Effects of spinal cord injury associated exosomes delivered tRF-41 on the progression of spinal cord injury progression.

BACKGROUND: Spinal cord injury (SCI) is a devastating neurological and pathological condition. Exosomal tsRNAs have reported to be promising biomarkers for cancer diagnosis and therapy. This study aimed to investigate the roles of SCI-associated exosomes, and related tsRNA mechanisms in SCI. METHODS: The serum of healthy controls and SCI patients at the acute stage were collected for exosomes isolation, and the two different exosomes were used to treat human astrocytes (HA). The cell viability, apoptosis, and cycle were determined, and the expression of the related proteins were detected by western blot. Then, the two different exosomes were sent for tsRNA sequencing, and four significant known differentially expressed tsRNAs (DE-tsRNAs) were selected for RT-qPCR validation. Finally, tRT-41 was chosen to further explore its roles and related mechanisms in SCI. RESULTS: After sequencing, 21 DE-tsRNAs were identified, which were significantly enriched in pathways of Apelin, AMPK, Hippo, MAPK, Ras, calcium, PI3K-Akt, and Rap1. RT-qPCR showed that tRF-41 had higher levels in the SCI-associated exosomes. Compared with the control HA, healthy exosomes did not significantly affect the growth of HA cells, but SCI-associated exosomes inhibited viability of HA cells, while promoted their apoptosis and increased the HA cells in G2/M phase; but tRF-41 inhibitor reversed the actions of SCI-associated exosomes. Additionally, SCI-associated exosomes, similar with tRF-41 mimics, down-regulated IGF-1, NGF, Wnt3a, and ß-catenin, while up-regulated IL-1ß and IL-6; but tRF-41 inhibitor had the opposite actions, and reversed the effects induced by SCI-associated exosomes. CONCLUSIONS: SCI-associated exosomes delivered tRF-41 may inhibit the growth of HA through regulating Wnt/ ß-catenin pathway and inflammation response, thereby facilitating the progression of SCI.

Elucidation of pericholangitis and periductal fibrosis in cholestatic liver diseases via extracellular vesicles released by polarized biliary epithelial cells.

Cholestatic liver diseases causes inflammation and fibrosis around bile ducts. However, the pathological mechanism has not been elucidated. Extracellular vesicles (EVs) are released from both the basolateral and apical sides of polarized biliary epithelial cells. We aimed to investigate the possibility that EVs released from the basolateral sides of biliary epithelial cells by bile acid stimulation induce inflammatory cells and fibrosis around bile ducts, and they may be involved in the pathogenesis of cholestatic liver disease. Human biliary epithelial cells (H69) were grown on cell culture inserts and stimulated with chenodeoxycholic acid + IFN-γ. Human THP-1-derived M1-macrophages, LX-2 cells, and KMST-6 cells were treated with the extracted basolateral EVs, and inflammatory cytokines and fibrosis markers were detected by RT-PCR. Highly expressed proteins from stimulated EVs were identified, and M1-macrophages, LX-2, KMST-6 were treated with these recombinant proteins. Stimulated EVs increased the expression of TNF, IL-1ß, and IL-6 in M1-macrophages, TGF-ß in LX-2 and KMST-6 compared with the corresponding expression levels in unstimulated EVs. Nucleophosmin, nucleolin, and midkine levels were increased in EVs from stimulated cells compared with protein expression in EVs from unstimulated cells. Leukocyte cell-derived chemotaxin-2 (LECT2) is highly expressed only in EVs from stimulated cells. Stimulation of M1-macrophages with recombinant nucleophosmin, nucleolin, and midkine significantly increased the expression of inflammatory cytokines. Stimulation of LX-2 and KMST-6 with recombinant LECT2 significantly increased the expression of fibrotic markers. These results suggest that basolateral EVs are related to the development of pericholangitis and periductal fibrosis in cholestatic liver diseases.NEW & NOTEWORTHY Our research elucidated that the composition of basolateral EVs from the biliary epithelial cells changed under bile acid exposure and the basolateral EVs contained the novel inflammation-inducing proteins NPM, NCL, and MK and the fibrosis-inducing protein LECT2. We report that these new results are possible to lead to the potential therapeutic target of cholestatic liver diseases in the future.

Elevated Midkine Serum Levels Are Associated with Long-Term Survival in Critically Ill Patients.

Midkine (Mdk) is a multifunctional protein involved in inflammatory processes. Hence, circulating Mdk is increased in sepsis and has been previously suggested as a potential biomarker in these patients. The aim of this study was to elucidate the role of Mdk serum concentrations in critical illness and sepsis and to verify its value as a prognostic biomarker. Thus, we analyzed the Mdk serum concentrations of 192 critically ill patients on admission to the medical intensive care unit (ICU). While the serum levels of Mdk at admission were similar in septic and nonseptic critical illness (362 vs. 337 ng/L, p = 0.727), we found several interesting correlations of Mdk to laboratory and clinical markers associated with ischemia or hypoxia, e.g., to renal failure and hepatic injury. Mdk serum concentrations at admission did not differ between various causes of sepsis or other critical illness. Most noticeable, we observed upregulated Mdk serum concentrations at admission in patients surviving in the long-term, which was only seen in nonseptic critical illness but not in sepsis. Our study suggests a relevant role of Mdk in critically ill patients in general and highlights the possible protective features of Mdk in critical illness.

Relationship between serum Midkine and Omentin-1 levels and the severity of sepsis in patients and their prognostic value.

To explore the relationship between serum levels of midkine and omentin-1 and the severity of sepsis in patients, and their prognostic value. A retrospective analysis was conducted on the clinical data of 180 sepsis patients. According to the severity of the patient's condition, they were separated into sepsis group (n = 76), severe sepsis group (n = 59), and sepsis shock group (n = 45). Based on the survival within 28 days of admission, they were grouped into survivors group (n = 128) and nonsurvivors group (n = 52). The serum Midkine level and APACHE II score in the sepsis shock group were higher than those in the severe sepsis group and sepsis group, while the Omentin-1 level was lower than that in the severe sepsis group and sepsis group (p < 0.05). The serum Midkine level and APACHE II score in the severe sepsis group were higher than those in the sepsis group, while the Omentin-1 level was lower than that in the sepsis group (p < 0.05). The Midkine and APACHE II score in the nonsurvivors group was higher than those in the survivors group, while the Omentin-1 score was lower than that in the survivors group (p < 0.05). Midkine and APACHE II score were independent risk factors for the prognosis of sepsis patients, while Omentin-1 was a protective factor for the prognosis of sepsis patients (p < 0.05). The AUC of the combined prediction of serum Midkine and Ommentin-1 for the prognosis of sepsis patients was 0.880, with a sensitivity of 90.38% and a specificity of 72.66%. The combined prediction of serum Midkine and Ommentin-1 was better than that of individual prediction of Midkine and Ommentin-1. Serum Midkine is highly expressed and Omentin-1 is lowly expressed in sepsis patients, and the combination of the two has a high predictive power for the prognosis of sepsis patients.

Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker.

Midkine (MDK) is a multifunctional secreted protein that can act as a cytokine or growth factor regulating multiple signaling pathways and being implicated in fundamental cellular processes, such as survival, proliferation, and migration. Although its expression in normal adult tissues is barely detectable, MDK serum levels are found to be elevated in several types of cancer, including hepatocellular carcinoma (HCC). In this review, we summarize the findings of recent studies on the role of MDK in HCC diagnosis and progression. Overall, studies show that MDK is a powerful biomarker for HCC early diagnosis, as it can differentiate not only between HCC patients and normal individuals but also between HCC patients and patients with other liver pathologies. It is correlated with high recurrence rates and was shown to be valuable for the diagnosis of early-stage HCC, even in patients negative for α-fetoprotein (AFP), the most commonly used biomarker for HCC diagnosis. A comparison with AFP reveals that MDK is inferior to AFP with regard to specificity but significantly superior with regard to sensitivity, which further indicates the need for using both biomarkers for more effective HCC diagnosis.

Midkine promotes renal fibrosis by stabilizing C/EBPß to facilitate endothelial-mesenchymal transition.

Numerous myofibroblasts are arisen from endothelial cells (ECs) through endothelial to mesenchymal transition (EndMT) triggered by TGF-ß. However, the mechanism of ECs transforms to a different subtype, or whether there exists an intermediate state of ECs remains unclear. In present study, we demonstrate Midkine (MDK) mainly expressed by CD31 + ACTA2+ECs going through partial EndMT contribute greatly to myofibroblasts by spatial and single-cell transcriptomics. MDK is induced in TGF-ß treated ECs, which upregulates C/EBPß and increases EndMT genes, and these effects could be reversed by siMDK. Mechanistically, MDK promotes the binding ability of C/EBPß with ACTA2 promoter by stabilizing the C/EBPß protein. In vivo, knockout of Mdk or conditional knockout of Mdk in ECs reduces EndMT markers and significantly reverses fibrogenesis. In conclusion, our study provides a mechanistic link between the induction of EndMT by TGF-ß and MDK, which suggests that blocking MDK provides potential therapeutic strategies for renal fibrosis.

Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis.

BACKGROUND: Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. METHODS: We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. FINDINGS: CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. INTERPRETATION: MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. FUNDING: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.

Novel Midkine Inhibitor Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

BACKGROUND/AIM: Multiple myeloma (MM), the second most common hematological malignancy, is characterized by the accumulation of malignant plasma cells within the bone marrow. Despite various drug classes for MM treatment, it remains incurable, necessitating novel and efficacious agents. This study aims to explore the anti-cancer activity of a midkine inhibitor, iMDK (C21H13FN2O2S), in myeloma cell lines. MATERIALS AND METHODS: This study assessed the antiproliferative activity using the MTT assay. Cell cycle and apoptosis were evaluated using flow cytometry. To further investigate the inhibitory mechanism, western blotting was used to detect cell cycle-related proteins, pro-apoptotic proteins, and anti-apoptotic proteins. RESULTS: iMDK inhibits MM cell proliferation in a dose- and time-dependent manner, inducing cell cycle arrest and apoptosis. The reduction in Cdc20 expression by iMDK treatment leads to G2/M phase cell cycle arrest. Furthermore, iMDK down-regulates anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1, and c-FLIP), thereby activating both intrinsic and extrinsic apoptosis pathways. CONCLUSION: iMDK could be a potential candidate for MM treatment.

RNA Interference based Midkine Gene Therapy for Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) arises from hepatocytes and accounts for 90% of primary liver cancer. Reasons for HCC prognosis remaining dismal are that HCC is asymptomatic in its early stages, leading to late diagnosis, and it is markedly resistant to conventional chemo- and radiotherapy. In this study, we investigated RNA interference (RNAi)-based treatment for HCC by targeting MDK. AIM: The present study aimed to evaluate MDK serum levels as a diagnostic biomarker for HCC detection and the effect of MDK silencing by RNAi on HCC. SUBJECTS AND METHODS: A total of 140 participants, including 120 patients diagnosed with HCC and 20 healthy volunteers were enrolled in this study, all patients who underwent liver resection were sampled for tumor and adjacent non-tumor liver tissues, in addition to 5 ml of blood sample. Midkine expression levels were evaluated by ELISA and by qRT-PCR. The in vitro transfection and gene knockdown efficiency of midkine by MDK-siRNA was detected by qRT-PCR and ELISA. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by cell counting. RESULTS: The results showed that the expression of MDK was significantly increased in the serum of HCC patients compared to control serum samples with P<0.001 and significant elevated expression levels of MDK in tumor tissues compared to non-tumor ones with P<0.001. It also showed that down-regulation of MDK using RNAi can significantly inhibit HepG2 cells. CONCLUSION: Molecular targeting of MDK using RNAi interference decreases proliferation and could be a therapeutic target.

Precise Photodynamic Therapy by Midkine Nanobody-Engineered Nanoparticles Remodels the Microenvironment of Pancreatic Ductal Adenocarcinoma and Potentiates the Immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is notorious for its resistance against chemotherapy and immunotherapy due to its dense desmoplastic and immunosuppressive tumor microenvironment (TME). Traditional photodynamic therapy (PDT) was also less effective for PDAC owing to poor selectivity, insufficient penetration, and accumulation of photosensitizers in tumor sites. Here, we designed a light-responsive novel nanoplatform targeting the TME of PDAC through tumor-specific midkine nanobodies (Nbs), which could efficiently deliver semiconducting polymeric nanoparticles (NPs) to the TME of PDAC and locally produce abundant reactive oxygen species (ROS) for precise photoimmunotherapy. The synthesized nanocomposite can not only achieve multimodal imaging of PDAC tumors (fluorescence and photoacoustic imaging) but also lead to apoptosis and immunogenic cell death of tumor cells via ROS under light excitation, ultimately preventing tumor progression and remodeling the immunosuppressive TME with increased infiltration of T lymphocytes. Combined with a PD-1 checkpoint blockade, the targeted PDT platform showed the best antitumor performance and markedly extended mice survival. Conclusively, this work integrating Nbs with photodynamic NPs provides a novel strategy to target formidable PDAC to achieve tumor suppression and activate antitumor immunity, creating possibilities for boosting efficacy of immunotherapy for PDAC tumors through the combination with precise local PDT.

NF1 mutation-driven neuronal hyperexcitability sets a threshold for tumorigenesis and therapeutic targeting of murine optic glioma.

BACKGROUND: With the recognition that noncancerous cells function as critical regulators of brain tumor growth, we recently demonstrated that neurons drive low-grade glioma initiation and progression. Using mouse models of neurofibromatosis type 1 (NF1)-associated optic pathway glioma (OPG), we showed that Nf1 mutation induces neuronal hyperexcitability and midkine expression, which activates an immune axis to support tumor growth, such that high-dose lamotrigine treatment reduces Nf1-OPG proliferation. Herein, we execute a series of complementary experiments to address several key knowledge gaps relevant to future clinical translation. METHODS: We leverage a collection of Nf1-mutant mice that spontaneously develop OPGs to alter both germline and retinal neuron-specific midkine expression. Nf1-mutant mice harboring several different NF1 patient-derived germline mutations were employed to evaluate neuronal excitability and midkine expression. Two distinct Nf1-OPG preclinical mouse models were used to assess lamotrigine effects on tumor progression and growth in vivo. RESULTS: We establish that neuronal midkine is both necessary and sufficient for Nf1-OPG growth, demonstrating an obligate relationship between germline Nf1 mutation, neuronal excitability, midkine production, and Nf1-OPG proliferation. We show anti-epileptic drug (lamotrigine) specificity in suppressing neuronal midkine production. Relevant to clinical translation, lamotrigine prevents Nf1-OPG progression and suppresses the growth of existing tumors for months following drug cessation. Importantly, lamotrigine abrogates tumor growth in two Nf1-OPG strains using pediatric epilepsy clinical dosing. CONCLUSIONS: Together, these findings establish midkine and neuronal hyperexcitability as targetable drivers of Nf1-OPG growth and support the use of lamotrigine as a potential chemoprevention or chemotherapy agent for children with NF1-OPG.

The Association of Serum Midkine Level with Invasion in Placenta Previa: A Case-Control Study from a Tertiary Reference Center.

We aimed to examine the relationship between serum midkine levels and placental invasion in pregnant women with placenta previa. The study group consisted of 43 pregnant women diagnosed with placenta previa, whereas the control group consisted of 60 healthy pregnant women. Serum midkine levels were compared between pregnant women with placenta previa and the control group in this study's first part. Thereafter, the utility of midkine in the prediction of the abnormally invasive placenta (AIP) was investigated and optimal cutoff values were calculated. Significantly higher serum midkine level was observed in placenta previa cases than in the controls (1.16 ng/mL vs. 0.18 ng/mL, P < 0.001). Serum midkine level was also significantly higher in the AIP group among the placenta previa cases (P = 0.004). In the receiver operating characteristic analysis, the cutoff value of the midkine level in predicting AIP was 1.19 ng/mL. This study revealed that the serum midkine level is higher in pregnant women with AIP. Maternal serum midkine level may be used as a complementary biomarker to the radiological and clinical findings for the prediction of the AIP in placenta previa cases.

The role of midkine in health and disease.

Midkine (MDK) is a neurotrophic growth factor highly expressed during embryogenesis with important functions related to growth, proliferation, survival, migration, angiogenesis, reproduction, and repair. Recent research has indicated that MDK functions as a key player in autoimmune disorders of the central nervous system (CNS), such as Multiple Sclerosis (MS) and is a promising therapeutic target for the treatment of brain tumors, acute injuries, and other CNS disorders. This review summarizes the modes of action and immunological functions of MDK both in the peripheral immune compartment and in the CNS, particularly in the context of traumatic brain injury, brain tumors, neuroinflammation, and neurodegeneration. Moreover, we discuss the role of MDK as a central mediator of neuro-immune crosstalk, focusing on the interactions between CNS-infiltrating and -resident cells such as astrocytes, microglia, and oligodendrocytes. Finally, we highlight the therapeutic potential of MDK and discuss potential therapeutic approaches for the treatment of neurological disorders.

Niveles plasmáticos de midkina en pacientes con trastorno por uso de cocaína en abstinencia / Plasma midkine levels in patients with cocaine use disorder during abstinence

Diversos estudios preclínicos han sugerido que la midkina endógenapodría jugar un papel modulador clave sobre los efectos neurotóxicosy adictivos de distintas drogas, incluidos los psicoestimulantes. Estahipótesis no ha sido aún explorada en humanos. Como primer pasoen esta dirección, en el presente trabajo hemos medido los nivelesplasmáticos de midkina en 75 pacientes con trastorno por uso decocaína en abstinencia y 26 controles apareados con los anteriorespor sexo, edad e índice de masa corporal. Los pacientes fueronademás divididos en un grupo de abstinencia temprana (menos deun mes, n = 30) y otro de abstinencia tardía (más de un mes, n =45). Se cuantificaron los niveles plasmáticos de midkina de todoslos participantes mediante un ensayo por inmunoabsorción ligadoa enzimas. Los pacientes en abstinencia temprana mostraron unincremento del 60% en su concentración plasmática de midkina conrespecto a los controles que tendió a desaparecer en los pacientes conperiodos de abstinencia más prolongados. Los resultados demuestranque los niveles periféricos de midkina están estrechamenterelacionados con el uso de cocaína y apoyan la idea de que dichacitoquina podría jugar un papel protector limitando la actividadbiológica de los psicoestimulantes. (AU)

Preclinical evidence suggests that endogenous midkine couldplay a key modulatory role on the neurotoxic and addictive effectsof different kinds of drugs of abuse, including psychostimulants.However, this hypothesis has not yet been explored in humans. As afirst approach to progress in this knowledge, we have comparativelystudied plasma midkine levels in 75 patients with cocaine use disorderunder abstinence and 26 control subjects matched for sex, ageand body mass index. Patients were further segmented into earlyabstinent (up to one month of abstinence, n = 30) and late-abstinent(more than one month of abstinence, n = 45). Midkine levels werequantified in plasma samples of all the participants by enzyme-linkedimmunosorbent assays. Early-abstinent patients exhibited a 60%increase of midkine plasma concentration in comparison with thecontrols. This elevation tended to normalize upon the progressionof abstinence. The results obtained demonstrate that peripheralmidkine levels are closely related to cocaine use and are consistentwith the idea that this cytokine could play a protective role by limitingthe biological activity of psychostimulants. (AU)

H. sinensis mycelium inhibits epithelial-mesenchymal transition by inactivating the midkine pathway in pulmonary fibrosis / 医学前沿

The medical fungus Hirsutella sinensis has been used as a Chinese folk health supplement because of its immunomodulatory properties. Our previous studies established the antifibrotic action of Hirsutella sinensis mycelium (HSM) in the lung. The epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of idiopathic pulmonary fibrosis. The present study investigates the role of HSM in mediating EMT during the development of pulmonary fibrosis. HSM significantly inhibits bleomycin (BLM)-induced pulmonary fibrosis by blocking the EMT. In addition, the expression levels of midkine are increased in the lungs of the BLM-induced group. Further analysis of the results indicates that the mRNA level of midkine correlated positively with EMT. HSM markedly abrogates the transforming growth factor β-induced EMT-like phenotype and behavior in vitro. The activation of midkine related signaling pathway is ameliorated following HSM treatment, whereas this extract also caused an effective attenuation of the induction of EMT (caused by midkine overexpression) in vitro. Results further confirm that oral medication of HSM disrupted the midkine pathway in vivo. Overall, findings suggest that the midkine pathway and the regulation of the EMT may be considered novel candidate therapeutic targets for the antifibrotic effects caused by HSM.

The expressions of TP, MK and CD105 in laryngeal squamous cell carcinoma and their clinical significance / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To explore the expressions of thymidine phosphorylase (TP), midkine (MK) and MVD marked with CD105 antibody in laryngeal squamous cell carcinoma (LSCC) and their clinical significance.@*METHOD@#The expressions of TP, MK and CD105 in LSCC tissues of 43 cases were studied by immunohistochemical staining.@*RESULT@#The positive expression rates of TP and MK in LSCC were 67.4% and 60.5% respectively, the mean value of MVD was 6.01 +/- 1.78. MVD was significantly higher in tumor tissue with both positive TP and MK than in that with both negative TP and MK (7.07 +/- 3.26 vs. 4.03 +/- 1.90, P < 0.05). The expression of TP, MK and CD105 were all correlated with T-stage and lymph node metastasis. Positive TP, MK expression and high MVD were all associated with a poor survival, and positive expression of both TP and MK in tumors conferred a poorer prognosis than negative expression of those factors in tumors, but only the lymph node metastasis and MVD were independent prognostic factors on multivariate analysis.@*CONCLUSION@#Both TP and MK are important for angiogenesis in LSCC. TP, MK and angiogenesis are all closely correlated with the progress of LSCC and the lymph node metastasis. The lymph node metastasis and MVD marked with CD105 antibody were independent prognostic factors. TP and MK may affect the progression and prognosis of tumor by promotion of angiogenesis. A combinative detection of TP, MK and CD105 can be as valuable tumor marker and prognostic factor for LSCC.