Empagliflozin ameliorates vascular calcification in diabetic mice through inhibiting Bhlhe40-dependent NLRP3 inflammasome activation.

Type 2 diabetes mellitus (T2DM) patients exhibit greater susceptibility to vascular calcification (VC), which has a higher risk of death and disability. However, there is no specific drug for VC therapy. NLRP3 inflammasome activation as a hallmark event of medial calcification leads to arterial stiffness, causing vasoconstrictive dysfunction in T2DM. Empagliflozin (EMPA), a sodium-glucose co-transporter 2 inhibitor (SGLT2i), restrains hyperglycemia with definite cardiovascular benefits. Given the anti-inflammatory activity of EMPA, herein we investigated whether EMPA protected against VC in the aorta of T2DM mice by inhibiting NLRP3 inflammasome activation. Since db/db mice receiving a normal diet developed VC at the age of about 20 weeks, we administered EMPA (5, 10, 20 mg·kg-1·d-1, i.g) to 8 week-old db/db mice for 12 weeks. We showed that EMPA intervention dose-dependently ameliorated the calcium deposition, accompanied by reduced expression of RUNX2 and BMP2 proteins in the aortas. We found that EMPA (10 mg·kg-1·d-1 for 6 weeks) also protected against VC in vitamin D3-overloaded mice, suggesting the protective effects independent of metabolism. We showed that EMPA (10 mg·kg-1·d-1) inhibited the abnormal activation of NLRP3 inflammasome in aortic smooth muscle layer of db/db mice. Knockout (KO) of NLRP3 significantly alleviated VC in STZ-induced diabetic mice. The protective effects of EMPA were verified in high glucose (HG)-treated mouse aortic smooth muscle cells (MOVASs). In HG-treated NLRP3 KO MOVASs, EMPA (1 µM) did not cause further improvement. Bioinformatics and Western blot analysis revealed that EMPA significantly increased the expression levels of basic helix-loop-helix family transcription factor e40 (Bhlhe40) in HG-treated MOVASs, which served as a negative transcription factor directly binding to the promotor of Nlrp3. We conclude that EMPA ameliorates VC by inhibiting Bhlhe40-dpendent NLRP3 inflammasome activation. These results might provide potential significance for EMPA in VC therapy of T2DM patients.

Transformation to small cell lung cancer is irrespective of EGFR and accelerated by SMAD4-mediated ASCL1 transcription independently of RB1 in non-small cell lung cancer.

OBJECTIVES: Histological transformation to small cell lung cancer (SCLC) has been identified as a mechanism of TKIs resistance in EGFR-mutant non-small cell lung cancer (NSCLC). We aim to explore the prevalence of transformation in EGFR-wildtype NSCLC and the mechanism of SCLC transformation, which are rarely understood. METHODS: We reviewed 1474 NSCLC patients to investigate the NSCLC-to-SCLC transformed cases and the basic clinical characteristics, driver gene status and disease course of them. To explore the potential functional genes in SCLC transformation, we obtained pre- and post-transformation specimens and subjected them to a multigene NGS panel involving 416 cancer-related genes. To validate the putative gene function, we established knocked-out models by CRISPR-Cas 9 in HCC827 and A549-TP53-/- cells and investigated the effects on tumor growth, drug sensitivity and neuroendocrine phenotype in vitro and in vivo. We also detected the expression level of protein and mRNA to explore the molecular mechanism involved. RESULTS: We firstly reported an incidence rate of 9.73% (11/113) of SCLC transformation in EGFR-wildtype NSCLC and demonstrated that SCLC transformation is irrespective of EGFR mutation status (P = 0.16). We sequenced 8 paired tumors and identified a series of mutant genes specially in transformed SCLC such as SMAD4, RICTOR and RET. We firstly demonstrated that SMAD4 deficiency can accelerate SCLC transition by inducing neuroendocrine phenotype regardless of RB1 status in TP53-deficient NSCLC cells. Further mechanical experiments identified the SMAD4 can regulate ASCL1 transcription competitively with Myc in NSCLC cells and Myc inhibitor acts as a potential subsequent treatment agent. CONCLUSIONS: Transformation to SCLC is irrespective of EFGR status and can be accelerated by SMAD4 in non-small cell lung cancer. Myc inhibitor acts as a potential therapeutic drug for SMAD4-mediated resistant lung cancer. Video Abstract.

Mechanistic and Clinical Comparison of the Erythropoietic Effects of SGLT2 Inhibitors and Prolyl Hydroxylase Inhibitors in Patients with Chronic Kidney Disease and Renal Anemia.

Renal anemia is treated with erythropoiesis-stimulating agents (ESAs), even though epoetin alfa and darbepoetin increase the risk of cardiovascular death and thromboembolic events, including stroke. Hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitors have been developed as an alternative to ESAs, producing comparable increases in hemoglobin. However, in advanced chronic kidney disease, HIF-PHD inhibitors can increase the risk of cardiovascular death, heart failure, and thrombotic events to a greater extent than that with ESAs, indicating that there is a compelling need for safer alternatives. Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of major cardiovascular events, and they increase hemoglobin, an effect that is related to an increase in erythropoietin and an expansion in red blood cell mass. SGLT2 inhibitors increase hemoglobin by ≈0.6-0.7 g/dL, resulting in the alleviation of anemia in many patients. The magnitude of this effect is comparable to that seen with low-to-medium doses of HIF-PHD inhibitors, and it is apparent even in advanced chronic kidney disease. Interestingly, HIF-PHD inhibitors act by interfering with the prolyl hydroxylases that degrade both HIF-1α and HIF-2α, thus enhancing both isoforms. However, HIF-2α is the physiological stimulus to the production of erythropoietin, and upregulation of HIF-1α may be an unnecessary ancillary property of HIF-PHD inhibitors, which may have adverse cardiac and vascular consequences. In contrast, SGLT2 inhibitors act to selectively increase HIF-2α, while downregulating HIF-1α, a distinctive profile that may contribute to their cardiorenal benefits. Intriguingly, for both HIF-PHD and SGLT2 inhibitors, the liver is likely to be an important site of increased erythropoietin production, recapitulating the fetal phenotype. These observations suggest that the use of SGLT2 inhibitors should be seriously evaluated as a therapeutic approach to treat renal anemia, yielding less cardiovascular risk than other therapeutic options.

Phase I LITESPARK-001 study of belzutifan for advanced solid tumors: Extended 41-month follow-up in the clear cell renal cell carcinoma cohort.

BACKGROUND: Accumulation of the HIF-2α transcription factor is an oncogenic event implicated in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). In the phase I LITESPARK-001 study, the first-in-class HIF-2α inhibitor belzutifan demonstrated antitumor activity and an acceptable safety profile for pretreated patients with advanced ccRCC. Updated data with additional follow-up of > 40 months are presented. METHODS: LITESPARK-001 is an ongoing open-label study with a 3 + 3 dose-escalation design followed by an expansion phase. Patients with ccRCC enrolled at 7 sites received belzutifan 120 mg orally once daily until disease progression, unacceptable toxicity, or patient withdrawal. The data cutoff date was July 15, 2021. The primary end point was identifying the maximum tolerated dose and/or the recommended phase II dose. Secondary end points included objective response rate (ORR) and duration of response (DOR) per RECIST v1.1 by investigator assessment and safety. RESULTS: Median follow-up was 41.2 months (range, 38.2-47.7). Patients received a median of 3 (range, 1-9) prior systemic therapies. Of 55 patients, 14 (25 %) achieved an objective response. Median DOR was not reached (range, 3.1 + to 38.0 + months). Adverse events (AEs) attributed to study treatment by investigator assessment were reported in 53 patients (96 %). 22 patients (40 %) had grade 3 treatment-related AEs; the most common were anemia (n = 13; 24 %) and hypoxia (n = 7; 13 %). No grade 4 or 5 treatment-related AEs occurred. CONCLUSION: After a median follow-up of 41.2 months, belzutifan monotherapy demonstrated durable antitumor activity in patients with advanced ccRCC and acceptable safety. CLINICALTRIALS: gov. NCT02974738.

Decoupling NAD+ metabolic dependency in chondrosarcoma by targeting the SIRT1-HIF-2α axis.

Chondrosarcomas represent the second most common primary bone malignancy. Despite the vulnerability of chondrosarcoma cells to nicotinamide adenine dinucleotide (NAD+) depletion, targeting the NAD+ synthesis pathway remains challenging due to broad implications in biological processes. Here, we establish SIRT1 as a central mediator reinforcing the dependency of chondrosarcoma cells on NAD+ metabolism via HIF-2α-mediated transcriptional reprogramming. SIRT1 knockdown abolishes aggressive phenotypes of chondrosarcomas in orthotopically transplanted tumors in mice. Chondrosarcoma cells thrive under glucose starvation by accumulating NAD+ and subsequently activating the SIRT1-HIF-2α axis. Decoupling this link via SIRT1 inhibition unleashes apoptosis and suppresses tumor progression in conjunction with chemotherapy. Unsupervised clustering analysis identifies a high-risk chondrosarcoma patient subgroup characterized by the upregulation of NAD+ biosynthesis genes. Finally, SIRT1 inhibition abolishes HIF-2α transcriptional activity and sensitizes chondrosarcoma cells to doxorubicin-induced cytotoxicity, irrespective of underlying pathways to accumulate intracellular NAD+. We provide system-level guidelines to develop therapeutic strategies for chondrosarcomas.

Clinical Efficacy of Bushen Yiqi Fuzheng Decoction Combined with Sunitinib in the Treatment of Postoperative Patients with Renal Cell Carcinoma and Its Influence on Their Immune Function.

BACKGROUND: This study aimed to investigate the effect of Bushen Yiqi Fuzheng decoction combined with sunitinib on the prognosis, clinical efficacy and immune function of patients with renal cell carcinoma (RCC) after surgery. METHODS: A total of 120 patients who experienced RCC after surgery were randomly divided into the observation and control groups in this prospective study, with 60 cases in each group. The therapeutic effect, improvement of clinical symptoms, changes of immune function-related indicators and adverse reactions during medication were recorded. The changes in immune cell population, midkine (MK), interleukin 35 (IL-35), hypoxia-inducible factor 2alpha (HIF-2α), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), carcinoembryonic antigen (CEA), osteopontin (OPN), ferritin (FERR) and beta2-microglobulin (ß2-MG) levels were measured. The Karnofsky performance status (KPS) score of patients was recorded. RESULTS: The total effective rate of the observation group (95%) was better than that of the control group (85%, p < 0.05). After treatment, the changes of immune function indexes in the control group were not obvious. The indexes related to immune function in the observation group significantly decreased. Significant differences were observed in the cluster of differentiation 3+ (CD3+), cluster of differentiation 4+ (CD4+), cluster of differentiation 8+ (CD8+) and CD4+/CD8+ between the two groups after treatment. The incidence of adverse reactions in the observation group was lower than that of the control group. The KPS of the observation group was higher than that of the control group. Before treatment, no differences were observed in the MK, IL-35, HIF-2α, CEA, OPN, FERR, ß2-MG, MMP-9 and TIMP-1 levels between the two groups. After treatment, the levels of the above parameters were lower than those before treatment, especially in the observation group. CONCLUSIONS: Bushen Yiqi Fuzheng decoction combined with sunitinib can significantly improve the clinical efficacy and postoperative immune function of RCC patients after surgery and down-regulate MMP-9 and TIMP-1 levels in the serum, which is beneficial to the prognosis of patients.

[Clinical Significance of Minimal Residual Disease in Pediatric Patients with TCF3/PBX1+ B-cell Acute Lymphoblastic Leukemia].

OBJECTIVE: To explore the consistency of flow cytometry (FCM) method and polymerase chain reaction (PCR) technique in the detection of minimal residual disease (MRD) at different treatment stages in pediatric patients with TCF3/PBX1+ B-cell acute lymphoblastic leukemia (B-ALL) and the correlations between the detection results and prognosis. METHODS: The clinical data of 64 newly diagnosed pediatric patients with TCF3/PBX1+ B-ALL admitted to the Department of Pediatrics of Peking University People's Hospital from January 2005 to December 2017 were retrospectively analyzed. FCM and PCR methods were used to monitor the MRD level in bone marrow samples from 64 children during the same period of treatment on d33 and d90 respectively, and the detection results were analyzed. RESULTS: There were 37 males and 27 females in the 64 patients, with a median age of 8 years(range 0.8 to 16 years). The complete remission (CR) rate after the first cycle of induction chemotherapy was 98.4% (62/63), with overall CR rate of 100%. 12 patients experienced recurrence, with a median recurrence time of 16.9 (5.3-46.3) months. The median follow-up time of the 64 patients was 77.2 (1.0-184.8) months , and the 5-year overall survival (OS) rate and event-free survival (EFS) rate were 82.8%±4.7% and 75.0%±5.4%, respectively. On d90, the concordance rate of the MRD results from the two methods was 98.4%, and the related kappa value was 0.792 (P < 0.001), which were significantly higher than those on d33. After induction chemotherapy (d33), the 5-year EFS rate of MRD-FCM- group (79.3%±5.3%) was significantly better than that of MRD-FCM+ group (40.0%±21.9%) (P =0.028), there were no significant differences in the 5-year OS rate and EFS rate between MRD-PCR+ group and MRD-PCR- group, and the 5-year EFS rate of MRD-FCM-/PCR- group (85.4%±5.5%) was significantly better than that of MRD-FCM+/PCR+ group (40.0 %±21.9%) (P =0.026). CONCLUSION: In children with TCF3/PBX1+ B-ALL, the MRD results detected by FCM and PCR methods show good consistency, especially in consolidation therapy period (d90). The MRD level at the end of induction therapy (d33) is an important factor affecting the long-term prognosis, especially the MRD results detected by FCM method, which is significantly associated with prognosis.

Current Drugs and their Therapeutic Targets for Hypoxia-inducible Factors in Cancer.

Hypoxia, a prevalent characteristic of both solid and liquid malignancies, is found to regulate how genes are expressed in a way that promotes cellular adaptability and survival. Metastasis is controlled by hypoxia-inducible factors (HIFs). HIFs are dimeric protein molecules made up of an oxygen (O2) responsive HIF-1α, HIF-2α, or HIF-3α domain and a periodically produced HIF-1ß portion (also known as ARNT). Nevertheless, it is important to note that HIFs degrade under normoxic conditions. A large multitude of different biological operations, including vessels generation, oxygen delivery, stemness, pluripotency, multiplication, epithelial to mesenchymal shift, metastatic prevalence and intrusion, redox equilibrium, and programmed cell death, are strictly controlled by over 70 immediate HIF target genes that have been reported. Metabolic reprogramming, which modulates cellular energy generation aside from oxidative phosphorylation and concerning glycolysis, is among the core tasks of HIF target genes. As a result, choosing HIFs as a primary target in the treatment of various tumors is essential. We have a very limited understanding of this extremely complex topic, which is characterised by hypoxia- induced resistance. In order to combat this, scientists are investigating numerous cutting-edge approaches. Traditional chemotherapeutic drugs used to treat cancer are frequently linked to unfavourable side effects and the development of chemoresistance. The use of natural compounds in conjunction with chemotherapy drugs is rising as a result of their capacity to alter a number of molecular practices with a lower detrimental impact. Experimental and clinical research is accumulating evidence that phytochemicals can influence the genesis and progression of cancer by favourably modulating a number of signalling pathways. Combinations of phytochemicals are potent cancer treatment options because they incentivise apoptosis, limit cell prevalence, make cancerous cells more susceptible, and escalate immunity. Despite being characterised, HIF-1-independent mechanisms for medication resistance in hypoxia are still infrequently reported. The prime aim of the article is to summarise the most recent research on the molecular basis of hypoxia-induced chemoresistance and how chemotherapy and phytochemicals can be used to treat cancer patients who are resistant to drugs.

Belzutifan (MK-6482): Biology and Clinical Development in Solid Tumors.

PURPOSE OF REVIEW: To review the biology, drug development, and clinical data regarding the efficacy and safety of belzutifan (MK-6482), a small molecule inhibitor of HIF-2α. RECENT FINDINGS: Belzutifan, a second-generation HIF-2α inhibitor, was shown to provide clinically meaningful benefit in the treatment of VHL-associated tumors (including ccRCC, pancreatic lesions as well as neuroendocrine tumor, and CNS hemangioblastomas). The recommended dose of belzutifan is 120 mg orally daily and half-life is 14 h. In pretreated ccRCC, belzutifan achieved disease control rate of 80% in phase I trial. The most common side effects include anemia and hypoxia related symptoms. Investigation into the important role HIF-2α plays in the expression of genes associated with angiogenesis, erythropoiesis, carcinogenesis, and progression of tumors and the discovery of structural vulnerability within HIF-2α have resulted in the development of a new therapy that has demonstrated efficacy and safety in recent clinical trials. Further research is ongoing to optimize therapeutic benefits from this new exciting therapeutic modality and to improve the outcome of HIF-2α-driven tumors.

Empagliflozin enhances neuroplasticity in rotenone-induced parkinsonism: Role of BDNF, CREB and Npas4.

AIMS: Parkinsonism is characterized by degeneration of dopaminergic neurons and impairment in neuroplasticity. Empagliflozin (EMPA) is an anti-diabetic drug that has been shown to improve cognitive dysfunctions and exerted antioxidant and anti-inflammatory effects in different models. This study aimed to determine the neuroprotective effects of EMPA against rotenone (ROT)-induced parkinsonism. MAIN METHODS: ROT (1.5 mg/kg) was injected subcutaneously three times per week for two successive weeks. Mice were treated with EMPA (3 and 10 mg/kg, orally) for one week prior ROT administration and for another two weeks along with ROT. After that, motor functions and histopathological changes were assessed, and brains were isolated for biochemical analyses and immunohistochemical investigation. KEY FINDINGS: Results indicated that, in a dose dependent manner, EMPA improved motor functions and histopathological changes induced by ROT, increased brain content of reduced glutathione (GSH), dopamine (DA), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear factor erythroid 2-related factor 2 (Nrf2), inositol trisphosphate (IP3), calcium (Ca2+), calcium/calmodulin-dependent protein kinase type IV (CaMKIV) and phospho-Protein kinase B (p-Akt) levels compared to ROT group. Additionally, EMPA decreased the levels of malondialdehyde (MDA), and tumor necrosis factor-α (TNF-α), and inactivated glycogen synthase kinase-3 beta (GSK-3ß). Improvement in neuroplasticity was also observed indicated by elevation in brain derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB), and neuronal PAS domain Protein 4 (Npas4). SIGNIFICANCE: EMPA improved motor functions possibly through improving neuroplasticity markers and antioxidant, anti-inflammatory, and neuroprotective effects in a dose dependent manner.

Clinical Significance of Minimal Residual Disease in Pediatric Patients with TCF3/PBX1+ B-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the consistency of flow cytometry (FCM) method and polymerase chain reaction (PCR) technique in the detection of minimal residual disease (MRD) at different treatment stages in pediatric patients with TCF3/PBX1+ B-cell acute lymphoblastic leukemia (B-ALL) and the correlations between the detection results and prognosis.@*METHODS@#The clinical data of 64 newly diagnosed pediatric patients with TCF3/PBX1+ B-ALL admitted to the Department of Pediatrics of Peking University People's Hospital from January 2005 to December 2017 were retrospectively analyzed. FCM and PCR methods were used to monitor the MRD level in bone marrow samples from 64 children during the same period of treatment on d33 and d90 respectively, and the detection results were analyzed.@*RESULTS@#There were 37 males and 27 females in the 64 patients, with a median age of 8 years(range 0.8 to 16 years). The complete remission (CR) rate after the first cycle of induction chemotherapy was 98.4% (62/63), with overall CR rate of 100%. 12 patients experienced recurrence, with a median recurrence time of 16.9 (5.3-46.3) months. The median follow-up time of the 64 patients was 77.2 (1.0-184.8) months , and the 5-year overall survival (OS) rate and event-free survival (EFS) rate were 82.8%±4.7% and 75.0%±5.4%, respectively. On d90, the concordance rate of the MRD results from the two methods was 98.4%, and the related kappa value was 0.792 (P < 0.001), which were significantly higher than those on d33. After induction chemotherapy (d33), the 5-year EFS rate of MRD-FCM- group (79.3%±5.3%) was significantly better than that of MRD-FCM+ group (40.0%±21.9%) (P =0.028), there were no significant differences in the 5-year OS rate and EFS rate between MRD-PCR+ group and MRD-PCR- group, and the 5-year EFS rate of MRD-FCM-/PCR- group (85.4%±5.5%) was significantly better than that of MRD-FCM+/PCR+ group (40.0 %±21.9%) (P =0.026).@*CONCLUSION@#In children with TCF3/PBX1+ B-ALL, the MRD results detected by FCM and PCR methods show good consistency, especially in consolidation therapy period (d90). The MRD level at the end of induction therapy (d33) is an important factor affecting the long-term prognosis, especially the MRD results detected by FCM method, which is significantly associated with prognosis.

HEY1-mediated cisplatin resistance in lung adenocarcinoma via epithelial-mesenchymal transition.

Lung cancer is one of the most common malignancies and the leading cause of cancer-related death in the world. In patients with advanced lung adenocarcinoma who are negative for driver gene mutations, platinum-based chemotherapy represented by cisplatin remain the standard of care. Therefore, studying the mechanism behind inevitable cisplatin resistance in lung adenocarcinoma is still important. In this study, the potentially related differential expression gene for cisplatin resistance in lung adenocarcinoma was screened in the GEO database. The expression level of HEY1 in cell lines of lung adenocarcinoma was detected and HEY1 expression was up-regulated in cisplatin-resistant lung adenocarcinoma tissues and cell lines A549/DDP. Patients with high HEY1 expression have poor prognosis after cisplatin therapy. Gain and loss function assays uncovered that HEY1 could regulate the cisplatin sensitivity of NSCLC cells. In vivo experiments have confirmed that silence of HEY1 expression can induce cisplatin resistance, and epithelial-mesenchymal transition (EMT) changes occur during this process. Mechanically, HEY1 silencing significantly up-regulated E-cadherin expression and down-regulated Vimentin in A549/DDP cells. While up-regulation of HEY1 resulted in down-regulation of E-cadherin and up-regulation of Vimentin in A549 cells. Immunohistochemical experiments confirmed that E-cadherin was significantly decreased, and Vimentin expression was significantly up-regulated in cisplatin-resistant lung adenocarcinoma tissues. HEY1 can mediate the occurrence of cisplatin-acquired resistance in lung adenocarcinoma, and the possible mechanism is to regulate the EMT. The results of this study can provide a new direction and target for clinical research on the reversal of cisplatin resistance in lung adenocarcinoma.

[Expression of TCF3 in Burkitt's Lymphoma and Its Proliferative Effect and Prognostic Significance].

OBJECTIVE: To observe the mutation and expression of TCF3 gene in Burkitt's lymphoma (BL), and explore its effect on the proliferation of BL cells and clinical efficacy and prognosis. METHODS: The mutation and expression of TCF3 in tumor tissues from BL patients were observed by the second-generation sequencing and real-time quantitative PCR. The proliferation and apoptosis of lymphoma cells after TCF3 knocked down were observed by siRNA interference technique and CCK-8 method. Survival analysis was used to observe the relationship between TCF3 mutation and the treatment efficacy and prognosis of BL patients. RESULTS: There were high frequency mutation rate (mutation rate was 23.7%) and high expression of TCF3 in BL patients. After TCF3 knocked down, cell proliferation was inhibited and apoptosis was promoted. In TCF3-siRNA group and control group, the cell proliferation rate at 48 h was (50.2±5.9)% and (96.6±11.4)%, and apoptosis rate was 30.1% and 1.5%, respectively, which showed significantly different between the two groups (P<0.001, P=0.005). The complete remission rate of patients with TCF3 mutation was low. The complete remission rate of mutant group and wild-type group was 44.4% and 82.8%, respectively (P=0.023). The 2-year progression-free survival rate and overall survival rate of the patients with TCF3 mutation was 55.6% and 61.0%, respectively, which was lower than 83.2% and 85.2% of the patients without mutation, but the differences were not statistically significant. CONCLUSION: There are mutation and abnormal expression of TCF3 in patients with BL. Patients with TCF3 mutations have low remission rate and poor prognosis.

MiR-6924-5p-rich exosomes derived from genetically modified Scleraxis-overexpressing PDGFRα(+) BMMSCs as novel nanotherapeutics for treating osteolysis during tendon-bone healing and improving healing strength.

Osteolysis at the tendon-bone interface can impair pullout strength during tendon-bone healing and lead to surgery failure, but the effects of clinical treatments are not satisfactory. Mesenchymal stem cell (MSC)-derived exosomes have been used as potent and feasible natural nanocarriers for drug delivery and have been proven to enhance tendon-bone healing strength, indicating that MSC-derived exosomes could be a promising therapeutic strategy. In this study, we explored Scleraxis (Scx) dynamically expressed in PDGFRα(+) bone marrow-derived mesenchymal stem cells (BMMSCs) during natural tendon-bone healing. Then, we investigated the role of PDGFRα(+) BMMSCs in tendon-bone healing after Scx overexpression as well as the underlying mechanisms. Our data demonstrated that Scx-overexpressing PDGFRα(+) BMMSCs (BMMSCScx) could efficiently inhibit peritunnel osteolysis and enhance tendon-bone healing strength by preventing osteoclastogenesis in an exosomes-dependent manner. Exosomal RNA-seq revealed that the abundance of a novel miRNA, miR-6924-5p, was highest among miRNAs. miR-6924-5p could directly inhibit osteoclast formation by binding to the 3'-untranslated regions (3'UTRs) of OCSTAMP and CXCL12. Inhibition of miR-6924-5p expression reversed the prevention of osteoclastogenic differentiation by BMMSCScx derived exosomes (BMMSCScx-exos). Local injection of BMMSCScx-exos or miR-6924-5p dramatically reduced osteoclast formation and improved tendon-bone healing strength. Furthermore, delivery of miR-6924-5p efficiently inhibited the osteoclastogenesis of human monocytes. In brief, our study demonstrates that BMMSCScx-exos or miR-6924-5p could serve as a potential therapy for the treatment of osteolysis during tendon-bone healing and improve the outcome.

Mash-1 modified neural stem cells transplantation promotes neural stem cells differentiation into neurons to further improve locomotor functional recovery in spinal cord injury rats.

BACKGROUND: Spinal cord injury (SCI) leads to severe motor and sensory dysfunctions. Neural stem cells (NSCs) transplantation therapy plays a positive role in functional recovery after SCI, but the effectiveness of this therapy is limited by inadequate differentiation ability of transplanted NSCs. Mammalian achaete-scute homologue-1 (Mash-1) has been reported to improve differentiation of NSCs. Thus, this study modified NSCs with Mash-1 to repair SCI. METHODS: NSCs isolated from rat embryo hippocampus were cultured and identified in vitro and further transfected with the lentiviral vectors (Lv-Mash-1). After establishing a SCI rat model, the rats were transplanted with Mash-1 modified NSCs, the histopathological changes of rat spinal cord were detected by hematoxylin-eosin (HE) staining, and the locomotor activity of rats was evaluated with the Basso, Beattie and Bresnahan (BBB) scale. The NSCs cultured in vitro or extracted from SCI rat spinal cord were identified by immunofluorescence (IF). Mash-1, ß3-Tubulin, and NeuN expressions in those cells were determined by Western blotting and reverse transcription­quantitative polymerase chain reaction (RT­qPCR). RESULTS: NSCs isolated from rat embryo hippocampus were Nestin- and NeuN-positive. NSC transplantation modified by Mash-1 increased BBB score of SCI rats and promoted recovery in lesion site of SCI rats. Mash-1 overexpression also promoted ß3-Tubulin and NeuN expressions in NSCs cultured in vitro or extracted from spinal cord of SCI rats. CONCLUSION: Mash-1 overexpression promoted NSC differentiation into neurons, and further improved locomotor functional recovery of SCI rats.

Neuroprotective effect of olfactory ensheathing cells co-transfected with Nurr1 and Ngn2 in both in vitro and in vivo models of Parkinson's disease.

AIMS: The aim of the study is to evaluate the neuroprotective effects of olfactory ensheathing cells (OECs) with the overexpression of nuclear receptor-related factor 1 (Nurr1) and neurogenin 2 (Ngn2) in experimental models of Parkinson's disease (PD) and to elucidate the potential mechanism underlying the neuroprotective effects of OECs-Nurr1-Ngn2. MATERIALS AND METHODS: In vitro study, OECs-Nurr1-Ngn2 conditioned medium (CM) was added to MPP+-treated PC12 cells for 24h, and then the viability of PC12 cells, oxidative stress and apoptosis were detected. In vivo study, 48 male Sprague-Dawley (SD) rats were randomly divided into four groups. OECs/VMCs and OECs-Nurr1-Ngn2/VMCs groups were transplanted with 2×105 cells each of OECs or OECs-Nurr1-Ngn2 and VMCs into the right striatum one week after a unilateral 6-OHDA lesion. Control and PD groups were injected with 0.9% NaCl and 0.2% ascorbic acid into the same region. Rotational behavior was determined at 2, 4, 6 and 8weeks after injection or implantation in all groups. Neuronal differentiation markers, oxidative stress- and apoptosis-related indicators were detected at 8weeks post-grafting. KEY FINDINGS: OECs-Nurr1-Ngn2 increased the viability of PC12 cells, inhibited oxidative stress and apoptosis, and these effects could be reversed by pre-treatment of k252a, a TrkB receptor inhibitor. The behavioral deficits of PD rat were ameliorated by the transplantation of OECs-Nurr1-Ngn2/VMCs. SIGNIFICANCE: These results suggest that OECs-Nurr1-Ngn2 exhibits substantial neuroprotective, anti-oxidant, and anti-apoptotic effects against PD via the up-regulation of the neurotrophic factor-TrkB pathway.

Lentivirus carrying the NeuroD1 gene promotes the conversion from glial cells into neurons in a spinal cord injury model.

In this study, we aimed to reprogram reactive glial cells into neural stem cells and neurons in vivo, by up-regulating NeuroD1. 60 rats were randomly divided into three groups with equal numbers. All rats received a spinal cord injury. Group A received only normal saline injection. Group B received unloaded lentivirus injection. Group C received NeuroD1 recombinant lentivirus injection. The BBB scores of the animals were documented at 3, 7, 14, 21days after SCI. 5 random rats of each group were removed at those timings after SCI and sacrificed. In the spinal cord slices, the numbers and morphology of neural stem cells, immature neurons and mature neurons were examined after immunohistochemical staining. Except day 3 after SCI, the BBB scores of group C on days 7, 14 and 21 were significantly higher than the other two groups (P<0.05). Compared with the group A and B, a remarkable increase was noticed in the number of positive cells in the group C (P<0.05). These findings suggest that NeuroD1 up-regulated cells can be reprogrammed into neural stem cells, immature and functional neurons. It may be possible to replace neurons after injury by endogenous means, as a first step toward spinal cord repair.

Targeted delivery of neurogenin-2 protein in the treatment for cerebral ischemia-reperfusion injury.

Neurogenin-2 (Ngn2), as a proneural gene that promotes the survival and differentiation of neural precursor cells, is an attractive candidate for therapy against cerebral ischemia-reperfusion injury. However, the delivery approach limits its clinical application. To deliver Ngn2 protein into the cerebral ischemic region and exert a therapeutic effect on injured neurons after ischemia, we here reported that the fusion protein TAT-LBD-Ngn2 was constructed by fusing a transactivator of transcription (TAT) domain and a laminin-binding domain (LBD) to Ngn2. TAT-LBD-Ngn2 promoted the outgrowth of neuronal neurite, increased the survival rate and alleviated apoptosis of hippocampal neurons exposed to oxygen glucose deprivation in vitro. Furthermore, a focal cerebral ischemia model in C57BL/6 mice showed that TAT-LBD-Ngn2 efficiently crossed the blood brain barrier, aggregated in the ischemic zone and was consistently incorporated into neurons. Moreover, TAT-LBD-Ngn2 transduced into brains attenuated neuronal degeneration and apoptosis in the ischemic zone. TAT-LBD-Ngn2 treatment resulted in a reduction of infarct volume that was associated with a parallel improvement in neurological functional outcomes after reperfusion. In conclusion, the targeted delivery of TAT-LBD-Ngn2 into the ischemic zone attenuated cerebral ischemia-reperfusion injury through the inhibition of neuronal degeneration and apoptosis, suggesting that TAT-LBD-Ngn2 is a promising target candidate for the treatment of ischemic stroke.

Atoh1 induces auditory hair cell recovery in mice after ototoxic injury.

OBJECTIVES/HYPOTHESIS: To evaluate the ability of the Ad28.gfap.atoh1 to promote hair cell regeneration and hearing recovery in cochlea injured with kanamycin and furosemide. STUDY DESIGN: In vivo model of hair cell ablation and subsequent treatment with Atoh1. METHODS: The hair cells of C57BL/6 mice were ablated with systemic administration of kanamycin and furosemide. The left ears were treated with Ad28.gfap.atoh1. The right ears were not treated. Preablation audiograms and distortion product otoacoustic emissions (DPOAEs) were compared to 1- or 2-month postablation studies. Harvested cochleae were examined for histologic evidence of hair cell regeneration and spiral ganglion cell survival. RESULTS: Delivery of Ad28.gfap.atoh1 results in development of auditory hair cells. There was no recovery of DPOAEs at 1 or 2 months post-treatment. Two months after delivery of Ad28.gfap.atoh1, the left ear exhibited a moderate recovery of hearing at 4 and 8 kHz (P < .01). There was no significant difference at 16 or 32 kHz. One month after treatment, myosin VII-positive immunohistochemical staining can be seen in both the inner and outer hair cells of the treated ear. In the untreated ear, minimal myosin VII-positive debris is seen, with no indication of normal hair cells. Two months after ablation, there is evidence of hair cell recovery on the treated side, whereas the untreated cochlea demonstrates a flattened epithelium. Untreated ears showed decreased spiral ganglion cell density at the basal turn compared to treated ears. CONCLUSIONS: Ad28.gfap.atoh1 promotes hair cell regeneration in cochlea ablated with kanamycin and furosemide resulting in moderate hearing recovery.

Epigenetic regulation of hypoxia inducible factor in diseases and therapeutics.

Hypoxia-inducible factors (HIFs) are master regulators of angiogenesis and cellular adaptation in hypoxic microenvironments. Accumulating evidence indicates that HIFs also regulate cell survival, glucose metabolism, microenvironmental remodeling, cancer metastasis, and tumor progression, and thus, HIFs are viewed as therapeutic targets in many diseases. Epigenetic changes are involved in the switching 'on' and 'off' of many genes, and it has been suggested that the DNA hypermethylation of specific gene promoters, histone modifications (acetylation, phosphorylation, and methylation) and small interfering or micro RNAs be regarded epigenetic gene targets for the regulation of disease-associated cellular changes. Furthermore, the hypoxic microenvironment is one of the most important cellular stress stimuli in terms of the regulation of cellular epigenetic status via histone modification. Therefore, drug development and therapeutic approaches to ischemic diseases or cancer for targeting HIFs by modulation of epigenetic status become an attractive area. Here, the authors provide a review of the literature regarding the targeting of HIF, a key modulator of hypoxic-cell response under various disease conditions, by modulating histone or DNA using endogenous small RNAs or exogenous chemicals.