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Atherosclerosis constitutes the pathological basis of life-threatening events, including heart attack and stroke. Midkine is a heparin-binding growth factor and forms a small protein family with pleiotrophin. Under inflammatory or hypoxic conditions, midkine expression is up-regulated. Upon binding to its receptors, midkine can activate multiple signal pathways to regulate cell survival and migration, epithelial-to-mesenchymal transition, and oncogenesis. Circulating midkine levels are significantly increased in patients with essential hypertension, obesity or severe peripheral artery disease. Importantly, midkine exerts a proatherogenic effect by altering multiple pathophysiological processes involving atherogenesis, including macrophage lipid accumulation, vascular inflammation, neointima formation, insulin resistance and macrophage apoptosis. Midkine represents a potential therapeutic target for atherosclerosis-associated diseases. This review described the structure characteristics, expression patterns and signal transduction pathways of midkine with an emphasis on its role in atherosclerosis.

Objective: To explore the clinical significance of serum midkine (MDK) levels for the diagnosis of hepatocellular carcinoma (HCC) and evaluate the efficacy of interventional therapy. Methods: Eighty-four patients with HCC were enrolled in this retrospective study. They received an interventional treatment. A follow-up was performed every 2 months, using magnetic resonance imaging, to determine whether the treatment should be continued. Serum alpha-fetoprotein (AFP) and MDK levels were measured at the first diagnosis and during the follow-ups, and the HCC detection rates based on the cutoff values of these two measurements were compared. The relationships between AFP and MDK and the clinical tumor characteristics and changes in APK and MDK before and after treatment were also compared using a rank sum test and χ2 test, respectively. The prognostic significance of MDK for HCC was determined through regression analysis. A two-sided P ​

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Background DNA damage repair (DDR) alterations are important events in cancer initiation, progression, and therapeutic resistance. However, the involvement of DDR alterations in glioma malignancy needs further investigation. This study aims to characterize the clinical and molecular features of gliomas with DDR alterations and elucidate the biological process of DDR alterations that regulate the cross talk between gliomas and the tumor microenvironment. Methods Integrated transcriptomic and genomic analyses were undertaken to conduct a comprehensive investigation of the role of DDR alterations in glioma. The prognostic DDR-related cytokines were identified from multiple datasets. In vivo and in vitro experiments validated the role of p53, the key molecule of DDR, regulating M2 polarization of microglia in glioma. Findings DDR alterations are associated with clinical and molecular characteristics of glioma. Gliomas with DDR alterations exhibit distinct immune phenotypes, and immune cell types and cytokine processes. DDR-related cytokines have an unfavorable prognostic implication for GBM patients and are synergistic with DDR alterations. Overexpression of MDK mediated by p53, the key transcriptional factor in DDR pathways, remodels the GBM immunosuppressive microenvironment by promoting M2 polarization of microglia, suggesting a potential role of DDR in regulating the glioma microenvironment. Interpretation Our work suggests that DDR alterations significantly contribute to remodeling the glioma microenvironment via regulating the immune response and cytokine pathways. Fund This study was supported by: 1. The National Key Research and Development Plan (No. 2016YFC0902500); 2. National Natural Science Foundation of China (No. 81702972, No. 81874204, No. 81572701, No. 81772666); 3. China Postdoctoral Science Foundation (2018M640305); 4. Special Fund Project of Translational Medicine in the Chinese-Russian Medical Research Center (No. CR201812); 5. The Research Project of the Chinese Society of Neuro-oncology, CACA (CSNO-2016-MSD12); 6. The Research Project of the Health and Family Planning Commission of Heilongjiang Province (2017–201); and 7. Harbin Medical University Innovation Fund (2017LCZX37, 2017RWZX03)., Highlights • Gliomas with DNA damage repair alterations had distinct genomic variation spectrum. • DDR alterations exhibit distinct immune phenotypes, cytokine processes and immune cell types in glioma. • DDR-related cytokines in GME have an unfavorable prognostic implication for GBM patients. • P53-mediated midkine expression derived from glioma cells promotes M2 polarization of microglia.

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

The growth factor Midkine is a heparin-binding cytokine originally discovered during the differentiation process induced by the retinoic acid in embryonal carcinoma cells. Several studies pointed out the key role of this protein in tumour progression and its elevated expression in different malignancies, including pancreatic cancer. New diagnostic and therapeutic tools are urgently required to treat this highly aggressive and incurable disease capable of metastasising, evading diagnosis, and resisting therapy. Serum midkine promises to be a very functional tumour marker and a target for cancer treatment as an elevated concentration of serum midkine is consistently reported in patients with various tumours. Here, we identified high levels of midkine in extracellular vesicles isolated from pancreatic cancer cell lines and showed that it stimulates the growth of pancreatic cancer cells not expressing midkine.

BACKGROUND New biomarkers for diagnosis and monitoring the COVID-19 disease are the most important topics to be studied recently. We aimed to investigate the association between midkine levels and disease severity in pregnant women with COVID-19. METHODS Totally 186 pregnant women were participated in this study. 96 of them were healthy pregnant women, 90 of them were pregnant women with COVID19. Pregnant women were evaluated according to their trimesters. Serum midkine level, biochemical profile clinical and disease severity outcomes of pregnant women were obtained. RESULTS Our results showed that pregnant women with COVID19 have significantly increased serum midkine level compared to healthy pregnant women (1.801 ± 0.977 vs 0.815 ± 0.294 ng/dL). According to the data among each trimester, it was shown that there were significant increase in serum midkine level during all pregnancy trimesters (1st trimester Control Group: 0.714 ± 0.148, COVID-19 group 1.623 ± 0.824, p

Midkine (Mdk) is a heparin-binding growth factor that is involved in regulating cell growth, differentiation and migration. Here, we report the isolation and characterization of duplicated mdk genes in blunt snout bream (Megalobrama amblycephala). The mdka and -b genes encode 146 aa and 147 aa peptides, respectively, sharing a sequence identity of 64%. During embryogenesis, mdka mRNA is detectable after 12 h post-fertilization (hpf) and mdkb mRNA can be detected after 8 hpf, about 4 h prior to mdka mRNA. Whole-mount in situ hybridization demonstrated that two paralogs of mdk mRNA were detected in the brain and dorsal neural tube at 16 hpf. At 22 hpf, mdka mRNA was abundant in the brain and dorsal neural tube, whereas mdkb mRNA were transcribed in the brain and tailbud. Later, at 55 hpf, both paralogs were mainly expressed in the brain. Furthermore, both the mdk genes were highly expressed in multiple adult tissues except in the skin and a low expression of mdka in the muscle. In addition, they were differentially inhibited in the liver and intestine with exogenous recombinant human growth hormone, while their mRNA levels were up-regulated in the brain. During starvation, both the mdk genes were significantly up-regulated in the intestine, brain and liver and returned to the control levels following 6 days of refeeding. Our results suggest that duplicated mdk genes may play conserved and divergent roles in embryonic development and tissue growth regulation in blunt snout bream.

Midkine is a pleiotropic factor, which is involved in angiogenesis. However, its mode of action in this process is still ill defined. The function of midkine in arteriogenesis, the growth of natural bypasses from pre-existing collateral arteries, compensating for the loss of an occluded artery has never been investigated. Arteriogenesis is an inflammatory process, which relies on the proliferation of endothelial cells and smooth muscle cells. We show that midkine deficiency strikingly interferes with the proliferation of endothelial cells in arteriogenesis, thereby interfering with the process of collateral artery growth. We identified midkine to be responsible for increased plasma levels of vascular endothelial growth factor A (VEGFA), necessary and sufficient to promote endothelial cell proliferation in growing collaterals. Mechanistically, we demonstrate that leukocyte domiciled midkine mediates increased plasma levels of VEGFA relevant for upregulation of endothelial nitric oxide synthase 1 and 3, necessary for proper endothelial cell proliferation, and that non-leukocyte domiciled midkine additionally improves vasodilation. The data provided on the role of midkine in endothelial proliferation are likely to be relevant for both, the process of arteriogenesis and angiogenesis. Moreover, our data might help to estimate the therapeutic effect of clinically applied VEGFA in patients with vascular occlusive diseases., Highlights • Leukocyte domiciled midkine is decisive for collateral endothelial cell proliferation in arteriogenesis. • Midkine controls the bioavailability of VEGFA mediating endothelial Nos1 and Nos3 expression. • Nos1 and Nos3, relevant for endothelial cell proliferation, can substitute for each other. Arteriogenesis is a life and tissue saving process as it compensates for the loss of an occluded artery. Decoding the underlying molecular mechanisms is a prerequisite for the development of novel therapeutic options to treat patients with vascular occlusive diseases. Lautz et al. identified midkine to be responsible for the increased bioavailability of VEGFA during arteriogenesis, necessary and sufficient to promote endothelial cell proliferation. These data might help to estimate the therapeutic effect of clinically applied VEGFA. As the identified mechanisms might also apply for angiogenesis, they are likely to be of broader relevance, e.g. in terms of tumor treatment.

The overexpression level of Midkine (MDK) indicates the malignant degree of cancer cells, being a potential indicator and biomarker for cancer diseases. However, traditional biological methods such as Enzyme-Linked ImmunoSorbent Assay (ELISA) suffers inevitable disadvantages of long measurement time and false positive problems. In this study, the metasurfaces with electromagnetically induced transparency (EIT) resonant mode are utilized as metasensors to detect the MDK concentration, and its biosensing properties are resolved from time-frequency double domains. The resonant frequency shift and amplitude difference are demonstrated as indicators for MDK concentration level. A set of pseudo-spectra obtained by implementing Fourier transform twice on full-spectral amplitude difference (FSAD) further confirm that the limit of detection (LOD) of metasensors at least reaches 0.5 µg/mL, and the measurement time shortens to smaller than 5 minutes. The 2D mappings derived from continuous wavelet transform (CWT) clearly recognize different solutions without arousing confusion. Moreover, these label-free mappings with double domains similar to fluorescence pictures of traditional ELISA serve as an effective visualized output of MDK expression level and supply unified measurement standard, showing vigorous functionality of EIT metasensors in future label-free detection in cancer diseases.

Facial nerves are frequently injured during cosmetic or other types of facial surgery. However, information on the genes involved in the damage and recovery of the facial nerves is limited. Here, we aimed to identify the genes affected by facial nerve injury and repair using next-generation sequencing. We established a rat axotomy model and a parallel epineurial neurorrhaphy model, in which gene expression was analyzed from 3 days to 8 weeks after surgery. We discovered that ARRB1, SGK1, and GSK3B genes associated with neuronal cell death were upregulated in the axotomy model. In contrast, MFRP, MDK, and ACE genes involved in neural recovery and regeneration exhibited higher expression in the neurorrhaphy model. In the present study, the analysis of the big data obtained from the next-generation sequencing (RNA-seq) technology reveals that the expression of genes involved in neuronal cell death is induced during nerve damage, and those associated with neural recovery are more abundantly expressed during repair processes. These results are considered to be useful for the establishment of the treatment of related diseases and basic research in various neuroscience fields by utilizing damage and recovery mechanism of facial nerves.

Background Midkine (MDK) is a tumor-promoting factor that is often overexpressed in various human carcinomas, and the role of MDK has not yet been fully investigated in prostate cancer stem cells (PCSCs). Materials and Methods Prostate cancer CD133 + stem cells (PCSCs) were isolated from human castration-resistant PC3 cells. PCSCs were treated with different concentrations of MDK inhibitor, iMDK, for 24-72 hours. The IC 50 values were determined by the MTT test. Endogenous MDK messenger RNA expression was knocked down by small interfering RNA. Quantitative reverse transcription polymerase chain reaction, western blot analyses and image-based cytometry were used to investigate apoptosis and cell cycle progression as well as their underlying molecular mechanisms. Cell migration was evaluated by the wound healing test. Results iMDK caused dose- and time-dependent inhibition of PCSC survival. Similar growth inhibition was also obtained by small interfering RNA–mediated knockdown of endogenous MDK expression. iMDK was shown to preferentially induce cell cycle arrest at the S and G2/M phases. Suppressed PCSC growth was also accompanied by increases in p53 and the cell cycle inhibitor p21 genes. Combinatorial treatment of iMDK with docetaxel significantly inhibited cell proliferation versus either of the agents used alone. Inhibition of MDK expression strongly suppressed the migration of PCSCs compared to untreated and docetaxel-treated cells. iMDK and the knockdown of MDK decreased p-Akt and significantly upregulated the expression of PI3K/phosphatase/tensin homolog. Conclusions Our data indicate that MDK plays a crucial role in controlling PCSC proliferation and migration. Therefore, suppression of endogenous expression of MDK would, in combination with traditional chemotherapy drugs, be a potential treatment for PCSCs.

Contrast-induced acute kidney injury (CI-AKI) is an underdiagnosed complication of percutaneous coronary intervention (PCI). Midkine (MK) is a heparin-binding growth factor that regulates cell growth, survival and migration. Serum MK is considered an early predictor of CI-AKI. The study aimed to

Midkine (MK) is a heparin-binding growth factor, which behaves like a cytokine, involved in various cellular processes such as cellular proliferation, differentiation, survival, adhesion, and migration. Studies provided evidence for a role of MK in acute and chronic inflammatory processes. The association between midkine and Henoch-Schönlein purpura (HSP) has not yet been explored. The aim of our study was to investigate the potential role of midkine in children with HSP.A total of 152 cases consisting of 92 children with HSP and 60 age- and sex-matched healthy control children were enrolled in this prospective study. Circulating midkine, IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A was measured in all of the 92 patients and 60 healthy controls. Midkine diagnostic value was evaluated by receiver operating characteristic (ROC) analysis.Renal involvement occurred in 36 of the 92 patients. Circulating midkine level was elevated in children with HSPN than those of patients without renal involvement and of the controls (326.58 (266.58-459.25) pg/ml versus 280.72 (233.67-384.36) pg/ml and 217.3 (198.98-243.65) pg/ml, respectively; P0.05). Midkine positively correlated with IL-4, IL-6, IL17A, IgA and IgE. The threshold MK concentration of HSPN was 295.58pg/ml, with the sensitivity and specificity of 80.6% and 88.3%, respectively. The area under the receiver operating characteristic (ROC) curve (AUCROC) of MK was 0.902.MK seems to be involved in the development of HSP. Measurement of serum levels of MK is helpful in confirming the diagnosis of HSP and predicting HSPN.

Midkine (MK) is a low molecular-weight protein that was first identified as the product of a retinoic acid-responsive gene involved in embryonic development. Recent studies have indicated that MK levels are related to various diseases, including cardiovascular disease (CVD), renal disease and autoimmune disease. MK is a growth factor involved in multiple pathophysiological processes, such as inflammation, the repair of damaged tissues and cancer. The pathophysiological roles of MK are diverse. MK enhances the recruitment and migration of inflammatory cells upon inflammation directly and also through induction of chemokines, and contributes to tissue damage. In lung endothelial cells, oxidative stress increased the expression of MK, which induced angiotensin-converting enzyme (ACE) expression and the consequent conversion from Ang I to Ang II, leading to further oxidative stress. MK inhibited cholesterol efflux from macrophages by reducing ATP-binding cassette transporter A1 (ABCA1) expression, which is involved in lipid metabolism, suggesting that MK is an important positive factor involved in inflammation, oxidative stress and lipid metabolism. Furthermore, MK can regulate the expansion, differentiation and activation of T cells as well as B-cell survival; mediate angiogenic and antibacterial activity; and possess anti-apoptotic activity. In this paper, we summarize the pathophysiological roles of MK in human disease.

Protein-tyrosine phosphatase receptor type Z (PTPRZ) is predominantly expressed in the developing brain as a CS proteoglycan. PTPRZ has long (PTPRZ-A) and short type (PTPRZ-B) receptor forms by alternative splicing. The extracellular CS moiety of PTPRZ is required for high-affinity binding to inhibitory ligands, such as pleiotrophin (PTN), midkine, and interleukin-34; however, its functional significance in regulating PTPRZ activity remains obscure. We herein found that protein expression of CS-modified PTPRZ-A began earlier, peaking at approximately postnatal days 5-10 (P5-P10), and then that of PTN peaked at P10 at the developmental stage corresponding to myelination onset in the mouse brain. Ptn-deficient mice consistently showed a later onset of the expression of myelin basic protein, a major component of the myelin sheath, than wild-type mice. Upon ligand application, PTPRZ-A/B in cultured oligodendrocyte precursor cells exhibited punctate localization on the cell surface instead of diffuse distribution, causing the inactivation of PTPRZ and oligodendrocyte differentiation. The same effect was observed with the removal of CS chains with chondroitinase ABC but not polyclonal antibodies against the extracellular domain of PTPRZ. These results indicate that the negatively charged CS moiety prevents PTPRZ from spontaneously clustering and that the positively charged ligand PTN induces PTPRZ clustering, potentially by neutralizing electrostatic repulsion between CS chains. Taken altogether, these data indicate that PTN-PTPRZ-A signaling controls the timing of oligodendrocyte precursor cell differentiation in vivo, in which the CS moiety of PTPRZ receptors maintains them in a monomeric active state until its ligand binding.

Preconditioning exercise can exert neuroprotective effects after stroke. However, the mechanism underlying these neuroprotective effects by preconditioning exercise remains unclear. We investigated the neuroprotective effects of preconditioning exercise on brain damage and the expression levels of the midkine (MK) and brain-derived neurotrophic factor (BDNF) after brain ischemia. Animals were assigned to one of 4 groups: exercise and ischemia (Ex), no exercise and ischemia (No-Ex), exercise and no ischemia (Ex-only), and no exercise and intact (Control). Rats ran on a treadmill for 30 min once a day at a speed of 25 m/min for 5 days a week for 3 weeks. After the exercise program, stroke was induced by a 60 min left middle cerebral artery occlusion using an intraluminal filament. The infarct volume, motor function, neurological deficits, and the cellular expressions levels of MK, BDNF, GFAP, PECAM-1, caspase 3, and nitrotyrosine (NT) were evaluated 48 h after the induction of ischemia. The infarct volume, neurological deficits and motor function in the Ex group were significantly improved compared to that of the No-Ex group. The expression levels of MK, BDNF, GFAP, and PECAM-1 were enhanced in the Ex group compared to the expression levels in the No-Ex group after brain ischemia, while the expression levels of activated caspase 3 and NT were reduced in the area surrounding the necrotic lesion. Our findings suggest that preconditioning exercise reduced the infract volume and ameliorated motor function, enhanced expression levels of MK and BDNF, increased astrocyte proliferation, increased angiogenesis, and reduced neuronal apoptosis and oxidative stress.

Background and Aims Midkine (MDK) is a heparin-binding growth factor and is overexpressed in various types of human cancer. However, little is known about the clinical significance of MDK in non-small cell lung cancer (NSCLC). The aim of this study was to measure MDK protein levels in patients with NSCLC and to explore its clinical significance. Methods The expression status of MDK in NSCLC at Gene Expression Omnibus (GEO accession number: GSE19804 ) was observed. The expression of MDK mRNA and protein was examined in NSCLC tissues and normal lung tissues through real-time PCR and Western blot. Meanwhile, the relationship of MDK protein expression levels with clinical characteristics of 186 NSCLC patients was analyzed by immunohistochemistry. Results MDK expression was increased in NSCLC tissues compared with paired normal lung tissues in microarray data ( GSE19804 ). MDK mRNA and protein expression were obviously increased in NSCLC tissues than in paired adjacent normal lung tissues. Using immunohistochemistry, MDK protein overexpression was positively correlated with status of clinical stage, T classification, N classification, and M classification in NSCLC patients. In survival analysis, patients with higher MDK protein expression had a significantly shorter overall survival time than did patients with lower MDK protein expression. Multivariate analysis indicated that the MDK protein overexpression was an independent poor prognostic indicator for patients with NSCLC. Conclusions MDK plays an important role in NSCLC progression and prognosis and may act as a convincing prognostic indicator for NSCLC patients.

Immune cells, such as natural killer (NK) cells, recognize virally infected and transformed cells, and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells. Shedding of NKG2D ligands is thought to be a type of counter-mechanism employed by pathogenic cells to evade from NKG2D-mediated immune surveillance. MHC class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. We previously reported that, in soluble form, MICA expression levels are significantly associated with hepatitis virus-induced hepatocellular carcinoma. Here, we report a MICA shedding assay that utilizes membrane-bound MICA tagged at its N-terminus with a nano-luciferase reporter to quantify MICA shedding into culture media. Using this method, we screened a compound library and identified putative regulators of MICA shedding that have the potential to enhance the immune reaction by simultaneously increasing cell surface MICA levels and decreasing soluble MICA levels. This shedding assay may be useful for screening regulators of cell surface molecule shedding.

Using an in vivo reporter for lymphangiogenesis, a recent study in Nature from Olmeda et al. ( 2017) describes a new subset of melanomas that induce systemic pre-conditioning of distant organs for formation of tumor metastatic niches, and identifies the responsible factor as the pleiotropic cytokine midkine. Non

Cardiac hypertrophy is a heart reaction to the increase of cardiac load, with the characteristics of increased expression of cardiac hypertrophy markers, enhanced protein synthesis, and enlarged cell area. However, molecular mechanisms in cardiac hypertrophy are still poorly substantiated. It has been reported that miRNAs can modulate human diseases, among which miR-326 has been reported as a biological regulator in human cancers, but its role in cardiac hypertrophy is rarely explored. This study focused on the exploration of the potential of miR-326 in cardiac hypertrophy. Our data revealed the downregulation of miR-326 in the TAC-induced hypertrophic mice and the Ang II-induced hypertrophic H9c2 cells. Functionally, miR-326 attenuated the effect of Ang II on cardiac hypertrophy in vitro. In addition, miR-326 negatively regulated JAK/STAT and MAPK signaling pathways. Mechanistically, miR-326 targeted and inhibited MDK to induce JAK/STAT and MAPK pathways. Rescue assays certified that miR-326 attenuated cardiac hypertrophy through targeting MDK and inhibiting JAK/STAT and MAPK signaling pathways. In brief, our study unveiled that miR-326 targets MDK to regulate the progression of cardiac hypertrophy through blocking JAK/STAT and MAPK signaling pathways, indicating that targeting miR-326 as a potential approach for cardiac hypertrophy treatment.

Contrast-induced acute kidney injury (CI-AKI), is a crucial complication of percutaneous coronary intervention (PCI) that is associated with significant morbidity and mortality. Midkine (MK) is a heparin-binding growth factor regulating cell growth, survival and migration. The pathophysiological

Contrast-induced acute kidney injury (CI-AKI), is an important complication of percutaneous coronary intervention (PCI). The pathophysiological roles of Midkine (MK) range from AKI occurrence to chronic kidney disease (CKD) progression, so we aimed to detect the role of serum MK as an early

Midkine is a recently identified new growth factor/cytokine with pleiotropic functions in the human organism. First discovered in the late eighties, midkines have now become the subject of numerous studies in cardiovascular, neurologic, renal diseases and also various types of cancers. We summarize here the most important functions of midkine in cardiovascular diseases, emphasizing its role in inflammation and its antiapoptotic and proangiogenetic effects. Midkine has multiple roles in the organism, with the specific feature of being either beneficial or harmful depending on which tissue it acts on. Even though midkine has been shown to have cardiac protective effects against acute ischemia/reperfusion injury and to inhibit cardiac remodeling, it also promotes intimal hyperplasia and vascular stenosis. As such, different therapeutic strategies are currently being evaluated, consisting of administering either midkine proteins or midkine inhibitors depending on the desired outcome. More data is gathering to suggest that these novel therapies could become an adjunctive to standard cardiovascular therapy. Nonetheless, much is still to be learned about midkine. The encouraging results up till now require further studying in order to fully understand the complete profile of its mechanism of action and the clinical safety and efficacy of novel therapeutic opportunities offered by midkine molecular targeting.

Purpose This study aimed to assess whether synergism could be achieved when combining midkine (MK) antisense oligodeoxynucleotides (anti-MK ODN) and recombinant human hepatocyte growth factor (HGF) in diabetic nephropathy (DN) rat models. Methods Rats were randomized into 6 groups: control, DN rats without treatment, DN rats treated with scrambled ODN, DN rats treated with anti-MK ODN, DN rats treated with HGF and DN rats treated with anti-MK ODN plus HGF. DN models were created by intraperitoneal injection of streptozotocin. Two weeks later, treatments commenced. ODN (1 mg/kg) was intravenously injected weekly for 4 weeks. HGF (500 μg/kg) was subcutaneously injected daily for 4 weeks. Eight weeks later, rats were euthanized. Serum and urine parameters, kidney histopathological injury scores, immunohistochemistry and protein expressions were measured. Results Blood glucose, creatinine, blood urea nitrogen and urine albumin were significantly elevated in DN rats. Any single treatment markedly reduced their levels, yet combined treatment decreased them significantly further. Any monotherapy could decrease renal injury score and immunohistochemistry positive percentage, although the most prominent change was displayed in combinational therapy. Western blot showed the expression of MK was significantly elevated in DN rats. Anti-MK ODN suppressed MK significantly. The protein expressions and serum concentrations of transforming growth factor-β1 and connective tissue growth factor between monotherapy and the combined therapy were significant. Conclusions This study demonstrated that combining MK gene suppressing ODN and HGF protein synergistically attenuates renal injury in DN rats. This study may provide a novel avenue for designing future therapeutic regimens against DN.

Kallikrein-related peptidases (KLKs) are a group of serine proteases widely expressed in various tissues and involved in a wide range of physiological and pathological processes. Although our understanding of the pathophysiological roles of most KLKs has blossomed in recent years, identification of the direct endogenous substrates of human KLKs remains an unmet objective. In this study we employed a degradomics approach to systemically investigate the endogenous substrates of KLK7 in an effort to understand the molecular pathways underlying KLK7 action in skin. We identified several previously known as well as novel protein substrates. Our most promising candidates were further validated with the use of targeted quantitative proteomics (selected reaction monitoring methods) and in vitro recombinant protein digestion assays. Our study revealed midkine, CYR61, and tenascin-C as endogenous substrates for KLK7. Interestingly, some of these substrates (e.g. midkine) were prone to proteolytic cleavage only by KLK7 (and not by other skin-associated KLKs), whereas others (e.g. CYR61 and tenascin-C) could be digested by several KLKs. Furthermore, using melanoma cell line, we show that KLK7-mediated cleavage of midkine results in an overall reduction in the pro-proliferative and pro-migratory effect of midkine. An inverse relation between KLK7 and midkine is also observed in human melanoma tissues. In summary, our degradomics approach revealed three novel endogenous substrates for KLK7, which may shed more light on the pathobiological roles of KLK7 in human skin. Similar substrate screening approaches could be applied for the discovery of biological substrates of other protease.

The role of angiogenesis in haematological malignancies such as chronic lymphocytic leukaemia (CLL) is difficult to envision, because leukaemia cells are not dependent on a network of blood vessels to support basic physiological requirements. Regardless, CLL cells secrete high levels of major angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet derived growth factor (PDGF). Nonetheless, it remains unclear how most angiogenic factors regulate accumulation and delayed apoptosis of CLL cells. Angiogenic factors such as leptin, granulocyte colony-stimulating factor (G-CSF), follistatin, angiopoietin-1 (Ang1), angiogenin (ANG), midkine (MK), pleiotrophin (PTN), progranulin (PGRN), proliferin (PLF), placental growth factor (PIGF), and endothelial locus-1 (Del-1), represent novel therapeutic targets of future CLL research but have remained widely overlooked. This review aims to outline our current understanding of angiogenic growth factors and their relationship with CLL, a still uncured haematopoietic malignancy.

Background Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of the patients with HCC lose the surgical opportunity at the time of diagnosis. Some novel therapeutic modalities, like gene therapy, are promising for the treatment of HCC. However, the success of gene therapy depends on two aspects: efficient gene materials and gene delivery vectors. The present study was to develop new chitosan-based nanoparticles for a midkine-siRNA (anti-HCC gene drug) delivery. Methods The novel gene delivery vector (MixNCH) was synthesized by hybrid-type modification of chitosan with 2-chloroethylamine hydrochloride and N, N-dimethyl-2-chloroethylamine hydrochloride. The chemical structure of MixNCH was characterized by FT-IR and 1 HNMR. The cytotoxicity of MixNCH was determined by MTS assay. The gene condensation ability and size, zeta potential and morphology of MixNCH/MK-siRNA nanoparticles were measured. The in vitro transfection and gene knockdown efficiency of midkine by MixNCH/MK-siRNA nanoparticles was detected by qRT-PCR and Western blotting. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by MTS assay. Results MixNCH was successfully acquired by aminoalkylation modification of chitosan. The MixNCH could condense MK-siRNA well above the weight ratio of 3. The average size of MixNCH/MK-siRNA nanoparticles was 100-200 nm, and the surface charge was about +5 mV. Morphologically, MixNCH/MK-siRNA nanoparticles were in regular spherical shape with no aggregation. Regarding to the in vitro transfection of nanoparticles, the MixNCH/MK-siRNA nanoparticles reduced MK mRNA level to 14.03%±4.03%, which were comparable to Biotrans (8.94%±3.77%). MixNCH/MK-siRNA effectively inhibited the proliferation of HepG2 in vitro. Conclusion MixNCH/MK-siRNA nanoparticles could be effective for the treatment of hepatocellular carcinoma.

The aim of this study was to investigate whether co-expression of midkine (MK) and pleiotrophin (PTN) has prognostic relevance in human gliomas. Immunohistochemistry was used to investigate the expression of MK and PTN proteins in 168 patients with gliomas. The levels of MK and PTN mRNA in glioma tissues and paratumor tissues were evaluated in 45 paired cases by quantitative real-time polymerase chain reaction (qRT-PCR). Kaplan-Meier survival analysis was performed to assess prognostic significance. The expression levels of MK and PTN proteins in glioma tissue were both significantly higher (both p

Opiates cause persistent restructuring in the mesolimbic reward system. Although a possible role for midkine and pleiotrophin cytokines in the field of synaptic plasticity has been proposed, it has not been assessed whether morphine administration regulates astrogliosis and midkine and pleiotrophin transcription. We observed that single morphine injection and chronic morphine increased glial fibrillary acidic protein expression in the ventral tegmental area (VTA). Interestingly, single morphine injection and chronic morphine increased VTA midkine and pleiotrophin mRNA expression. Given these results, we hypothesize a role for these cytokines in mediating, at least in part, acute neuroprotective effects and chronic neurotrophic adaptations that contribute to drug dependence.

Estrogens are well known steroid hormones necessary to maintain bone health. In addition, mechanical loading, in which estrogen signaling may intersect with the Wnt/β-catenin pathway, is essential for bone maintenance. As osteocytes are known as the major mechanosensory cells embedded in mineralized bone matrix, osteocyte ERα deletion mice (ERα(ΔOcy/ΔOcy)) were generated by mating ERα floxed mice with Dmp1-Cre mice to determine the role of ERα in osteocytes. Trabecular bone mineral density of female, but not male ERα(ΔOcy/ΔOcy) mice was significantly decreased. Bone formation parameters in ERα(ΔOcy/ΔOcy) were significantly decreased while osteoclast parameters were unchanged. This suggests that ERα in osteocytes exerts osteoprotective function by positively controlling bone formation. To identify potential targets of ERα, gene array analysis of Dmp1-GFP osteocytes sorted by FACS from ERα(ΔOcy/ΔOcy) and control mice was performed. Gene expression microarray followed by gene ontology analyses revealed that osteocytes from ERα(ΔOcy/ΔOcy) highly expressed genes categorized in 'Secreted' when compared to control osteocytes. Among them, expression of Mdk and Sostdc1, both of which are Wnt inhibitors, was significantly increased without alteration of expression of the mature osteocyte markers such as Sost and β-catenin. Moreover, hindlimb suspension experiments showed that trabecular bone loss due to unloading was greater in ERα(ΔOcy/ΔOcy) mice without cortical bone loss. These data suggest that ERα in osteocytes has osteoprotective functions in trabecular bone formation through regulating expression of Wnt antagonists, but conversely plays a negative role in cortical bone loss due to unloading.

Midkine is a multifunctional growth factor, and its serum levels are increased with the functional severity of heart failure. This study aimed to examine the role of midkine in heart failure pathogenesis. Midkine expression levels were increased in the kidney and lung after transverse aortic constriction (TAC) surgery, but not sufficiently increased in the heart. After TAC, phosphorylation of extracellular signal-regulated kinase1/2 and AKT, and the expression levels of foetal genes in the heart were considerably increased in transgenic mice with cardiac-specific overexpression of midkine (MK-Tg) compared with wild-type (WT) mice. MK-Tg mice showed more severe cardiac hypertrophy and dysfunction, and showed lower survival rate after TAC than WT mice. We conclude that midkine plays a critical role in cardiac hypertrophy and remodelling.

Mid regional pro-adrenomedullin (MR-proADM), midkine and ST2 are novel cardiac biomarkers associated with heart failure and atherosclerotic diseases as stable ischemic disease and acute coronary syndrome. The potential association between these three biomarkers and peripheral atherosclerosis (PA)

Midkine is a multifunctional cytokine found to be a promising cancer biomarker, however, its suitability in colorectal cancer (CRC) has not been evaluated yet. We assessed midkine circulating levels immunoenzymatically in 105 CRC patients, 86 individuals with increased risk for CRC (56 with inflammatory bowel disease (IBD) and 30 with adenomas), and 70 healthy controls and compared its performance as CRC biomarker to carcinoembryonic antigen (CEA). Midkine was higher in CRC (807 ng/L) than in IBD (477 ng/L; 633 ng/L in active and 335 ng/L in inactive), adenomas (418 ng/L) or controls (245 ng/L). Its levels increased along with advancing CRC stage, being significantly higher compared to controls already in stage I, and dedifferentiation (higher in grade 3 than 1 and 2). Lymph node or distant metastases were associated with significant midkine elevation as well. Midkine positively correlated with IL-1β, IL-6, IL-8, TNF-α, MCP-1, MIP-1α, G-CSF, GM-CSF, VEGF-A, and PDGF-BB with IL-1β and PDGF-BB explaining 40% in its variability. Midkine was better marker of CRC than CEA with 80% accuracy, 83% sensitivity and 68% specificity as compared to 60%, 37%, and 88% of CEA, also in its early stages (74% vs. 52% accuracy). Midkine better differentiated CRC from inactive while CEA from active IBD. Midkine was included in the multimarker panel and significantly contributed to efficient (Λ=0.16) differentiation of healthy controls, adenomas and CRC. Concluding, midkine may lack sufficient specificity to be a sole CRC marker but seems to constitute a valuable addition to multimarker panels devised for CRC screening and/or surveillance.

The neurotrophic factors Midkine (MK) and Pleiotrophin (PTN) have been suggested to modulate drugs of abuse-induced effects. To test this hypothesis, cocaine (10 and 15 mg/kg)-induced conditioned place preference (CPP) was rendered in PTN knockout (PTN−/−), MK knockout (MK−/−) and wild type (WT+/+) mice, and then extinguished after repeated saline injections (distributed in 4 extinction sessions). Cocaine induced a similar CPP in all the three genotypes. We found a significantly increased percentage of MK−/− mice that did not extinguish cocaine CPP at the end of the extinction sessions. Particularly, 40% of MK−/− mice did not extinguish cocaine (15 mg/kg)-induced CPP compared to WT+/+ and PTN−/− mice (∼0–6%). Interestingly, we found that a greater magnitude of extinction of CPP after the first extinction session (5 days after last administration of cocaine) correlates with increased tyrosine phosphorylation of the enzyme peroxiredoxin 6 in the dorsal striatum of MK−/− mice. On the other hand, a greater magnitude of CPP extinction correlates with increased tyrosine phosphorylation of aconitase 2 in the prefrontal cortex of WT+/+ mice. In contrast, a lower magnitude of CPP extinction correlates with increased phosphorylation of aconitase 2 in the prefrontal cortex of PTN−/− mice, suggesting that the correlation between the tyrosine phosphorylation levels of aconitase 2 and magnitude of CPP extinction depends on the genotype considered. The data demonstrate that MK is a novel genetic factor that plays a role in the extinction of cocaine-induced CPP by mechanisms that may involve specific phosphorylation of striatal peroxiredoxin 6.

Injuries in the mammalian central nervous system induce a variety of factors which promote or inhibit neuronal axon regeneration/sprouting. However, the inhibitory activities are much stronger, and indeed are the major obstacle to functional recovery. Chondroitin sulfate proteoglycans (CSPGs) are produced by activated glial cells, and are among the strongest inhibitors. Here, we investigated the role of the growth factor midkine (MK), which binds to CSPGs, in neuronal injury. MK expression was induced by spinal cord injury, and was mainly produced by activated astrocytes. A prolonged culture of neurons also produced MK. MK not only enhanced neurite outgrowth on the substratum coated with poly-l-lysine, but also overcame the neurite growth inhibition by the CSPG substratum. Moreover, we found that MK activated neither astrocytes nor microglia as evaluated by morphological changes and cell proliferation or nitric oxide production. These properties would be advantageous for the treatment of neuronal injuries in vivo. Therefore, we next explored the therapeutic effect of MK in a rat spinal cord injury model. MK or vehicle was administered intrathecally for 2 weeks using an osmotic pump after spinal cord contusion injury. Rats treated with MK showed significantly better functional recovery after 5 weeks. These results suggest that MK may offer a potent alternative for the treatment of neuronal injuries without activating glial cells.

The neurotrophic factors pleiotrophin (PTN) and midkine (MK) have been shown to modulate amphetamine-induced neurotoxicity. Accordingly, PTN-/- and MK-/- mice show an increased vulnerability to amphetamine-induced neurotoxic effects. In an effort to uncover new pharmacological targets to prevent amphetamine neurotoxic effects, we have now used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN-/-, MK-/- and wild type (WT) mice treated with amphetamine. We identified 13 differentially expressed phosphoproteins that are judged to be relevant in the neuroprotective roles of PTN and MK against amphetamine-induced neurotoxicity. It is very interesting to note that 4 of these phosphoproteins, annexin A7 (ANXA7), COP9 signalosome subunit 5 (COPS5), aldehyde dehydrogenase family 1 member A1 (ALDH1A1) and creatine kinase U-type (CKMT1), are known to be involved in Parkinson's disease, a result of significant importance since PTN and MK have been also demonstrated to limit Parkinson's disease (PD) progress and have been suggested to be among the important genetic factors possibly preventing the development of PD in methamphetamine abusers. The data identify phosphoproteins differentially regulated by amphetamine treatment and/or the presence of endogenous PTN/MK which may be relevant mediators of PTN/MK neuroprotective effects against amphetamine-induced neurotoxicity. The data support further studies to validate the phosphoproteins here identified as possible new pharmacological targets to prevent amphetamine neurotoxic effects.

The midkine antisense oligonucleotide (MK-ASODN, 5′-CCC CGG GCC GCC CTT CTT CA-3′) nanoliposomes have been identified to suppress hepatocellular carcinoma (HCC) growth effectively, and have a great potential to be an effective target drug for HCC. In this study, a facile and reproducible method for large-scale preparation of MK-ASODN nanoliposomes followed by lyophilization has been developed successfully. Meanwhile, the MK-ASODN nanoliposomes characteristics, storage stability and their antitumor efficiency were studied. The mean particle size of MK-ASODN nanoliposomes were 229.43 ± 15.11 nm, and the zeta potential were 29.7 ± 1.1 mV. High entrapment efficiency values were achieved around 90%. Transmission electron microscopy images revealed spherical shaped nanoliposomes. Nanoliposomes allowed sustained MK-ASODN release for as long as 14 days. During 180 days of storage, freeze-dried nanoliposomes showed no significant change in the mean size, zeta potential, entrapment efficiency and drug release ratio. Regarding their antitumor efficiency, the in vitro proliferation of human liver cancer cells were significantly inhibited by the MK-ASODN nanoliposomes. Furthermore, the MK-ASOND nanoliposomes also significantly inhibited the growth of HCC in the mouse model. In summary, the results confirmed that this large-scale preparation of MK-ASOND nanoliposomes was facile and reproducible, and potentially, could speed up the application process of our MK-ASOND nanoliposomes for HCC therapy.

Adaptive immunity depends on the production and maintenance of a pool of mature peripheral lymphocytes throughout life. The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Lasting B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. Cytokines have been shown to play a critical role in maintaining lymphocyte homeostasis. This review focuses on the role of cytokines and their receptors in the regulation of peripheral B cell survival, with an emphasis on those that have received relatively less attention in the literature.

Objective Midkine (MK) is a heparin-binding growth factor that is overexpressed in various human cancers. The aim of this study was to investigate the expression of MK in ameloblastomas and correlate the results with clinicopathologic parameters. Study Design Cases of ameloblastoma seen between 1999 and 2010 were identified. Clinical information was collected regarding age, gender, race, and location of tumor. Cases were classified as solid/multicystic, unicystic, and peripheral. The expression of midkine was assessed using immunohistochemistry. A significant difference was considered present at P Results A total of 34 cases of ameloblastoma and 4 cases of ameloblastic carcinomas were identified. MK was expressed in 67% of lesions (23.5% weak expression; 14.7% moderate expression; 29.4% strong expression). A significant difference was seen between solid/multicystic and unicystic lesions. Conclusions MK is expressed in the majority of ameloblastomas, suggesting a role of the protein in the tumor's development, progression, and behavior.

Midkine (MDK) and Pleiotrophin (PTN) belong to a class of heparin-binding growth factors and are highly expressed in a number of cancers. Bioactive and recombinant MDK and PTN are critical reagent for cancer drug discovery studies. MDK and PTN belong to a newly evolving family of secreted neurotrophic and developmentally regulated heparin-binding molecules. PTN is related to MDK with 45% sequence identity and both proteins have been shown to be involved in promoting neurite outgrowth. MDK is a cysteine-rich 13kDa protein containing five disulfide bonds and PTN is 19kDa protein containing ten disulphide bonds. In this study, we expressed recombinant human MDK (rhMDK), mouse MDK (rmMDK) and human pleiotrophin (rhPTN) in Escherichia coli BL21(DE3)pLysS strain. Soluble rhMDK, rmMDK and rhPTN were expressed at a high-level in this strain and the protein was purified (∼90%) by a one-step purification using heparin affinity chromatography. A total of 4mg purified MDK and 7mg of purified PTN were obtained with the overall yield from 1L of bacterial culture. Activity of purified rhMDK and rhPTN was confirmed by a cell proliferation assay using NIH3T3 cells.

Background Midkine is a heparin-binding growth factor that promotes the proliferation, survival, migration and differentiation of various target cells. Midkine plays an important role in tumorigenesis and tumor progression, and is overexpressed in many human malignant tumors. Patients with high tumor midkine expression frequently have a worse prognosis than those with low expression. The present study was designed to investigate the interaction network of midkine in hepatic cancer cells, and to elucidate its role in hepatocellular carcinoma. Methods DNA encoding full-length midkine was cloned into pDBLeu vector to serve as bait in yeast two-hybrid screening to identify interacting proteins. Candidate proteins were examined on SC-Leu-Trp-His+3-AT (20 mmol/L) plates and assayed for X-gal activity, then sequenced and classified according to the GenBank. Finally, identified proteins were expressed by the in vitro expression system pCMVTnT, and protein interactions were confirmed by co-immunoprecipitation. Results Using the yeast two-hybrid system, we found 6 proteins that interacted with midkine: NK-kappa-B inhibitor alpha (I-κ-B-α), Dvl-binding protein naked cuticle 2, granulin, latent active TGF-β binding protein 3, latent active TGF-β binding protein 4, and phospholipid scramblase 1. In vitro co-immunoprecipitation demonstrated that all identified proteins directly interacted with midkine. Conclusions The identification of midkine-interacting proteins in hepatic cancer cells indicates that midkine is a multifunctional factor that may participate in cell migration, differentiation, and proliferation, and is also associated with the multicellular response feedback during the development of hepatocellular carcinoma.

Genetic deletion of pleiotrophin (PTN) impairs spinal nociceptive transmission suggesting that this heparin binding growth factor could play roles in acute pain processing. Despite the high functional redundancy between PTN and midkine (MK), the only other member of this family of growth factors, we now demonstrate that genetic inactivation of MK does not alter acute nociceptive transmission since pain responses of female MK genetically deficient (MK-/-) and wild type (WT+/+) mice were found to be similar in the hot-plate and tail-immersion tests. It has also been shown that morphine administration significantly regulates MK levels within the brain, suggesting that MK could play a role in morphine-induced antinociceptive effects. To test this hypothesis, we have now studied morphine-induced antinociceptive effects in female MK-/- and WT+/+ mice. We did not find differences among genotypes using different doses of morphine (2.5, 5 and 10 mg/kg) in the hot-plate test. In contrast, we found that morphine significantly delayed pain responses in MK-/- mice compared to WT+/+ mice in the tail-immersion test. In confirmation of previous results from our group, we also found significantly enhanced morphine-induced antinociceptive effects in PTN-/- mice in the tail-immersion test. In addition, we now demonstrate that enhanced morphine analgesic effects in PTN-/- and MK-/- mice are not caused by a different contribution of descending noradrenergic inhibitory pathways since the α(2)-adrenergic antagonist yohimbine failed to alter morphine-induced analgesia in all genotypes. The data demonstrate that MK is an endogenous modulator of morphine antinociceptive effects, identify significant differences between PTN and MK in the control of pain processing at the spinal level, and support the hypothesis that inhibitors of the PTN/MK signaling pathway could potentiate opioid analgesia which may be relevant in opioid-refractory pain cases.