Multiple myeloma (MM) is a plasma cell malignancy. Novel therapies have improved outcomes in MM patients; however, MM still remains incurable. MM cells present genomic instability, whose molecular basis is not fully understood. Recently, it has been reported that the DNA damage response (DDR) may influence genomic changes and drug resistance in MM. An abnormal DNA repair function may provide an alternative explanation for genomic instability. In this review, we show an overview of the DNA repair pathways and discuss the role of the tumor microenvironment in governing the DNA repair mechanisms. The tumor microenvironment factors, such as hypoxia and inflammation, and chemotherapeutic agents profoundly influence the DNA repair pathways in MM.Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms. Multiple myeloma (MM), which accounts for 1% of all cancers and approximately 10% of all hematologic malignancies, is characterized by the clonal proliferation of plasma cells . The devMM cells present genomic instability , whose mGenomic stability is important for ensuring the inheritance of correct genetic information. The occurrence of genomic instability can result in harmful consequences, which might lead to severe diseases, such as cancers. Endogenous and exogenous environmental factors can act as DNA-damaging agents, causing genetic mutations ,24. EndoMost cancers are characterized by genomic instability ,28,29, aIGH locus on chromosome 14 and one of the five recurrent translocation partners on chromosomes 4, 6, 11, 16, and 20. Five chromosomal partners account for the majority of primary IGH translocations: 11q13 (CCND1), 6p21 (CCND3), 4p16 (FGFR3 and NSD2), and 16q23 (MAF) [MYC, KRAS, NRAS, and TP53, which are involved in the DDR. Secondary events are detected in the late stage of the disease and affect its progression [MM patients are broadly grouped into hyperdiploid or non-hyperdiploid groups, depending on the number of chromosomes present in the MM cells. Hyperdiploid tumors are characterized by trisomies of one or more of the odd-numbered chromosomes 3, 7, 9, 11, 15, or 17, while the majority of non-hyperdiploid tumors display a translocation involving the 23 (MAF) ,38. Thes23 (MAF) . These sgression ,40. Amongression . The comgression ,42.PARP1), RAD51, PCNA, OGG1, XPC, BRCA1, ERCC1, BARD1, and TP53BP1, were associated with the outcome and overall survival [Current treatment modalities of MM include alkylating agents, PIs, and anthracyclines, all of which induce excessive DNA damage . DNA-damsurvival . These gsurvival . Recent survival ,47,48. Isurvival ,50. In csurvival . In the survival . Doxorubsurvival . This chsurvival  and ROS survival .BRCA1/BRCA2-mutated cancers [DNA damage requires effective DNA repair capacity, which may be limited in tumor cells. Compared to normal cells, cancer cells present a higher accumulation of DNA damage and replication stress (RS) due to faulty cell cycle checkpoint activation ,25. Sour cancers . Thus, D cancers ,61. NextThe DDR refers to a network of intracellular pathways that sense and resolve damaged DNA, but can also be a major source of genomic instability, particularly when cell death pathways are deactivated. This mechanism involves DNA repair, activation of cell cycle checkpoints, transcriptional modulation, apoptotic pathways, senescence, and DNA damage tolerance  62,63,6,65,66,67Molecular “sensors” of DNA damage lesions, such as DSBs, are early players in the DDR. The MRE11/RAD50/NBN (MRN) complex is a DSB sensor in the ATM pathway. This complex recruits ATM to DSB sites, and the interaction with NBN activates ATM, resulting in the phosphorylation of target proteins, such as the histone variant H2AX, which leads to γ-H2AX. Ku (comprising the KU70/KU80 protein heterodimer) is another DNA damage sensor that binds to DSBs and recruits DNA-PKcs. Autophosphorylation of DNA-PKcs serves as a platform for the subsequent recruitment of classical non-homologous end joining (NHEJ) proteins. RPA, a sensor in the ATR pathway, binds to ssDNA generated at the site of stalled replication forks. In turn, ATR-interacting protein (ATRIP) binds to the RPA–ssDNA complex to recruit ATR. The RAD9A/RAD1/HUS1 (9-1-1) complex is another DNA damage sensor in the ATR pathway. Regarding “transducers”, ATM/ATR and the downstream CHEK1/CHEK2 kinases are well-studied players in the DDR pathway. The DDR is controlled by ATM and ATR, both of which are activated by DNA damage and RS, but their functions differ. ATM is mainly implicated in the repair of DSBs, whereas ATR responds to various types of DNA damage that have in common the presence of ssDNA ,69. ATM MM cells present constitutive, ongoing DNA damage, as evidenced by the high number of γ-H2AX foci in their nuclei . FurtherMammalian cells have six major DNA repair pathways involved in the DDR: the base excision repair (BER), NER, and mismatch repair (MMR) pathways repair nucleotide lesions on ssDNA; the HR and NHEJ pathways are involved in DSB repair; and the Fanconi anemia (FA) pathway repairs ICL lesions in co-operation with the NER and HR pathways ,24,25.Macrophages in the tumor microenvironment release proinflammatory cytokines and lead to genomic instability through the production of ROS and reactive nitrogen species. Such chemicals can attack DNA, leading to adducts that impair base pairing and/or block DNA replication, base loss, or DNA SSBs . The latSSBs occur more than 10,000 times per mammalian cell each day, being the most common type of DNA damage in cells . These l2O2 and HOCl, and ROS-generated injuries, such as those caused by UV radiation. Increased expression of APEX1 correlates with an increase in its endonuclease and redox activities [APEX1 sensitizes them to melphalan treatment [APEX1 in RKO cell lines using shRNA reduces cell proliferation. In addition, inhibition of APEX1 in CHO cells under acidic conditions increases oxidative DNA damage related to increased intracellular ROS [APEX1 gene or protein by shRNA or an APEX inhibitor, respectively, reduces the proliferation of the MM cells. Noteworthy, APEX1 contributes to the dysregulation of other DNA repair pathways, such as HRR in MM cells [APEX1 gene are also associated with shorter survival of patients with MM [APEX2 genomic alterations are estimated to occur with a frequency of ~17% in 14 cancer types, based on data available at The Cancer Genome Atlas. APEX2 expression is upregulated in several cancers, including kidney, breast, lung, liver, and uterine cancers. Furthermore, APEX2 mRNA levels positively correlated with PCNA, APEX1, XRCC1, PARP1, CHEK1, and CHEK2 across these tumor tissues. Both APEX1 and APEX2 are upregulated in MM cell lines and samples from patients with MM [APEX1 and APEX2 also inhibits HR activity. Based on this, upregulation of BER in cancers may represent an adaptive survival response [APEX1 is a multifunctional protein involved both in the BER of DNA lesions and regulation of gene expression as a redox co-activator of different transcription factors, such as EGR1, TP53, NFKB1, HIF1A, PAX5, PAX8, and AP-1 ,80. APEXtivities ,79. APEXtivities , ovariantivities , and protivities  cancer ctivities ,85, and tivities ,87. Melpreatment ; also, dular ROS . Inhibit with MM . In addi with MM . APEX2 g with MM , and in  with MM . shRNA-mresponse ,83.PARP1 is a nuclear DNA-dependent ADP-ribosyltransferase that catalyzes the poly ADP-ribosylation (PAR) of nuclear proteins. PARP enzymes are broadly involved in the cellular response to DNA damage . PARP1 hBRCA1- or BRCA2-deficient breast and ovarian cancers [PARP1 knockdown or olaparib was shown to result in significant inhibition of tumor growth using xenografts of human MM cells [High expression of PARP1 is associated with a higher grade of some cancers, including gastric, ovarian, and breast cancers ,96,97. T cancers ,103. In  cancers . OlapariMM cells .The NER pathway removes the bulky DNA lesions induced by UV radiation, smoking, environmental mutagens, and alkylating agents . This DNThe MMR pathway corrects replication errors, such as mismatched bases and insertion/deletion loops. MMR recognizes and repairs erroneous insertion, deletion, and misincorporation of bases that can arise during DNA replication, recombination, and repair of DNA damage. This pathway recruits DNA repair proteins to damaged sites and increases the accuracy of the DNA replication . MMR proDSBs are created by genotoxic agents commonly used in the treatment of cancer. Radiation, oxidative free radicals, topoisomerase inhibitors, and DNA replication inhibitors can all lead to DSBs ,116. FaiHR is widely used to ensure genomic stability when a homologous template is available for repair. The MRN complex plays an important role in this DDR pathway, regulating both signaling and damage responses. MRN recognizes DSBs and activates ATM and ATR signaling, which orchestrate cell cycle progression and damage responses ,118,119.NHEJ is another major pathway that repairs DSBs in mammalian cells. NHEJ mediates the direct re-ligation of the broken DNA molecule ,122. ThiBRCA1 and BRCA2 mutations result in increased sensitivity to DNA-damaging agents [BRCA2 that corresponds to a Lys 3326 Stop substitution predicted to cause the loss of the final 93 amino acids of the BRCA2 protein. This seems to be a genetic basis for at least a small fraction of MM cases [PARP, RAD51, MUTYH, OGG1, PCNA, TPMT, and XPC were associated with disease progression in patients with MM who underwent HDM treatment and ASCT [Defects in HRR are observed in some cancers, including breast and ovarian cancers ,127. Herg agents . These og agents . In contMM cases . MoreoveMM cases . RecombiMM cases . High acMM cases . The acqMM cases . A cliniMM cases . Furtherand ASCT .TP53 double knockout mice develop lymphomas with IGH/MYC translocations and aneuploidy [ERCC1, ERCC5, or XRCC5 (encoding KU80). A longer OS was associated with SNPs in ERCC1 and XRCC5 [XRCC4, XRCC6 (encoding KU70), DCLRE1C, and LIG4 genes and risk of MM development has also been reported [XRCC5 and DCLRE1C genes was associated with poor prognosis in patients with MM [RAD51, TP53BP1, and XRCC4, and enhanced γ-H2AX. Conversely, NSD2 overexpression is associated with increased DNA repair efficiency [A large number of murine studies have demonstrated the critical role of NHEJ core proteins as well as HR mediators in maintaining genomic integrity. NHEJ core protein/euploidy . NHEJ aceuploidy . Howevernd XRCC5 . In addireported ,136. Hig with MM , and upr with MM . NSD2 isficiency . Given tAlt-NHEJ is crucial for genomic instability and survival of MM cells. LIG3 mRNA expression in MM patients correlates with shorter survival and even increases with more advanced stage of disease. Higher LIG3 protein levels were detected in AMO1 bortezomib-resistant cells as compared to the AMO1 bortezomib-sensitive cells. LIG3 knockdown strongly increased DNA damage and finally inhibits MM cell growth in vitro and in vivo. Further, expression of miR-22 impairs growth and survival of MM cells, via targeting LIG3 .RAD51C, that make up the FA pathway. This pathway orchestrates the detection and removal of ICLs through the combined actions of NER and HR, with minor contributions of other DNA repair pathways [The FA pathway is a DNA damage-activated pathway required for the repair of ICLs. FA is a recessively inherited disorder characterized by bone marrow failure, developmental abnormalities, inability to repair ICLs, and a high incidence of malignancies . ICLs arpathways ,141. Melpathways , and deppathways . NF-κB tNSD2, RIF1, and XRCC5 (KU80), and two with good prognostic value: PNKP and POLL); six encoding HR proteins ; three encoding FA proteins with bad prognostic value ; eight encoding NER proteins ; two encoding MMR proteins with bad prognostic value (EXO1 and MSH2); and one encoding a BER protein with bad prognostic value (LIG3). These findings demonstrate that these DNA repair pathways are important mechanisms in myeloma cells for melphalan treatment [Gene expression profile analyses of patients treated with HDM and ASCT revealed the prognostic value of genes involved in the NHEJ, HR, FA, NER, MMR, and BER pathways . Seventereatment .DNMT1 was reported to show an increasing median expression from the controls (101.8) to MM cells (143.3) and myeloma cell lines (1101) [Cancer cells exhibit a high level of epigenetic modifications, which modulate expression of genes, as well as cellular processes and tumorigenesis. Epigenetic changes, such as aberrant DNA methylation and histone modification profiles, and abnormal non-coding RNA expression are considered as important in contributing to the pathogenesis of MM . DNA mets (1101) . The cyts (1101) ,149. Firs (1101) .HDACs are involved in many biological processes, such as apoptosis, senescence, differentiation, and angiogenesis. Progression-free survival was significantly shorter in MM patients with higher levels of class I HDAC expression. Panobinostat, a pan-HDAC inhibitor, in combination with a PI and dexamethasone, has improved survival in relapsing/refractory MM patients . The exaMicroRNAs (miRNAs) are small non-coding RNAs of 19–25 nucleotides, which regulate the gene expression by degrading or inhibiting the translation of target mRNAs, primarily via base pairing to partially or fully complementary sites in the 3′ UTR. Deregulated miRNA expression in MM patients has been associated with tumor progression, prognosis, and drug response ,152. Und2*) free radicals and nonradical molecules, such as H2O2, which are less reactive than the majority of ROS [Inflammation is a protective mechanism of the body against invading pathogens or tissue damage. However, persistent inflammation leads to a plethora of diseases, including autoimmune and metabolic disorders, atherosclerosis, cardiovascular diseases, Alzheimer’s disease, and cancer ,157,158.y of ROS . These ay of ROS . ROS-indy of ROS ,167. ROSy of ROS . Downstry of ROS . ATR–CHKy of ROS . Furthery of ROS . Increasy of ROS . MM celly of ROS . Expressy of ROS . Cyclin y of ROS . An incry of ROS . In cancy of ROS . Bortezoy of ROS . Furthery of ROS . Hence, y of ROS ,177. TheVLA4, CXCR4, IL6, and TGFB in MM cells [Hypoxia is a non-physiological level of oxygen tension that is common in the majority of malignant tumors and a common feature of the BM microenvironment. A hypoxic tumor microenvironment plays a significant role in cancer progression and therapy resistance ,179. HIFMM cells . miR-210MM cells . miR-210As for ROS, hypoxia is known to increase its production in cells. ROS generated at mitochondrial complex III stabilize HIF1-α during hypoxia ,183,184.Cancer has its cellular changes contributing to tumor growth and cell proliferation. Both glycolysis and glutaminolysis result in energy production and nucleotide synthesis. Enhancement of glycolysis and glutaminolysis are found in MM cells compared to normal cells. Hexokinase II (HKII), which is one of four HKs isoforms, is a widely overexpressed enzyme in several cancers, including MM . 3-bromoEZH2 is a histone methyltransferase acting primarily at H3K27, where it catalyzes the conversion to a tri-methylated mark (H3K27me3), which is overexpressed in multiple myeloma. High EZH2 mRNA expression in MM patients is associated with poor outcomes and high-risk clinical features. This posttranslational modification by EZH2 enhances H3K27 trimethylation, an important determinant in NHEJ repair. Histone H3K27 methylation modulates the dynamics of FANCD2 on chromatin to facilitate NHEJ and genome stability ,192.Furthermore, recent studies have shown the interplay between cellular metabolism and the DDR in cancer. Cancer cells show an alteration of the DDR coupled with modifications in cellular metabolism. Glutamine, aspartate, and other nutrients are essential for de novo nucleotide synthesis, which dictates the availability of the nucleotide pool, and thereby influences DNA repair and replication. The increased proliferation of cancer cells can deplete the dNTP pools, causing replication stress and, consequently, DSBs. Imbalances in nucleotide pools cause misincorporations during replication, which can lead to mutations .Repair of DNA is critical for normal cellular function. However, tumor cells use DNA repair pathways to develop resistance to chemotherapy. High expression of DNA repair genes is frequently associated with poor prognosis. Therefore, inhibiting the DNA repair pathway may overcome this drug resistance and poor prognosis. Many inhibitors targeting the DNA repair pathways  and cell-cycle checkpoints (CHEK1 and CHEK2) have now been developed and might be useful to induce MM cells apoptosis in combination with DNA damage-inducing drugs .The tumor microenvironment and abnormal DNA repair function affect genetic instability in MM. Moreover, tumor microenvironment factors, such as hypoxia and inflammation, as well as chemotherapeutic agents, profoundly influence the DNA repair pathways. MM cells with dysregulated DNA repair pathways are thus dependent on the remainder functional DNA repair mechanisms. As demonstrated in this review, the relationship between tumor microenvironment and the DDR is complex; however, novel strategies in MM may be possible based on changes in DDR in MM.


Following its discovery in Wuhan, China, in December 2019, COVID-19 has attained pandemic status in mere months. It is caused by SARS-CoV-2, an enveloped beta coronavirus. This infection causes a prothrombogenic state by interplay of inflammatory mediators, and endothelial, microvascular, and possible hepatic damage and tissue tropism of the virus. This leads to frequent pulmonary and cerebral thromboembolism as well as occasional involvement of other organs. We present a 71-year-old man who initially presented with 2 weeks of fever, cough, and shortness of breath and was diagnosed with COVID-19 pneumonia. He required readmission due to worsened hypoxia and was later found to have left renal artery thrombosis with left kidney infarction, associated with an ascending aortic thrombus. He was anticoagulated and recovered uneventfully. We suggest that physicians have a high degree of suspicion to diagnose and manage the novel manifestations of this disease. He was diagnosed with COVID-19 multifocal pneumonia and required readmission due to worsening hypoxia. During his second hospitalization, he had left flank pain and was found to have left renal artery thrombi with left kidney infarction associated with an ascending aortic thrombus. He was anticoagulated with intravenous heparin infusion and transitioned to oral apixaban on discharge after an uneventful recovery.After its discovery in Wuhan, China, in December 2019, COVID-19 has become a pandemic in just months. COVID-19 commonly presents with fever (88.7%), cough (67.8%), fatigue (38.1%), sputum production (33.7%), shortness of breath (18.7%), and myalgia (14.9%), whereas nausea or vomiting (5%) and diarrhea (3.8%) are uncommon.A 71-year-old man with no significant past medical history presented with 2 weeks of fever, cough, shortness of breath, and chest discomfort with coughing in early April 2020 at a tertiary care hospital in New Brunswick, NJ. He was exposed to his daughter who was diagnosed with COVID-19 pneumonia a week before he fell ill. On presentation, he was afebrile at 99.7°F (37.6°C), blood pressure was 134/67 mmHg, tachycardic with a pulse of 102/minute, tachypneic at 34 breaths/minute, and hypoxic at 78% breathing room air. Examination was notable for faint crackles over all lung fields. Supplemental oxygen via nasal cannula (NC) was initiated at 4 L/minute with improvement of saturation to 94%. Initial laboratory investigations revealed absolute lymphopenia and elevated D-dimer, lactate dehydrogenase (LDH), ferritin, and C-reactive protein (CRP) . His cheHowever, he was readmitted 2 days later with worsened hypoxia. Vitals revealed he was afebrile at 98.9°F (37.1°C), blood pressure was 134/80 mmHg, tachycardic with a pulse of 112/minute, tachypneic at 34 breaths/minute, and hypoxic to 88% on 4 L/minute of oxygen via NC. Examination still noted bilateral crackles in all lung fields. Repeat investigations revealed positive COVID-19 test, persistent absolute lymphopenia, and further elevation of D-dimer, LDH, ferritin, and CRP . A repea2 This manifests with a prominent elevation of D-dimer and fibrin/fibrinogen degradation products, whereas abnormalities in other coagulation parameters and platelet counts are relatively uncommon in the initial phases.7 Later, the complement pathway and cytokines like interleukin-6 (IL-6) are also involved, further activating the procoagulant pathway.4 In addition, SARS-CoV-2 has been detected in the pharynx, heart, liver, brain, kidney, and blood.8 It might infect endothelial cells via cluster of differentiation 13 (CD13) and angiotensin converting enzyme 2 (ACE2) receptors, resulting in diffuse endothelial inflammation and microvascular damage, which can result in widespread thrombosis; however, endothelial infection has been contested.11 Other proteins, such as transmembrane serine protease 2, sialic acid receptors, extracellular matrix metalloproteinase inducer (CD147 or basigin), and cathepsins B and L are all known as possible mediators facilitating SARS-CoV-2 entry in host cells, and are also expressed in endotheliocytes.7 This prothrombotic state may be further potentiated by an underlying viral hepatic dysfunction/damage with decreased antithrombin production.6 This results in disruption of the natural antithrombotic state.4 Occasionally, because of unclear mechanisms, the inflammatory response is severe, resulting in a dysregulated release of mediators like IL-6 (“cytokine storm”), resulting in magnified pathophysiology. Antiphospholipid antibodies (APLAs) have also been detected in some COVID-19 patients, which may lead to thrombotic events similar to antiphospholipid syndrome.12 Hence, we can infer how the interplay between the aforementioned factors leads to COVID-19–induced thromboembolic complications.COVID-19 is caused by SARS-CoV-2, an enveloped beta coronavirus. Our knowledge regarding the pathophysiology of SARS-CoV-2 infection is still evolving. The SARS-CoV-2 infection activates an inflammatory response, releasing inflammatory mediators and activating the endothelium and hemostatic systems, with a concomitant increase of the von Willebrand factor and tissue factor.14 Risk factors include male gender, hypertension, peripheral arterial disease, hyperlipidemia, and smoking.15 Etiology includes embolization, in situ thrombosis of the renal artery, renal artery dissection, fibromuscular dysplasia, hypercoagulability, and following endovascular interventions.15 Most patients have abdominal or flank pain, and, occasionally, fever, nausea, uncontrolled hypertension, hematuria, or acute renal failure. The rarity of the condition along with the nonspecific presentation makes diagnosis challenging.15 Imaging is essential for diagnosis, with contrast-enhanced computed tomogram (CT) being preferable; however, angiography remains gold standard.13Renal infarction is relatively rare, with an incidence of 0.1–1.4%.16 It has also been found in a kidney transplant recipient without any arterial or venous thrombi.17 In another patient, the renal infarct was associated with renal artery thrombosis; however, the renal function remained normal, similar to our patient.18 Severe infections with viruses such as influenza, pandemic H5N1/H1N1 influenza, parvovirus B19, cytomegalovirus, Epstein–Barr virus, hepatitis C, dengue, and chikungunya have been associated with thromboembolism and associated renal dysfunction.23 COVID-19–associated aortic thrombosis has been found in association with PE, acute limb ischemia, DVT, acute mesenteric ischemia, and stroke.25 Severe and fatal cerebral venous thrombosis has also been noted.26Before, in a series of two cases with renal infarcts in patients with COVID-19, one presented with acute kidney injury (AKI), whereas the other had AKI with associated bowel ischemia.31 Corticosteroid therapy can lower mortality.32 Prone positioning of awake, non-intubated patients may improve oxygenation.34 Antiphospholipid antibodies were not tested in our patient. For anticoagulation, the guidelines proposed by the anticoagulation forum, the American College of Cardiology, and the International Society of Thrombosis and Haemostasis were followed.5 For noncritically ill hospitalized patients, a standard regimen of venous thromboembolism prophylaxis is recommended.5 For critically ill patients, higher doses are recommended based on expert opinion.3 Regimens may include enoxaparin 0.5 mg/kg subcutaneous twice daily, heparin 7,500 units subcutaneous three times daily, or low-intensity heparin infusion.3 This was instituted in our patient’s second hospitalization. For the treatment of arterial thrombosis, the patient was put on a heparin infusion.15 On discharge, a 45- to 90-day course of therapeutic anticoagulation is recommended depending on risk.5In our patient, we hypothesized that severe COVID-19 pneumonia induced a prothrombotic state, resulting in the ascending aortic thrombosis. This thrombus likely embolized, or there could have been an isolated neo-thrombosis in the renal artery as well. This thromboembolic pathology manifested with renal infarction. We treated our patient with hydroxychloroquine and later lopinavir–ritonavir as per existing institutional protocols; however, this regimen was later deemed controversial in the literature.In conclusion, the thromboembolic consequences of COVID-19 can occasionally result in rare complications such as aortic thrombosis and renal infarction. During a pandemic, we advise that the physicians should maintain a high degree of clinical suspicion to diagnose rare manifestations of this novel disease for timely management.


How moxibustion improves chronic heart failure is extremely complex and still unclear. This study aimed to explore whether moxibustion inhibits autophagy and reduces inflammation by regulating mTOR expression to induce myocardial protective effects and alleviate symptoms associated with chronic heart failure. Echocardiography was used to detect cardiac function and cardiac structure of rats, including heart rate (HR), left atrium diameter (LA), left ventricular diameter (LV), left ventricular posterior wall (LVPW), interventricular septum (IVS), ejection fraction (EF), and fractional shortening (FS). BNP and NT-pro BNP levels were measured by enzyme-linked immunosorbent assay (ELISA). Autophagy-associated protein (ATG) genes and mTOR were detected by PCR. The expression of mTOR and phosphorylated-mTOR was detected through western blotting of proteins from myocardial tissue samples. The left ventricular inflammatory response was detected by immunohistochemistry and included ICAM-1, VCAM-1, MMP-2, and MMP-9 expression. The relationship between autophagy and inflammation was analyzed by correlation analysis. The results from echocardiography and ELISA showed that moxibustion could significantly improve heart function and structure. Western blot and PCR results showed that moxibustion treatment elevated mTOR expression. Further, moxibustion could inhibit autophagy and regulate the expression of key autophagy-related genes, including Vps34, ATG3, ATG5, ATG7, ATG12, and ATG13. By contrast, rapamycin could partially reduce the effects of moxibustion. Immunohistochemistry results indicated that moxibustion could reduce myocardial inflammation. Moreover, there was a positive correlation between autophagy and inflammation. Moxibustion can protect cardiac function in rats with heart failure, possibly inhibiting excessive autophagy of cardiomyocytes and reducing inflammatory reactions through the elevation of mTOR expression. Chronic heart failure (CHF) is the ultimate outcome of different cardiovascular diseases, which represents a public health problem requiring urgent attention all over the world due to its high morbidity, mortality, and rehospitalization rates , 2. The Autophagy is a protective mechanism through which cells strive to survive under adverse conditions. Conversely, excessive autophagy may lead to accelerated cell death, resulting in a variety of acute and chronic diseases , 5. BaseCHF is associated with inflammation , which iin vivo animal model. Our objective was to explore the molecular mechanisms of moxibustion in the treatment of CHF.Moxibustion, a treatment derived from traditional Chinese medicine (TCM), has been widely applied for treating various diseases, including cardiovascular disease, and exhibits good therapeutic effects , 21. CurThis study was performed in accordance with the animal welfare regulations for the Anhui Medical University . A total of 90 male Sprague-Dawley (SD) rats weighing 200–300 g were obtained for this study, and all were maintained under specific-pathogen-free (SPF) conditions . All experimental procedures were approved by the Animal Ethics Committee of the Anhui University of Chinese Medicine. The rats were kept in separate cages (Kangwei IR60), with free access to air, food, and water. During the experiment, the handling of animals was in accordance with the guidelines for the handling of Experimental Animals issued by the Ministry of Science and Technology. Every effort was made to minimize the pain and discomfort to the animals; animals were euthanized using 3% pentobarbital sodium at a dose of 30 mg/kg intraperitoneal injection at the end of the experimental procedures.To explore the effects of moxibustion on cardiac structure and function in rats with CHF, rats were randomly divided into three groups: the control group, model group (Adriamycin- (ADR-) induced) and moxibustion group (ADR plus moxibustion treatment). We observed changes in the heart rate (HR), left atrium diameter (LA), left ventricular diameter (LV), left ventricular posterior wall (LVPW), interventricular septum (IVS), ejection fraction (EF), and fractional shortening (FS) via echocardiographic evaluation of cardiac structure and function. Serum BNP and NT-pro BNP levels were assayed using ELISA kits . To assess whether moxibustion mediates the autophagy-inflammation effects on CHF through elevating mTOR expression, rats were randomly divided into five groups: the control group, model group (ADR-induced CHF), rapamycin [RAPA] + moxibustion group (ADR plus RAPA-moxibustion treatment), RAPA group (ADR plus RAPA-treatment), and moxibustion group (ADR plus moxibustion-treatment). We observed changes in mTOR expression in myocardial tissue, in expression of autophagy-related genes Vp34, ATG3, ATG5, ATG7, ATG12, and ATG13, and in inflammatory factors ICAM-1, VCAM-1, MMP-2, and MMP-9. We also examined the correlation between autophagy and inflammation.The rat model of CHF is based on the assumption of induction of myocardial toxicity by ADR . ADR wasIntraperitoneal injection of 3% pentobarbital sodium was used for anesthesia at a dose of 30 mg/kg, and the left ventricular cardiac functions were noted via echocardiography in the 2D B-mode and M-mode . HR, LA, LV, LVPW, IVS, EF, and FS were tested for at least three nonstop cardiac cycles. At the end of the measurements, all experimental rats were anaesthetized with sodium pentobarbital and euthanized to obtain blood and heart tissue samples.μL volume of supernatant was added to each well and was incubated for 2 h at room temperature and then biotin antibody was added to each well and incubated for 1 h. Next, horseradish peroxidase was added to the wells and incubated for an additional 30 min. Subsequently, the substrate reagent was added to each well. The absorbance at 450 nm was immediately detected by a microplate reader .Levels of serum BNP and NT-pro BNP were measured by enzyme-linked immunosorbent assay kits  according to the manufacturer's protocols. First, a 50 Myocardial tissues were extracted from the left ventricle, and protein was extracted from the homogenized tissue using precooled RIPA lysis solution  supplemented with 1% phenylmethylsulphonyl fluoride . The BCA  method was used to determine protein concentrations. To investigate the expression of mTOR and its phosphorylated protein (p-mTOR) in heart tissues, 50 mg of protein was separated by SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% skim milk. The membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used were anti-mTOR , anti-phospho-mTOR , anti-GAPDH , and IgG-HRP . The membranes were then incubated with the appropriate secondary antibody for 1 h at room temperature and exposed to ECL  in the darkroom at room temperature.μL . To analyze the cDNA of Vp34, ATG3, ATG5, ATG7, ATG12, and ATG13, PCR conditions were 15 s at 95°C for denaturation and 1 min at 55°C for annealing and extension. Each gene was amplified by 40 cycles. To determine the relative quantities of mTOR, Vp34, ATG3, ATG5, ATG7, ATG12, and ATG13 mRNA, GAPDH was used as the internal standard. The primer sequences are reported in We used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect mRNA expression of mTOR, Vp34, autophagy-related gene (ATG) 3 (ATG3), ATG5, ATG7, ATG12, and ATG13 in left ventricular tissues. The total RNA from each sample was extracted using Trizol Reagent , and then the RNA was retrotranscribed to cDNA using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix . The reaction volume was 20 μm thick sections. After deparaffinization in xylene, the tissues were subjected to antigen retrieval using 10 mM citrate-phosphate buffer (pH 6.0) and incubated in 3% H2O2 for 25 min. Then, the sections were blocked with 1% bovine serum albumin in PBS for 10 min. Following incubation with a prediluted biotinylated pan-specific universal secondary antibody  for 50 min at room temperature, sections were incubated overnight with mouse monoclonal ICAM-1 , VCAM-1 , MMP-2 , and MMP-9  antibody at 4°C. The tissues were washed with PBS, incubated in the streptavidin/peroxidase complex for 5 min and then washed with PBS again, before being washed with 3, 3′-diaminobenzidine  for 7 min. Sections were stained with hematoxylin for 3 min, then washed, and mounted. Images were captured using a fluorescent microscope .Samples from the left ventriculum were fixed in 4% paraformaldehyde at 25°C, embedded in paraffin, and cut into 3 R version 4 software .Pearson correlation coefficients were used to compare levels of ICAM-1, VCAM-1, MMP-2, MMP-9 with Vp34, ATG3, ATG5, ATG7, ATG12, and ATG13 in left ventricular tissues, respectively. Plots were generated using the statistical programming language p < 0.05, p < 0.01, and p < 0.001.Data are expressed as mean ± standard deviation (SD). For parametric data, the comparison of different groups was performed by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test for multiple comparisons. Statistical analyses were performed using GraphPad Prism version 6 software . All results were considered statistically significant at p < 0.01, Figures p < 0.01, Figures p < 0.01, Figures p < 0.01, Figures p < 0.01, Figures p < 0.05, p < 0.05, Figures After the 8-week experimental period, echocardiography revealed that the CHF rat model had been successfully established, based on evidence of the marked reduction in EF and FS values in the model group compared to the control group  and inflammatory factors , respectively (p < 0.001).Correlation analysis was performed between autophagy-associated genes (Vps34, and ATG3, ectively . The expMoxibustion is an important component of traditional Chinese medicine and is utilized to prevent and treat a variety of chronic diseases. It has been increasingly applied in health preservation and the effectiveness and safety of moxibustion for the treatment of different diseases have been evaluated. For example, a pilot controlled clinical trial concluded that moxibustion was effective in modulating heart rate variability over the long term . MoxibusIn our previous studies examining moxibustion for the treatment of CHF, we also found that moxibustion played a key role in myocardial protection by regulating the neuroendocrine-immune response, by inhibiting excessive autophagy, and by improving myocardial hypertrophy and cardiac functions –32. ConsAs a distinct phenomenon of eukaryotic cells, autophagy is characterized by degradation and recycling of intracellular biological macromolecules and damaged organelles , 36. AutSeveral key autophagy genes, including Vps34, ATG3, ATG5, ATG7, ATG12, and ATG13, are involved in the autophagy process . Vps34 aThe mechanisms involved in CHF are complex, and many are still unclear, although various theories have been proposed. According to the cytokine hypothesis, the progression of CHF is mediated by a variety of cytokines, which induce cardiomyocyte hypertrophy and eventually lead to poor ventricular remodeling . Some stAutophagy and inflammation are functionally interrelated and lead to the induction of autophagy-inflammatory reactions . Under nIn conclusion, the present study provides evidence supporting moxibustion treatment for improving cardiac structure and function of CHF. The underlying mechanism may involve the regulation of autophagy and enhanced anti-inflammatory responses by inducing mTOR expression.


Acute kidney injury requiring renal replacement therapy (RRT) is common among critically ill COVID-19 patients. In centers with available dialysis, low-flow ECCO2R (<500 mL/min) using RRT platforms could be carried out by dialysis specialists and might be an option to efficiently allocate resources during the COVID-19 pandemic for patients with hypercapnia as the main indication. Here, we report the feasibility, safety, and efficacy of ECCO2R using an RRT platform to provide either standalone ECCO2R or ECCO2R combined with RRT in four hypercapnic patients with moderate ARDS. A randomized clinical trial is required to assess the overall benefit and harm.Coronavirus disease 2019 (COVID-19)-associated acute respiratory distress syndrome (ARDS) is associated with high mortality. Lung-protective ventilation is the current standard of care in patients with ARDS, but it might lead to hypercapnia, which is independently associated with worse outcomes. Extracorporeal carbon dioxide removal (ECCOClinical Trial Registration:ClinicalTrials.gov. Unique identifier: NCT04351906. In tseverity . Acute k levels <00 mL/min mL/min) . Of note2R in combination with a CRRT platform as a standalone therapy or combined with CRRT for ARDS patients with refractory hypercapnia  secondary to confirmed COVID-19 to effectively decrease CO2 levels and enhance lung-protective ventilation.We report results of a single-center study evaluating the feasibility and safety of ECCOhttp://clinicaltrials.gov (Identifier: NCT04351906). Patients were sedated with fentanyl, midazolam, and propofol. Other medications, including antibiotics, fluids, catecholamines, and transfusions, were left to the discretion of the attending physician.COVID-19 was diagnosed according to the World Health Organization (WHO) guidance . All pat2/FiO2) <300 mmHg with positive end-expiratory pressure >5 cmH2O on mechanical ventilation expected to last >24 h; hypercapnia >55 mmHg with or without metabolic acidosis (pH < 7.3); bilateral opacities on chest imaging; with or without AKI requiring dialysis. Exclusion criteria were age <18 years, pregnancy, patients with decompensated heart failure or acute coronary syndrome, respiratory acidosis with persistent partial pressure of blood carbon dioxide (PaCO2) levels >80 mmHg, acute brain injury, severe liver insufficiency (Child–Pugh scores > 7) or fulminant hepatic failure, decision to limit therapeutic interventions, catheter access to a femoral vein or jugular vein impossible, and pneumothorax.In-patients ≥18 years of age with confirmed COVID-19 admitted to the University Hospital Giessen and Marburg, Giessen Medical Center, were enrolled in the feasibility study. Inclusion criteria were mild-to-moderate ARDS according to the Berlin definition , 100 mmH2R was provided using a polymethylpentene, hollow fiber, gas-exchanger membrane , a labeled and certified European device to be used in conjunction with multiFiltrate CRRT platforms  for combined respiratory and renal support. The manufacturer determined the multiECCO2R membrane's maximum duration to be 72 h. A 13.5-Fr dual lumen hemodialysis catheter  was percutaneously inserted under in the femoral vein. Sweep gas flow was set at a gas/blood flow ratio of 15:1. Data were collected before starting ECCO2R (baseline) and 1, 4, 24, and 48 h after initiation of ECCO2R. A bloodline warmer (Barkey S-line) and a thermal pad  wrapped around the multiECCO2R, as well as a warming blanket, were used to avoid undercooling of the patient.ECCO2R setup used in this study, either as standalone therapy  mode, and the multiECCO2R was inserted in series after the hemofilter . ECCO2R + CRRT was commenced at a blood flow of 200 mL/min. CVVHD was delivered with an effluent dose of 25 mL/kg/h and regional citrate anticoagulation aiming a post-filter ionized calcium concentration of ~0.25–0.35 mMol/L.For ECCO2R was assessed using Bowen et al.'s ; however, in the later course of intensive care, all patients developed severe hypercapnia despite escalated ventilation parameters. In patients 1 and 3, hypercapnia was seen as the result of diffuse consolidations and fibrotic remodeling of the lungs as indicated by the low compliance (18.4 mL/mbar), whereas patients 2 and 4 developed hypercapnia, at least in part, secondary to underlying chronic obstructive pulmonary disease.We report data of four male patients  admitted to our ICU between April and May 2020 due to ARDS secondary to confirmed severe acute respiratory syndrome coronavirus 2 infection  was started at 38 h post-ECCO2R initiation due to oliguria. Tidal volume, plateau and driving pressure, as well as respiratory rate could be reduced during the second day of ECCO2R . There was no detectable impact of ECCO2R on hemodynamics and vasopressor support. A comparison of pre- and post-ECCO2R PCO2 values showed a ~30 mmHg decrease  days due to a sustained improvement in hypercapnia. In patient 4, however, CRRT was continued for another 4 days due to oliguria. Furthermore, patient 2 developed AKI stage 3, necessitating CRRT 6 days after the termination of ECCO2R as a sequel to septic shock.ECCOthin 1 h . Patientdecrease . No pati2R using CRRT platforms might be safe and feasible to provide either standalone ECCO2R or ECCO2R combined with CRRT. This minimally invasive approach leads to efficient CO2 removal in the setting of moderate ARDS. No patient- or ECCO2R/CRRT-related adverse events occurred. Importantly, these data also implicate that every ICU with available dialysis may apply RRT platform-driven ECCO2R to limit ventilator-induced lung injury or rescue uncontrollable respiratory acidosis even in situations where “standard” ECCO2R consoles are not available. To the best of our knowledge, this is the first description of ECCO2R in COVID-19. Although these data may provide the rationale for randomized clinical trials, the following limitations need to be acknowledged. Given the invasive nature of an ECCO2R therapy, future randomized trials are required to assess the overall benefit and harm before widespread implementation can be recommended. Also, eligibility criteria should be further examined, particularly in those without an indication for CRRT. Furthermore, if the ECCO2R is intended to be continuous, sustaining a blood flow of 400 mL/min with a temporary catheter may be challenging, particularly in patients with COVID-19 who are obese or require prone positioning. COVID-19 induces a hypercoagulable state in many patients, which may result in premature extracorporeal circuit failure  and complied with the Declaration of Helsinki. Legally authorized representatives of the patients provided written informed consent.FH-S, H-WB, CR, H-DW, WS, and IV: concept and design of the study. VR, BK, SK, KT, MH, RM, SH, and OK: literature research and clinical advice. FH-S, H-WB, JW, CR, VR, BK, SK, KT, MH, RM, SH, OK, H-DW, WS, and IV: acquisition, analyses, or interpretation of data, and critical revision of the manuscript for important intellectual content. FH-S and IV: manuscript drafting and had full access to all study data and had final responsibility for submitting for publication. FH-S, JW, and IV: figure illustration. H-WB, H-DW, WS, and IV: study supervision. All authors shared the study design, data collection, data analyses, data interpretation, as well as preparation, review, and approval of the manuscript.WS received personal fees for consulting from Bayer Pharma, Liquidia Technologies, and United Therapeutics outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Autophagy is an intracellular process that targets intracellular pathogens for lysosomal degradation. Autophagy is tightly controlled at transcriptional and post-translational levels. Nuclear receptors (NRs) are a family of transcriptional factors that regulate the expression of gene sets involved in, for example, metabolic and immune homeostasis. Several NRs show promise as host-directed anti-infectives through the modulation of autophagy activities by their natural ligands or small molecules (agonists/antagonists). Here, we review the roles and mechanisms of NRs  in linking immunity and autophagy during infection. We also discuss the potential of emerging NRs  as candidate antimicrobials. The identification of novel roles and mechanisms for NRs will enable the development of autophagy-adjunctive therapeutics for emerging and re-emerging infectious diseases. Mycobacterium tuberculosis (Mtb), Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Legionella pneumophila, Anaplasma phagocytophilum, Coxiella burnetii, Francisella tularensis, and Brucella spp.—can be targeted by autophagy [Autophagy, an intracellular process, is a defense against intracellular pathogens involving lysosomal degradation ,3,4,5,6.utophagy . These autophagy ,12,13,14The nuclear receptor (NR) superfamily proteins regulate genes involved in cell survival, signaling, metabolism, and reproduction ,16,17. NThe interaction of host autophagy with pathogens has been reviewed by others ,29,30,31A series of ATGs play critical roles in autophagy initiation, elongation, and maturation, and are also involved in other physiological responses, including membrane trafficking and signaling pathways ,33,34,35The roles of ATGs differ according to the type of autophagy. Rubicon plays opposite roles in (macro)autophagy and LAP; it is a negative regulator of autophagy, but promotes Beclin-1/VPS34 kinase activity in the phagosome . SelectiCaenorhabditis elegans and Drosophila) and involving tissue-specific ATG deletion or overexpression have provided pathogenetic insight by identifying autophagy-dependent and -independent functions of ATGs [Model studies using vertebrates (mice) and invertebrates , retinoid X receptor (RXR) heterodimers (Class II), homodimeric orphan receptors (Class III), and monomeric orphan receptors (Class IV) ,61. The A total of 48 intracellular proteins have been identified as NRs ; among tIn class I NRs or steroid receptors, ligand binding at the plasma membrane is followed by a signal transduction cascade including enzymatic phosphorylation, which results in the translocation of transcription factors into the nucleus ,88. ER, The class II receptor family includes the thyroid hormone receptor (TR), VDR, retinoic acid receptor (RAR), and PPAR . They arClass III and IV receptors include the dimeric and monomeric orphan receptors, respectively. ERRα, one of the orphan nuclear receptors, is not regulated by the presence of natural ligands, but is regulated by post-transcriptional modifications, such as phosphorylation, sumoylation, or acetylation of the N-terminal domain ,100, andMycobacterium marinum [VDR signaling ameliorates infection and inflammation ,103,104. marinum . In addi marinum ,112,113. marinum .Vitamin D supplementation in mice significantly induced VDR, cathelin-related antimicrobial peptide (CRAMP), and LC3B expression, but decreased the collagenase matrix metalloproteinase-1 . A strucIFN-γ, which is an important cytokine in the adaptive Th1 immune response, alone  or in coHelicobacter pylori in gastric epithelial cells, by activating— protein disulfide isomerase family A member 3 (PDIA3) receptor and upregulating mucolipin-3 (MCOLN3)-mediated Ca2+ release [Aspergillus fumigatus infection, vitamin D treatment led to autophagic homeostasis by reducing the number of autophagy-mediated lysosomes and regulatory T-cells, thus enhancing the antimicrobial response [Vitamin D treatment reversed the influenza A virus-induced inhibition of autophagic flux by inducing the expression of syntaxin-17 and the V-type proton ATPase subunit (ATP6V0A2) . In addi release . In an aresponse . Moreoveresponse . AlthougEstrogen—a female sex steroid hormone—and its receptors (ERα and ERβ) reportedly modulate autophagy, which is implicated in various human diseases and the determination of cell fate ,129. IndEstrogen modulators influence antimicrobial responses by influencing autophagy. The selective ER modulator bazedoxifene suppresses the intracellular growth of Mtb by activating autophagy via ROS generation and Akt/ mammalian target of rapamycin (mTOR) signaling . TamoxifERRs are orphan members of the NR family, and are involved in a variety of biological responses, including cellular metabolism and energy control ,136,137.The nutrient-sensing NRs, PPARα and FXR, play reciprocal functions in the regulation of autophagy; PPARα enhances and FXR suppresses autophagy to enhance lipolysis  and ciliMycobacterium bovis bacillus Calmette-Guérin (BCG), or Mycobacterium abscessus by activating transcription factor EB (TFEB) [M. bovis BCG, suggesting that PPARα contributes to lipid catabolism and reduces the foamy refuge during mycobacterial infection [Staphylococcus aureus [PPARα modulates antimicrobial responses to Mtb, B (TFEB) ,71,72. Is aureus .Numerous agents have been reported to activate PPARα and enhance TFEB, thereby promoting lysosomal biogenesis in models of chronic inflammatory and degenerative diseases ,150,151.PPARβ/δ inhibits the ER stress induced by palmitate in AC16 cardiomyocytes by inducing expression of the autophagy markers Beclin-1 and LC3 II, thus preventing the harmful cardiac effects of ER stress . The trePlasmodium falciparum agent, suppresses autophagolysosomal degradation and PPARγ activity [Trypanosoma cruzi [Amodiaquine, which is a selective anti-activity . The PPAma cruzi . INT131,ma cruzi . HoweverThe adopted orphan NR—REV-ERBα—is involved in adipogenesis, muscle differentiation, glucose/lipid metabolism, and the circadian rhythm ,162,163.During infection, REV-ERBα activation by GSK4112 exerted an anti-mycobacterial effect in macrophages by enhancing autophagy and lysosomal biogenesis and suppressing IL-10 synthesis . HoweverAll-trans retinoic acid and/or arsenic trioxide induced autophagy of the oncoprotein promyelocytic leukemia (PML)/RARA, suggesting RARα as a therapeutic target for acute PML ,172. In Similarly, little is known about the role of RARβ in the regulation of autophagy during infection. RARβ has a tumor suppressive function and is involved in cell differentiation and apoptosis. Interestingly, the human papillomavirus type 16 (HPV16) E7 oncoprotein upregulated the mRNA and protein levels of RARβ in cervical cancer cells and in the cervix of K14E7 transgenic mice . During RORs are important in the regulation of the circadian clock, metabolic homeostasis, and tumorigenesis ,181. AltAs a nutrient-sensing and autophagy-regulating NR, FXRα regulates hepatic autophagy to maintain the energy balance in the liver ,193 and In a model of cholestasis, the activation of autophagy maturation is inhibited in an FXR-dependent manner, partly as a result of the induction of Rubicon. However, ursodeoxycholic acid (UDCA), which is a non-FXR-agonistic bile acid, induced hepatic autophagy and reduced the expression of Rubicon, which is an inhibitor of autophagy . In an aSalmonella infection by inducing the expression of the multifunctional enzyme CD38 [LXRα and LXRβ are negative regulators of cholesterol metabolism and inflammation ,197. Botyme CD38 . LXRs aryme CD38 ,204. TheThe thyroid hormone is a regulator of the metabolic rate, oxidative phosphorylation (OXPHOS), and ROS production ,206, andGiven the role of autophagy in controlling intracellular pathogens, there is an urgent need for autophagy-directed therapeutics and prophylactics. The targeting of autophagy in monocytes/macrophages stimulates the innate immune response, the dampening of inflammation and suppression of innate immunity, and the promotion of pathogen escape . TherefoNR protects against a variety of infections and modulates autophagy during pathogen invasion. Early studies exploited VDR- or ERRα-targeted antimicrobial responses and were followed by several trials expanding the effects of other NRs. It is known that the ATGs involved in non-canonical autophagy are different from those involved in canonical autophagy, but only a few studies on NR-mediated non-canonical autophagy pathways have been reported to date . We are


Background: Glaucoma is a leading cause of irreversible blindness. Remodeling of the scleral extracellular matrix (ECM) plays an important role in the development of glaucoma. The aim of this study was to identify the key genes and pathways for the ECM remodeling of sclera in glaucoma by bioinformatics analysis and to explore potential therapeutic agents for glaucoma management.Methods: Genes associated with glaucoma, sclera and ECM remodeling were detected using the text mining tool pubmed2ensembl, and assigned Gene Ontology (GO) biological process terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the GeneCodis program. A protein-protein interaction (PPI) network was constructed by STRING and visualized in Cytoscape, module analysis was performed using the Molecular Complex Detection (MCODE) plugin, and GO and KEGG analyses of the gene modules were performed using the Database of Annotation, Visualization and Integrated Discovery (DAVID) platform. The genes that clustered in the significant module were selected as core genes, and functions and pathways of the core genes were visualized using ClueGO and CluePedia. Lastly, the drug-gene interaction database was used to explore drug-gene interactions of the core genes to find drug candidates for glaucoma.Results: We identified 125 genes common to “Glaucoma”, “Sclera”, and “ECM remodeling” by text mining. Gene functional enrichment analysis yielded 30 enriched GO terms and 20 associated KEGG pathways. A PPI network that included 60 nodes with 249 edges was constructed, and three gene modules were obtained using the MCODE. We selected 13 genes that clustered in module 1 as core candidate genes that were associated mainly with ECM degradation and cell proliferation and division. The HIF-1 signaling pathway, FOXO signaling pathway, PI3K-Akt signaling pathway and TGFB signaling pathway were found to be enriched. We found that 11 of the 13 selected genes could be targeted by 26 existing drugs.Conclusions: The results showed that VEGFA, TGFB1, TGFB2, TGFB3, IGF2, IGF1, EGF, FN1, KNG1, TIMP1, SERPINE1, THBS1, and VWF were potential key genes involved to scleral ECM remodeling. Furthermore, 26 drugs were identified as potential therapeutic agents for glaucoma treatment and management. Glaucoma is a progressive optic neuropathy characterized by progressive degeneration of retinal ganglion cells and optic nerve head (ONH) axons Currently, lowering of intraocular pressure (IOP)  is the only available option to delay the development and progression of glaucoma Retinal ganglion cell axons converge at the ONH, pass through the laminar cribrosa, enter the scleral canal and exit the eye in vivo experiment, mice with microfibril deficiency were shown to have increased susceptibility to glaucomatous damage Stiffening of the sclera leading to scleral rigidity is one of the primary structural changes in glaucoma The discovery of new drug therapies using conventional approaches can be time-consuming and costly, whereas repurposing an existing drug to treat a condition beyond its original intent may be a more effective and less expensive alternative http://pubmed2ensembl.ls.manchester.ac.uk/), a publicly available resource that links over 2,000,000 articles in PubMed to nearly 150,000 genes from 50 species in Ensembl Homo sapiens (GRCh37)” was selected as the species dataset and “filter on MEDLINE: PubMed ID” was set to constrain the query result. The unduplicated genes were extracted and the intersection of gene hits from the three gene sets was retrieved as the text mining genes (TMGs).Text mining was performed using pubmed2ensembl (http://genecodis.cnb.csic.es/) to perform an enrichment analysis of the TMGs related to scleral ECM remodeling in glaucoma. GeneCodis is a web‑based server for functional analysis of gene lists that integrates different sources of information We used GeneCodis (https://string-db.org/cgi/input.pl) is an online database resource that covers approximately 24.6 million proteins and more than 3.1 billion interactions originating from 5090 organisms The Search Tool for the Retrieval of Interacting Genes , an online tool for gene functional analysis http://www.geneontology.org) database contains terms for the functional classification for genomic data under three main categories: biological processes, cellular component, and molecular function http://www.genome.ad.jp/kegg/) is a knowledge base for systematic analysis, annotation, and visualization of gene functions The GO functional and KEGG pathway enrichment analyses of significant module genes were performed using Database for Annotation, Visualization and Integrated Discovery (DAVID)  is a web resource that consolidates disparate data sources that describe drug-gene interactions and gene druggability Using the text mining strategy described in the methods section Figure , we obtaThe GO biological process terms and KEGG pathway enrichment analyses using GeneCodis (with P=1.00E-07 as the cutoff) to identified the most enriched terms closely related to the pathology of scleral ECM remodeling in glaucoma. Thirty significantly enriched GO biological process annotations of 97 unique genes were identified. Among them, the five most enriched terms were “extracellular matrix organization” (P=3.47306E-27), “platelet degranulation” (P=2.44362E-19), “cytokine-mediated signaling pathway” (P=3.99419E-18), “response to drug” (P=3.82E-14), and “positive regulation of cell population proliferation” (P=1.45E-13), for 31, 20, 25, 22, and 27 TMGs, respectively Table .The KEGG pathway enrichment analysis identified 20 significant pathways that involved 64 TMGs. The five most significantly enriched pathways were “AGE-RAGE signaling pathway in diabetic complications” (P=1.04E-22), “Proteoglycans in cancer” (P=3.04E-19), “Pathways in cancer” (P=1.88327E-16), “HIF-1 signaling pathway” (P=1.17E-12), and “Relaxin signaling pathway” (P= 1.34E-12), involving 23, 26, 34, 16, and 17 TMGs, respectively Table .A PPI network was constructed for the 64 target genes using STRING with confidence score >0.900. The network had a total of 60 nodes with 249 edges Figure . ModularThe GO enrichment analysis showed that the three modules were related primarily to ECM degrading, cell proliferation and division which play crucial role in scleral ECM remodeling Figure A,C,D. ThTGFB1, TGFB2, TGFB3, VEGFA, IGF2, IGF1, EGF, FN1, KNG1, TIMP1, SERPINE1, THBS1 and VWF, as core candidate genes of the PPI network. The enrichment analysis indicated that these genes were significantly enriched in platelet alpha granule lumen, HIF-1 signaling pathway, and AGE-RAGE signaling pathway in diabetic complication  were predicted to be involved in drug-gene interactions. The primary connection among drugs, genes and pathways are shown in Figure We used the 13 core genes as potential targets in a druggs Table . Eleven Glaucoma is the leading cause of irreversible blindness globally. Reduction of IOP is the only proven method to treat glaucoma TGFB1, TGFB2, TGFB3, VEGFA, IGF2, IGF1, EGF, FN1, KNG1, TIMP1, SERPINE1, THBS1 and VWF, that were involved in the HIF-1 signaling pathway, TGFB signaling pathway, FOXO signaling pathway, and PI3K-Akt signaling pathway.In the present study, we identified 125 genes that might be involved in scleral ECM remodeling associated with glaucoma using text mining strategy , a glycoprotein that functions to bridge platelets with an exposed collagen surface, is associated with the severity of organ fibrosis Transforming growth factor β (TGFB) belongs to a family of fibrogenic cytokine that includes TGFB1, TGFB2 and TGFB3 VEGF-A expression and promote fibrinogenesis via the HIF-1α-VEGF pathway in bronchiolitis obliterans The HIF-1 signaling pathway, one of the enriched KEGG pathways, has been shown to promote scleral myofibroblast transdifferentiation by regulating the actin cytoskeleton pathway, as well as ECM remodeling via the ECM receptor interaction pathway in vivo animal experiments Potential drugs identified by the drug-gene interaction search were classified as mainly TGFB inhibitors, VEGFA inhibitors, IGF inhibitors, fibrinolytic agents, antineoplastic agents, or immunomodulating agents. The TGFB inhibitors were mainly of hyaluronidase, lerdelimumab and fresolimumab. Hyaluronidases are enzymes that regulate the remodeling of ECM. Hyaluronidases have been shown to inhibit the growth of pulmonary fibrosis and decrease TGFB production and collagen deposition in mice VEGFA, including ranibizumab, bevacizumab, aflibercept, carvedilol, dalteparin sodium, phenytoin and vandetanib. Ranibizumab, a monoclonal antibody fragment against VEGF-A, inhibits vascular permeability and has antiproliferative and antimetabolic effects on various cell lines. Several studies have indicated that ranibizumab may prevent scarring after glaucoma filtering surgery through the VEGF pathway More than a quarter of identified drugs target in vivo experiment demonstrated that dusigitumab inhibited tumor growth of mouse embryonic fibroblast EGF and TGFA in gastric mucosal cells THBS1 expression in human thyroid carcinoma FTC-133 cells through the JNK/ATF-2 pathway Dusigitumab is a human antibody of IGF1 and IGF2. An Scleral ECM remodeling also is a key factor in the pathogenesis of myopia. In myopic human eyes, the sclera was found to be thinner, and the scleral ECM was decreased, compared with those in healthy eyes VEGFA, TGFB1, TGFB2, TGFB3, IGF2, IGF1, EGF, FN1, KNG1, TIMP1, SERPINE1, THBS1 and VWF, that may be involved in the scleral ECM remodeling associated with glaucoma. These genes were enriched in the HIF-1 signaling pathway, FOXO signaling pathway, PI3K-Akt signaling pathway and TGFB signaling pathway. We also identified 26 potential drugs that may be help to guide future glaucoma therapies. The absence of experimental validation is a limitation of this study, and further experimental studies are required to verify the results.In conclusion, we identified 13 hub genes, Supplementary tables.Click here for additional data file.