylinositol 3-kinase (PtdIns3K)-dependent mecha-nism. It is well established that insulin sig-naling inhibits autophagy and this effect is mediated at least in part through acti-vation of class I PtdIns3K. In the heart, class I PtdIns3K signaling is associated with adaptive hypertrophy in response to physiological stimuli such as postnatal growth and exercise and is thought to be ‘cardio-protective.’ Although AT 2 signal-ing mechanisms are controversial, we have demonstrated that the prohypertrophic effects of AT 2 receptor stimulation in cardiomyocytes are PtdIns3K-dependent. This is consistent with a previously iden-tified mechanism involving coupling of the AT 2 receptor C terminus with the promyelocytic zinc finger protein (PLZF) transcription factor, which increases tran-scription of the p85α regulatory subunit of class I PtdIns3K. We propose that the pro-hypertrophic and anti-autophagic actions of the AT 2 receptor in cardiomyocytes are dependent on class I PtdIns3K activation.Autophagy is activated shortly after birth in neonatal tissues, including the heart. Neonatal autophagy is essential for survival because mice deficient in the autophagy-related genes Atg5 or Atg7 die within 1 day after birth. Autophagy is spec-ulated to have several important roles in the neonate including recycling amino acids in response to starvation, glycogen degra-dation, and programmed cell remodeling during the fetal-to-neonatal transition and in response to physiological oxidative stress that occurs at birth. Our work has shown that HHR rats, which are genetically pre-disposed to developing cardiac hypertro-phy in adulthood, display a heightened sensitivity to AT 1-induced autophagic vac-uolization during the neonatal period. We have previously demonstrated that HHR neonatal hearts are abnormally small at birth and contain fewer myocytes than their Normal Heart Rat (NHR) controls. HHR neonatal hearts are characterized by increased expression levels of the AT 2 receptor and PtdIns3K/Akt, suggesting that where there is an underlying disease predisposition for early myocyte deple-tion (i.e., in the HHR), the AT 2-mediated suppression of cardiomyocyte autophagy may be recruited as a preservation strat-egy to repress unrestrained cell loss in the neonatal heart. While further studies are required to determine the precise roles of AngII receptors and PtdIns3K signaling in autophagy modulation in the neonatal heart in vivo, we hypothesize that height-ened activation of the AT 2 receptor and ‘physiological’ growth signaling pathways (i.e., PtdIns3K/Akt) reduce autophagic cell losses and promote cardiac growth in the neonate.In summary, we have provided the first direct evidence of a link between AngII signaling and autophagy induction. The current finding that the AT 2 receptor is anti-autophagic could also lead to novel therapeutic strategies to enhance cell sur-vival in congenital and adult autophagic cardiomyopathies. Given the X chromo-some location of the AT 2 receptor gene (AGTR2), the potential role of this recep-tor in X-linked autophagic vacuolar car-diomyopathies should be investigated.。