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Midkine (MK), a heparin-binding growth factor, is associated with the poor prognosis of the pediatric tumor, neuroblastoma. MK would be a druggable target as many studies showed inhibition of its function in various cancers suppressed tumor developments. To establish the therapy targeting MK, identification of its binding partners, and elucidation of its intracellular signaling are needed. It was reported that exogenous MK induced phosphorylation of ribosomal protein S6 (RPS6) downstream of mTOR signaling. Using RPS6 phosphorylation as a marker of MK response, we searched for MK reactive cell lines. We found that MK cell lines expressing less MK tended to respond better to MK. Next, using an MK reactive neuroblastoma cell line, MK-knocked down SH-SY5Y cells, we employed a proximity-dependent biotin identification method, which was invented to evaluate protein-protein interactions by biotinylation. We confirmed that secreted MK fused to the biotin ligase BioID2 (MK-BioID2) was able to biotinylate proteins from the cells. Biotinylated proteins were identified by liquid chromatography-mass spectrometry analyses. Twenty five proteins were found to be overlapped after three independent experiments, among which insulin-like growth binding protein 2 (IGFBP2) was further analyzed. IGFBP2 was indeed detected with immunoblotting after streptavidin pull down of MK-BioID2 labeled cell extract of MK-knocked down SH-SY5Y cells. Our study suggests that the BioID2 method is useful to identify binding partners of growth factors.

Primary effusion lymphoma (PEL) is an aggressive B-cell non-Hodgkin lymphoma in immunocompromised in-dividuals such as AIDS patients. PEL shows a poor prognosis (median survival time < 6 months) compared with other AIDS-related lymphomas, and is generally resistant to conventional treatments. Novel drugs for PEL treatment are required. Midkine inhibitor (iMDK) was previously found to suppress midkine protein expression. Interestingly, iMDK suppressed cell proliferation in PEL cell lines in a time- and dose-dependent manner, regardless of midkine gene expression. We examined the mechanism of iMDK on PEL. Importantly, iMDK strongly induced cell cycle arrest at the G2/M phase within 12 h of incubation and suppressed the p-CDK1 protein level, which is associated with the cell cycle checkpoint at G2/M, resulting in mitotic catastrophe with observation of multipolar division. After mitotic catastrophe, iMDK-treated PEL showed apoptosis with caspase-3, 8, and 9 activation at 24 h incubation. However, iMDK showed no effects on viral protein-activated signaling pathways such as JAK-STAT, PI3K-Akt and NF-κB, and HHV-8/KSHV gene expression in PEL. These results indicate that iMDK is a novel CDK1 inhibitor and a promising lead compound for PEL chemotherapy treatment., Leukemia Research, 116, art. no. 106826; 2022

Background & Aims Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. Methods We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. Results We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. Conclusions This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. Lay summary We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.

Midkine (MK) is a small secreted heparin-binding protein highly expressed during embryonic/fetal development which, through interactions with multiple cell surface receptors promotes growth through effects on cell proliferation, migration, and differentiation. MK is upregulated in the adult central nervous system (CNS) after multiple types of experimental injury and has neuroprotective and neuroregenerative properties. The potential for MK as a therapy for developmental brain injury is largely unknown. This review discusses what is known of MK's expression and actions in the developing brain, areas for future research, and the potential for using MK as a therapeutic agent to ameliorate the effects of brain damage caused by insults such as birth-related hypoxia and inflammation.

Alpha fetoprotein (AFP) level is the gold standard diagnostic tool for detection and monitoring hepatocellular carcinoma (HCC) but with low sensitivity. Thus, the identification of alternative or combined serum markers of HCC is highly needed. Therefore, the aim of this work was to verify the value of serum midkine (MDK), Dickkopf-related protein 1 (DKK1), and alpha-L-fucosidase (AFU) in detection of HCC. We recruited 244 subjects to the present study; 89 with liver cirrhosis, 86 cirrhotic hepatitis C virus (HCV) induced HCC, and 69 apparently healthy volunteers as controls. Serum AFP, MDK, DKK1, and AFU were measured by ELISA. Patients with HCC showed significantly higher serum MDK, DKK1, and AFU levels compared with those patients with liver cirrhosis and healthy controls (X2 = 179.56, 153.94, and 90.07 respectively) (P 5 cm, those with portal vein thrombosis and advanced HCC stage. Receiver operator characteristic (ROC) curve analysis showed that serum MDK levels discriminated between cirrhosis and HCC at a sensitivity of 100%, a specificity of 90% at cut-off value of >5.1 ng/mL. Although our results showed that serum MDK, DKK-1, and AFU are increased in HCC cases only MDK may be considered as the most promising serological marker for the prediction of the development of HCC in cirrhotic HCV patients.

Hepatocellular carcinoma (HCC) is a fatal disease with limited therapeutic choices. The stroma-rich tumor microenvironment hinders the in vivo delivery of most nanomedicines. Ultra-small lipid nanoparticles (usLNPs) were designed for the selective co-delivery of the cytotoxic drug, sorafenib (SOR), and siRNA against the Midkine gene (MK-siRNA) to HCC in mice. The usLNPs composed of a novel pH-sensitive lipid, a diversity of phospholipids and a highly-selective targeting peptide. A microfluidic device, iLiNP, was used and a variety of factors were controlled to tune particle size aiming at maximizing tumor penetration efficiency. Optimizing the composition and physico-chemical properties of the usLNPs resulted in an enhanced tumor accumulation, selectivity and in vivo gene silencing. The optimized usLNPs exerted potent gene silencing in the tumor (median effective dose, ED50~0.1 mg/Kg) with limited effect on the healthy liver. The novel combination synergistically-eradicated HCC in mice (~85%) at a surprisingly-low dose of SOR (2.5 mg/Kg) which could not be achieved via individual monotherapy. Toxicity studies revealed the biosafety of the usLNPs upon either acute or chronic treatment. Furthermore, the SOR-resistant HCC established in mice was eradicated by 70% using this approach. We conclude that our strategy is promising for potential clinical applications in HCC treatment.

BackgroundMidkine is a multi-functional molecule participating in a various key pathological process. We aimed to evaluate the change of midkine in sepsis and its association with pulmonary function, as well as the mechanism by which midkine induced lung injury in sepsis. MethodsThe peripheral blood sample of septic patients on admission was obtained and measured for midkine, angiotensin-converting enzyme and angiotensin II. Cecal ligation and puncture (CLP) mouse model was used, and adeno-associated virus (AAV) was stilled trans-trachea for regional targeting midkine expression. The lung injury was evaluated, comparing the severity of lung injury. Furthermore, we studied the in vitro mechanism of midkine activates ACE system by using inhibitors targeting candidate receptors of midkine, and its effects on the vascular endothelial cells.ResultsPlasma midkine was significantly elevated in sepsis, and was closely associated with ACE system. Both circulating and lung midkine was increased in CLP mouse, and was related to severe lung injury. Regional interfering midkine expression in lung tissue by AAV could alleviate acute lung injury in CLP model. In vitro study elucidated that Notch 2 participated in the activation of ACE system and angiotensin II release, induced by midkine and triggered vascular endothelial injury by angiotensin II induced ROS production.ConclusionsMidkine participated in the process of sepsis induced lung injury, which was due to the activation of ACE system and angiotensin II release, triggering ROS production.Trial registration Clinicaltrials.gov NCT02605681. Registered 12 November 2015.

Background Early diagnosis of hepatocellular carcinoma enhances its effective management. Aim Evaluation of Midkine as a biomarker for diagnosis of hepatocellular carcinoma. Methods: 90 subjects devided into three groups, Group I included 40 HCV patients with liver cirrhosis, Group II included 40 HCV cirrhotic patients with hepatocellular carcinoma and Group III included 10 healthy subjects as a control group. Demographic, laboratory and imaging data were collected. All cirrhotic cases were evaluated by Child-Pugh and MELD scores while BCLC score and Okuda staging were applied for hepatocellular carcinoma cases. Serum Midkine was measured by ELISA technique. Results HCC group had significant elevation in Midkine level compared when to Cirrhotic and Control groups (3.5±2.5 ng/ml versus 1±0.7 ng/ml and 0.1±0.1 ng/ml) (p = 0.000). No significant correlations were found between Midkine and age, sex, site or size of focal lesion, Child classification, MELD score, Okuda staging or BCLC score. ROC analysis showed that the best cut-off value for Midkine was 1.33ng/ml and for AFP was 41.3ng/ml. Area Under the Curve was higher in Midkine than AFP (0.921 and 0.79 respectively) with higher specificity of AFP than Midkine (97.5% and 82.5% respectively) and higher sensitivity of Midkine than AFP (87.5 and 62.5% respectively). By combination of serum AFP and Midkine, AUC was 0.94 with specificity 97.5 % and sensitivity 87.5% Conclusion Midkine may be a sensitive biomarker for diagnosis of hepatocellular carcinoma and combination between alpha-fetoprotein and Midkine increases the accuracy in diagnosis.

Hypoxia-inducible factors (HIFs) induced by reduced O2 availability activate the transcription of target genes encoding proteins that play important roles in communication between cancer and stromal cells. Cancer cells were incubated under hypoxic conditions: H1299, A549 (NSCLC), Hep3B, HepG2 (HCC), HCT116, CT26 (Colon cancer), MCF-7, MDAMB231 (Breast cancer), MKN1, MKN5 (Gastric cancer), U87MG, SHSY5Y (Brain cancer), and SKOV3, SNU840 (Ovary cancer). All cells expressed HIF-1&alpha, and HIF-2&alpha, mRNA and proteins. However, cell proliferation of NSCLC, breast, gastric, and brain cancer cells under hypoxia was more dependent on HIF-1&alpha, except for HCC cells where it was more dependent on HIF-2&alpha, Among HIF-1&alpha, dependent cells H1299 was the most affected in terms of cell proliferation by HIF-1&alpha, knockdown. To examine which cytokines are secreted in NSCLC cells by HIF-1&alpha, to communicate with stromal cells, we performed a cytokine-profiling array with H1299. We screened the top 14 cytokines which were dependent on the HIF-1&alpha, expression pattern. Among them, midkine (MDK) expression was affected the most in response to HIF-1&alpha, MDK is a heparin-binding growth factor that promotes angiogenesis and carcinogenesis. Indeed, MDK significantly increased HUVEV endothelial cell migration and neo- vascularization in chick chorioallantoic membrane assay (CAM) assay via paracrine signaling. In addition, MDK secreted from NSCLC cells interacted with Notch2 which activated the Notch signaling pathway and induced EMT, upregulated NF-&kappa, B, and increased cancer promotion. However, in response to MDK knock down, siRNA or the MDK inhibitor, iMDK treatment not only decreased MDK-induced migration and angiogenesis of endothelial cells but also abrogated the progression and metastasis of NSCLC cells in in vitro and in vivo orthotopic and spontaneous lung metastasis models. Consequently, iMDK treatment significantly increased mice survival rates compared with the control or MDK expression group. MDK plays a very important role in the progression and metastasis of NSCLC cells. Moreover, the MDK targeting strategy provides a potential therapeutic target for the treatment of MDK-expressing lung cancers.

Uveal melanoma is a rare form of melanoma that originates in the eye, exerts widespread therapeutic resistance, and displays an inherent propensity for hepatic metastases. Because metastatic disease is characterized by poor survival, there is an unmet clinical need to identify new therapeutic targets in uveal melanoma. Here, we show that the pleiotropic cytokine midkine is expressed in uveal melanoma. Midkine expression in primary uveal melanoma significantly correlates with poor survival and is elevated in patients that develop metastatic disease. Monosomy 3 and histopathologic staging parameters are associated with midkine expression. In addition, we demonstrate that midkine promotes survival, migration across a barrier of hepatic sinusoid endothelial cells and resistance to AKT/mTOR inhibition. Furthermore, midkine is secreted and mediates mTOR activation by maintaining phosphorylation of the mTOR target RPS6 in uveal melanoma cells. Therefore, midkine is identified as a uveal melanoma cell survival factor that drives metastasis and therapeutic resistance, and could be exploited as a biomarker as well as a new therapeutic target. Implications: Midkine is identified as a survival factor that drives liver metastasis and therapeutic resistance in melanoma of the eye.

Colorectal carcinoma (CRC) is one of the most common carcinomas worldwide. Early detection is crucial for reducing morbidity and mortality. Several promising studies described the use of midkine (MK) as a tumor marker. This study aimed to investigate a larger collective to ascertain if the preoperative serum midkine level (S‐MK) is suitable as a marker for screening and if S‐MK correlates with tumor progression and localization. It was also investigated for the first time whether patients with high S‐MK show poor survival. This prospective single‐center study included 299 patients with CRC. The preoperative serum midkine level (S‐MK) was determined using ELISA. Established tumor markers Carcinoembryonic antigen (CEA) and Carbohydrate antigen 19‐9 (CA 19‐9) were collected for comparison. The median follow‐up period was 65 months. S‐MK was significantly elevated in patients with CRC (P, This study shows that preoperative serum midkine level (S‐MK) is suitable as marker for screening of patients with colorectal carcinoma. Moreover S‐MK correlates with poor survival.

Background: Pediatric dilated cardiomyopathy (DCM) is a disease with a poor prognosis that affects 1 in 100,000 children. Girls with DCM have worse outcomes than boys, and the mechanisms that lead to these differences are not clear. We have identified a cytokine, midkine (MDK) that is significantly upregulated in the serum from pediatric DCM patients. Circulating MDK is significantly higher in girls with DCM requiring heart transplantation compared to girls with DCM who are stable. Objective: The objective of this study is to determine if MDK impacts cardiomyocyte function in a sex-specific manner in juvenile male and female cells. Methods: Cardiomyocytes isolated from 3 week old juvenile male and female rats (JRVMs) were treated with 1μg MDK for 48 hours. Cardiomyocyte function and calcium dynamics were assessed using an IonOptix system. Myofibril mechanics and calcium sensitivity were measured. In addition, RNA sequencing was completed to identify differentially regulated pathways in juvenile male and female cardiomyocytes in response to MDK. Results: Female JRVMs treated with MDK had higher peak calcium and slower calcium reuptake compared to vehicle-treated female JRVMs. In contrast, male JRVMs treated with MDK did not demonstrate a change in peak calcium and had faster calcium reuptake compared to vehicle-treated male JRVMs. Myofibril calcium sensitivity was decreased in female JRVMs in response to MDK whereas calcium sensitivity was unchanged in male MDK-treated JRVMs. Analysis of the genes that were differentially expressed in cardiomyocytes in response to MDK demonstrated a sex-dependent regulation. Specifically, pathways which regulate calcium handling were only altered in female JRVMs treated with MDK but not in MDK-treated male JRVMs. Conclusions: This study demonstrates sex-specific differences in cardiomyocyte function in response to MDK. Particularly, female cardiomyocytes respond to MDK by regulating genes involved in calcium handling pathways. This suggests that elevated circulating MDK in pediatric DCM patients may lead to different cardiac responses in male and female patients. Therefore, elucidating these sex-specific disease mechanisms is critical to define therapies focused on male and female pediatric DCM patients.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease caused by pulmonary arterial remodeling. Midkine regulates cell proliferation and migration, and it is induced by hypoxia, but its roles in pulmonary arterial remodeling remain unclear. Serum midkine levels were significantly increased in PAH patients compared with control patients. Midkine expression was increased in lungs and sera of hypoxia-induced PAH mice. Hypoxia-induced pulmonary arterial remodeling and right ventricular hypertrophy were attenuated in midkine-knockout mice. Midkine-induced proliferation and migration of pulmonary arterial smooth muscle cells (PASMC) and epidermal growth factor receptor (EGFR) signaling were significantly increased under hypoxia, which also induced cell-surface translocation of nucleolin. Nucleolin siRNA treatment suppressed midkine-induced EGFR activation in vitro, and nucleolin inhibitor AS1411 suppressed proliferation and migration of PASMC induced by midkine. Furthermore, AS1411 significantly prevented the development of PAH in Sugen hypoxia rat model. Midkine plays a crucial role in PAH development through interaction with surface nucleolin. These data define a role for midkine in PAH development and suggest midkine-nucleolin-EGFR axis as a novel therapeutic target for PAH.

Background and Aims Introduction: Chronic kidney disease (CKD) patients are known to have greater risk of urothelial cancer (UC) than general population. Urine is the most non-invasive examination and is directly linked to UC. Urinalysis and urine cytology are the major screening tools; however, these tests have low sensitivity or specificity in early disease. In this study, we used both normal and Non-UC CKD patients as controls to discover novel and more specific protein biomarkers in urine for early UC diagnosis. Aims To discover novel and more specific urinary protein biomarkers for screening of urothelial cancer (UC) in CKD patients. Method This is a multicenter prospective cohort study, carried out at 10 nationwide hospitals covering different regions of Taiwan from 2013-2018. Subjects were Taiwanese healthy adults (healthy controls, n=210), patients with CKD but without UC (disease controls, n=273) and patients with ongoing UC (n=211). For non-UC patients, urine and blood specimens were collected at the time of the structured interview. For UC patients, urine and blood samples were collected before operation. iTRAQ-LC-MS/MS approach was applied to compare protein expression among normal, CKD and UC cohorts. Results A total of 3838 proteins in urine were identified and 2497 proteins of them can be quantified. By filtering with fold changes, published papers and Human ATLAS, 27 candidates were selected as potential biomarkers and were validated with ELISA assay in a large sample size of urine specimens. Among the 27 candidates, we found BRDT, CYBP, GARS and HDGF were highly-expressed in the UC cohort when compared to normal and CKD cohorts (Figure 1). To increase the diagnostic value of UC, we combined ROC of our newly discovered 4 protein biomarkers. The AUC values of our newly discovered 4-biomarker panel were higher than the AUC values of the published biomarker panel, including the FDA-approved protein biomarkers (Figure 2). Conclusion We discover and validate a highly specific urinary protein biomarker panel (BRDT, GARS, HDGF and CYBP) to distinguish UC from CKD and healthy controls. It may be used for high throughput screening of subjects, such as CKD patients, who are at high risk to develop UC.

Contrast-induced acute kidney injury (CI-AKI) is a common complication after intravascular injection of iodinated contrast media, and it is associated with a prolonged in-hospital stay and unfavorable outcome. CI-AKI occurs in 5% to 20% among hospitalized patients. Its diagnosis relies on the increase in serum creatinine levels, which is a late biomarker of kidney injury. Novel and early serum and urinary biomarkers have been identified to detect kidney damage before the expected serum creatinine increase.

The SARS-Cov-2 virus, which causes COVID 19, uses the cell surface protein ACE2 as receptor for entry into cells. Critically ill COVID-19 patients often require prolonged mechanical ventilation which can cause mechanical stress to lung tissue. In vitro studies have shown that expression of ACE2 in alveolar cells is increased following mechanical stretch and inflammation. Therefore, we analyzed transcriptome datasets of 480 (non-COVID-19) lung tissues in the GTex tissue gene expression database. We found that mechanical ventilation of the tissue donors increased the expression of ACE2 by more than two-fold (p

Brain tumors (gliomas) are heterogeneous cellular ecosystems, where non-neoplastic monocytic cells have emerged as key regulators of tumor maintenance and progression. However, relative to macrophages/microglia, comparatively less is known about the roles of neurons and T cells in glioma pathobiology. Herein, we leverage genetically engineered mouse models and human biospecimens to define the axis in which neurons, T cells, and microglia interact to govern Neurofibromatosis-1 (NF1) low-grade glioma (LGG) growth. NF1-mutant human and mouse brain neurons elaborate midkine to activate naïve CD8+ T cells to produce Ccl4, which induces microglia to produce a key LGG growth factor (Ccl5) critical for LGG stem cell survival. Importantly, increased CCL5 expression is associated with reduced survival in patients with LGG. The elucidation of the critical intercellular dependencies that constitute the LGG neuroimmune axis provides insights into the role of neurons and immune cells in controlling glioma growth, relevant to future therapeutic targeting., The role of neurons and T cells in glioma progression remains poorly understood. Here the authors show that midkine-dependent activation of a neuron-T cell-microglia axis promotes the growth of optic pathway gliomas.

Oral squamous cell carcinoma (OSCC) is an aggressive epithelial malignancy. Diagnosis at an early stage is a key for successful cancer therapy. Development of sensitive, specific, and non-invasive tumor markers, especially, in serum, is needed. Midkine (MK) is a plasma-secreted multifunctional peptide which is a heparin-binding growth factor.Blood samples were collected from 20 patients with OSCC, 20 patients with oral premalignancy and 10 healthy controls. Only histologically proven oral cancer and precancerous patients were taken as test subjects. Healthy individuals without predisposing habits were selected. The Human Midkine ELISA kit (Biovendor,Czech Republic) was stored at 2-80C.One way ANOVA was applied using SPSS software.Midkine Concentration in Poorly differentiated was significantly higher than Well differentiated OSCC. Midkine Concentration in stage II was significantly higher than stage I. There was a very strong positive and significant correlation between severity of disease and Midkine concentration. Also there was a strong positive and significant correlation between histological grades of oral squamous cell carcinoma and Midkine concentration.MK is a soluble, secreted cytokine and can be quantitated in blood. This is a merit of any biomarker compared to biopsy, as sampling of blood is minimally invasive, convenient, inexpensive and can be performed frequently for detecting, monitoring and managing illness. Increased MK expression in tissues, blood and urine has a strong relationship with higher malignant potential. Serum MK concentration may serve for cancer screening and monitoring the prognosis of the disease.

A growing deer antler contains a stem cell niche that can drive endochondral bone regeneration at up to 2 cm/day. Pleiotrophin (PTN), as a multifunctional growth factor, is found highly expressed at the messenger RNA level within the active antler stem cell tissues. This study aims to map the expression patterns of PTN protein and its receptors in a growing antler and investigate the effects of PTN on antler stem cells in vitro. Immunohistochemistry was employed to localise PTN/midkine (MDK) and their functional receptors, protein tyrosine phosphatase receptor type Z (PTPRZ), anaplastic lymphoma kinase (ALK), NOTCH2, and integrin αV β3, on serial slides of the antler growth centre. PTN was found to be the dominantly expressed growth factor in the PTN/MDK family. High expression of PTPRZ and ALK co-localised with PTN was found suggesting a potential interaction. The high levels of PTN and PTPRZ reflected the antler stem cell activation status during the regenerative process. When antler stem cells were cultured in vitro under the normoxic condition, no PTN protein was detected and exogenous PTN did not induce differentiation or proliferation but rather stem cell maintenance. Collectively, the antler stem cell niche appears to upregulate PTN and PTPRZ in vivo, and PTN-PTPRZ signalling may be involved in regulating antler stem cell behaviour during rapid antler regeneration.

Midkine (MDK), a heparin-binding growth factor cytokine, is involved in the pathogenesis of kidney diseases by augmenting leukocyte trafficking and activation. Animal models and small case control studies have implicated MDK as a pathological biomarker in chronic kidney diseases (CKD), however this is yet to be confirmed in prospective human studies. In a prospective study of 499 elderly, predominantly Caucasian women aged over 70 years the association between serum MDK collected in 1998, and renal function change and the risk of CKD-related hospitalisations and deaths at 5 and 14.5 years, respectively, was examined. Baseline serum MDK was not associated with 5-year change in estimated glomerular filtration rate using the CKD Epidemiology Collaboration creatinine and cystatin C equation (Standardised β = − 0.09, 95% confidence interval − 3.76–0.48, p = 0.129), 5-year rapid decline in renal function (odds ratio = 0.97, 95% confidence interval 0.46–2.02, p = 0.927) or the risk of 14.5-year CKD-related hospitalisations and deaths (hazard ratio = 1.27, 95% confidence interval .66–2.46, p = 0.470) before or after adjusting for major risk factors. In conclusion, in this cohort of elderly women with normal or mildly impaired renal function, serum MDK was not associated with renal function change or future CKD-related hospitalisations and deaths, suggesting that MDK may not be an early biomarker for progression of CKD.

Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.

Non-small cell lung cancer (NSCLC) accounts for over 80% of all diagnosed lung cancer cases. Lung cancer is the leading cause of cancer-related deaths worldwide. Most NSCLC cells overexpress vascular endothelial growth factor-A (VEGF-A) which plays a pivotal role in tumour angiogenesis. Anti-angiogenic therapies including VEGF-A neutralisation have significantly improved the response rates, progression-free survival and overall survival of patients with NSCLC. However, the median survival of these patients is shorter than 18 months, suggesting that NSCLC cells secrete VEGF-independent angiogenic factors, which remain unknown. We aimed to explore these factors in human NSCLC cell lines, A549, Lu99 and EBC-1 using serum-free culture, to which only EBC-1 cells could adapt. By mass spectrometry, we identified 1,007 proteins in the culture supernatant derived from EBC-1 cells. Among the identified proteins, interleukin-8 (IL-8), macrophage migration inhibitory factor (MIF), galectin-1, midkine (MK), IL-18, galectin-3, VEGF-A, hepatoma-derived growth factor (HDGF), osteopontin (OPN), connective tissue growth factor (CTGF) and granulin (GRN) are known to be involved in angiogenesis. Tube formation, neutralisation and RNA interference assays revealed that VEGF-A and HDGF function as angiogenic factors in EBC-1 cells. To confirm whether VEGF-A and HDGF also regulate angiogenesis in the other NSCLC cell lines, we established a novel culture method. NSCLC cells were embedded in collagen gel and cultured three-dimensionally. Tube formation, neutralisation and RNA interference assays using the three-dimensional (3D) culture supernatant showed that VEGF-A and HDGF were not angiogenic factors in Lu99 cells. By gene microarray in EBC-1 and Lu99 cells, we identified 61 mRNAs expressed only in Lu99 cells. Among these mRNAs, brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF-2) and FGF-5 are known to be involved in angiogenesis. Tube formation and neutralisation assays clarified that FGF-2 functions as an angiogenic factor in Lu99 cells. These results indicate that HDGF enhances VEGF-dependent angiogenesis and that FGF-2 is a VEGF-independent angiogenic factor in human NSCLC cells.

To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding expression changes by RNA-seq. To determine whether these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. These experiments validated new non-coding regulatory sequences near induced and/or essential genes during fin regeneration, including fgf20a, mdka, and cx43, identifying distinct domains of directed expression for each confirmed TREE. Whereas deletion of the previously identified LEN enhancer abolished detectable induction of the nearby leptin b gene during regeneration, deletions of enhancers linked to fgf20a, mdka, and cx43 had no effect or partially reduced gene expression. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.

Glioblastoma (GBM) is one of the most aggressive forms of cancer. It has been proposed that the presence within these tumors of a population of cells with stem-like features termed Glioma Initiating Cells (GICs) is responsible for the relapses that take place in the patients with this disease. Targeting this cell population is therefore an issue of great therapeutic interest in neuro-oncology. We had previously found that the neurotrophic factor MIDKINE (MDK) promotes resistance to glioma cell death. The main objective of this work is therefore investigating the role of MDK in the regulation of GICs. Methods: Assays of gene and protein expression, self-renewal capacity, autophagy and apoptosis in cultures of GICs derived from GBM samples subjected to different treatments. Analysis of the growth of GICs-derived xenografts generated in mice upon blockade of the MDK and its receptor the ALK receptor tyrosine kinase (ALK) upon exposure to different treatments. Results: Genetic or pharmacological inhibition of MDK or ALK decreases the self-renewal and tumorigenic capacity of GICs via the autophagic degradation of the transcription factor SOX9. Blockade of the MDK/ALK axis in combination with temozolomide depletes the population of GICs in vitro and has a potent anticancer activity in xenografts derived from GICs. Conclusions: The MDK/ALK axis regulates the self-renewal capacity of GICs by controlling the autophagic degradation of the transcription factor SOX9. Inhibition of the MDK/ALK axis may be a therapeutic strategy to target GICs in GBM patients. This work has been funded by the PI18/00442 grant integrated into the State Plan for R & D + I 2017-2020 and funded by the Instituto de Salud Carlos III (ISCIII) and confounded by the European Regional Development Fund (ERDF), “A way to make Europe” and by grants from ISCIII and ERDF, a way to make Europe (PS09/01401; PI12/02248 and PI15/00339 to GV, PI16/01580 to AM, PI16/01278 to JMS) by Ministerio de Economía y Competitividad (grant SAF2015-65175-R/ERDF to PSG), by Fundación Mutua Madrileña (AP101042012 to GV), “Fundació La Marató de TV3” (20134031 to GV), Voices Against Brain Cancer (US) (to GV), and donations by The Medical Cannabis Bike Tour Foundation (The Netherlands to GV) and Jeff Ditchfield (to GV). G Velasco's group is part of the COST Action CA15138 (Transautophagy). Israel López-Valero was supported by a predoctoral P-FIS contract from ISCIII, Cristina Sáiz was supported by a “Juan de la Cierva formación” contract of the Spanish Ministry of Economy and Competitiveness, Ander Matheu was recipient of a Miguel Servet contract (CP16/00039) from ISCIII. Work in JM Sepulveda group is supported by a grant from “Asociación Española contra el Cancer” (AECC) (GCTRA16015SEDA). Part of the work at G Velasco laboratory was funded by LYRAMID and Pfizer. Sí

Pleiotrophin (PTN) and midkine (MK) are cytokines that are up-regulated in the prefrontal cortex (PFC) after alcohol administration and have been shown to reduce alcohol intake and reward. Both cytokines are endogenous inhibitors of receptor protein tyrosine phosphatase (RPTP) β/ζ (a.k.a. PTPRZ1). Recently, a new compound named MY10 was designed with the aim of mimicking the activity of PTN and MK. MY10 has already shown promising results regulating alcohol-related behaviors in mice.We have now tested the effects of MY10 on alcohol operant self-administration and Drinking In the Dark-Multiple Scheduled Access (DID-MSA) paradigms in rats. Gene expression of relevant genes in the PTN/MK signaling pathway in the PFC was analyzed by real-time PCR.MY10, at the highest dose tested (100 mg/kg), reduced alcohol consumption in the alcohol operant self-administration paradigm (p = 0.040). In the DID-MSA paradigm, rats drank significantly less alcohol (p = 0.019) and showed a significant decrease in alcohol preference (p = 0.002). We observed that the longer the exposure to alcohol, the greater the suppressing effects of MY10 on alcohol consumption. It was demonstrated that the effects of MY10 were specific to alcohol since saccharin intake was not affected by MY10 (p = 0.804). MY10 prevented the alcohol-induced down-regulation of Ptprz1 (p = 0.004) and anaplastic lymphoma kinase (Alk; p = 0.013) expression.Our results support and provide further evidence regarding the efficacy of MY10 on alcohol-related behaviors and suggest the consideration of the blockade of RPTPβ/ζ as a target for reducing excessive alcohol consumption.

In a previous study, we have reported an increased plasma midkine (MK) and pleiotrophin (PTN) concentrations in patients with systemic lupus erythematosus (SLE) and the increase in MK and PTN associated with inflammatory cytokines interleukin (IL)-17 level and some clinical manifestations, suggesting the underlying association of MK and PTN with SLE. This study was conducted to investigate the association between common single-nucleotide polymorphisms (SNPs) in the MK and PTN gene and SLE susceptibility. A total of 989 subjects (496 SLE patients and 493 healthy controls) were included and genotyped for three MK SNPs and seven PTN SNPs in using improved multiple ligase detection reaction (iMLDR). Results have demonstrated no significant differences for genotype and allele frequencies in all 10 SNPs between SLE patients and healthy controls. Case-only analysis in SLE revealed that, in MK gene, the genotype frequency of AA/AG (rs35324223) was significantly lower in patients with photosensitivity than those without; the allele frequency of A/G (rs20542) was significantly higher in patients without serositis. In PTN gene, the A/G allele frequency (rs322236), C/T allele frequency, and TT/CT genotype frequency (rs6970141) showed significantly increased results in patients with immunological disorder compared to those without. Furthermore, no significant differences in plasma MK and PTN concentrations with its SNPs genotypes were found. MK and PTN SNPs showed no associations with SLE genetic susceptibility, but it may be associated with the course of this disease; further studies are needed to focus on the mechanism of MK and PTN genes in the pathogenesis of SLE.

Background Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinomas (HNSCCs). Midkine expression is restricted in adult tissues but is increased in several malignant tumors, including HNSCCs. Aim Here, we evaluated the antitumor effect of Midkine promoter–based conditionally replicative adenovirus expressing siRNA against EGFR for targeting HNSCCs expressing Midkine. Methods and results A conditionally replicative adenovirus vector controlled by the Midkine promoter, Ad‐MK‐siEGFR, was generated by integrating gene‐expressing siRNA against EGFR. Antitumor effect of Ad‐MK‐siEGFR was tested in vitro using established HNSCC cell line, T891 with strong Midkine expression. Expression of EGFR in T891 infected with Ad‐MK‐siEGFR was significantly lower than that of T891 infected with control. Cytotoxicity assays showed significant growth suppression of Ad‐MK‐siEGFR in T891 cells. Conclusions This study demonstrated the possibility of oncolytic therapy using the Midkine promoter–based conditional replication‐selective adenovirus containing siRNA against EGFR in HNSCC cell line T891. Further validation of the findings in more cell lines and in vivo should be performed to clarify the potential clinical application.

Neurotrophic growth factors were originally characterized for their support in neuronal differentiation, outgrowth, and survival during development. However, it has been acknowledged that they also play a vital role in the adult brain. Abnormalities in growth factors have been implicated in a variety of neurological and psychiatric disorders, including alcohol use disorder (AUD). This work focuses on the interaction between alcohol and growth factors. We review literature suggesting that several growth factors play a unique role in the regulation of alcohol consumption, and that breakdown in these growth factor systems is linked to the development of AUD. Specifically, we focus on the brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), and insulin growth factor 1 (IGF-1). We also review the literature on the potential role of midkine (MDK) and pleiotrophin (PTN) and their receptor, anaplastic lymphoma kinase (ALK), in AUD. We show that alcohol alters the expression of these growth factors or their receptors in brain regions previously implicated in addiction, and that manipulations on these growth factors and their downstream signaling can affect alcohol-drinking behaviors in animal models. We conclude that there is a need for translational and clinical research to assess the therapeutic potential of new pharmacotherapies targeting these systems.

Formation of a specialized wound epidermis is required to initiate salamander limb regeneration. Yet little is known about the roles of the early wound epidermis during the initiation of regeneration and the mechanisms governing its development into the apical epithelial cap (AEC), a signaling structure necessary for outgrowth and patterning of the regenerate. Here, we elucidate the functions of the early wound epidermis, and further reveal midkine (mk) as a dual regulator of both AEC development and inflammation during the initiation of axolotl limb regeneration. Through loss- and gain-of-function experiments, we demonstrate that mk acts as both a critical survival signal to control the expansion and function of the early wound epidermis and an anti-inflammatory cytokine to resolve early injury-induced inflammation. Altogether, these findings unveil one of the first identified regulators of AEC development and provide fundamental insights into early wound epidermis function, development, and the initiation of limb regeneration.