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Cell transfer therapy using mesenchymal stem cells (MSCs) has pronounced therapeutic potential, but concerns remain about immune rejection, emboli formation, and promotion of tumor progression. Because the mode of action of MSCs highly relies on their paracrine effects through secretion of bioactive molecules, cell‐free therapy using the conditioned medium (CM) of MSCs is an attractive option. However, the effects of MSC‐CM on tumor progression have not been fully elucidated. Herein, we addressed this issue and investigated the possible underlying molecular mechanisms. The CM of MSCs derived from human bone marrow greatly inhibited the in vitro growth of several human tumor cell lines and the in vivo growth of the SCCVII murine squamous cell carcinoma cell line with reduced neovascularization. Exosomes in the MSC‐CM were only partially involved in the inhibitory effects. The CM contained a variety of cytokines including insulin‐like growth factor binding proteins (IGFBPs). Among them, IGFBP‐4 greatly inhibited the in vitro growth of these tumors and angiogenesis, and immunodepletion of IGFBP‐4 from the CM significantly reversed these effects. Of note, the CM greatly reduced the phosphorylation of AKT, ERK, IGF‐1 receptor beta, and p38 MAPK in a partly IGFBP4‐dependent manner, possibly through its binding to IGF‐1/2 and blocking the signaling. The CM depleted of IGFBP‐4 also reversed the inhibitory effects on in vivo tumor growth and neovascularization. Thus, MSC‐CM has potent inhibitory effects on tumor growth and neovascularization in an IGFBP4‐dependent manner, suggesting that cell‐free therapy using MSC‐CM could be a safer promising alternative for even cancer patients. We found that conditioned medium (CM) of mesenchymal stem cells (MSCs) greatly inhibited in vitro and in vivo tumor growth and angiogenesis, and that insulin‐like growth factor binding protein‐4 (IGFBP‐4) in the MSC‐CM was critically involved in the inhibitory effects. This is the first report on the antitumor effects of MSC‐CM, of which IGFBP‐4 in the CM was shown to play important roles. Thus, cell‐free therapy using the MSC‐CM could be a safer and promising alternative for even cancer patients.

Therapies for type 2 diabetes primarily target hyperglycemia; however, complications are also triggered by advanced glycation end products (AGEs). We hypothesize that the anti-diabetic efficacy of insulin-like growth factor-binding protein 4 (IGFBP4) is enhanced when it assembles with a specific amino acid compound-2 (AAC2) into nanostructures. Their effects were examined in vitro and in ob/ob mice treated for 30 days with the AAC2–IGFBP4 complex or its individual components. IGFBP4-mediated glucose uptake in human and mouse preadipocytes was enhanced by complex formation with AAC2. This complex was confirmed by Fourier-transform mid-infrared spectroscopy, electrophoresis, and AFM. In ob/ob mice, the complex prolonged IGFBP4 circulation and amplified the effects of the individual components, resulting in reduced hyperphagia, body weight, and hyperinsulinemia, along with improved insulin sensitivity and glucose tolerance. Notably, HbA1c levels remained at 5.9% in the complex-treated group compared to > 7% in others, with lower plasma AGE levels than in AAC2-treated mice. Transcriptomic and pathway analyses revealed that the complex upregulated genes promoting the fenestrated phenotype of liver sinusoidal endothelial cells (LSECs), facilitating AGE and waste clearance, whereas free AAC2 inhibited this process. We propose a ‘scavenger-input’ hypothesis in which free AAC2 inhibits, while the AAC2–IGFBP4 complex activates fenestrated phenotype and waste‑clearance capacity in liver sinusoidal endothelial cells (LSECs). Based on our results, AAC2 could serve as an adaptable and inherently therapeutic nanofiber modality that enhances the functional properties of bound proteins, offering multidimensional treatment possibilities for diabetes and other degenerative disorders. Graphical Abstract

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease globally. Recent research has identified insulin-like growth factor-binding proteins 2 (IGFBP2) and 4 (IGFBP4) as potential biomarkers for DKD. Overactivation of the complement pathway in DKD remains poorly understood. Blood samples were collected from patients for proteomic analysis, complemented by both and experiments to investigate the roles of IGFBP2, IGFBP4, and the complement pathway in DKD. Elevated levels of IGFBP2 and IGFBP4 were observed in DKD patients. The levels of IGFBP2 and IGFBP4 increased in DKD mice, accompanied by the activation of the complement pathway, and a deterioration in renal function. High glucose and serum from DKD mice stimulated an increase in the levels of IGFBP2 and IGFBP4 in HK-2 cells. The supernatant from HK-2 cells was used to culture THP-1 cells, resulted in an increase in the M1 type of THP-1 cells, a decrease in the M2 type, and activation of the complement pathway. The supernatant from THP-1 cells affected the growth of primary human renal podocytes. The exogenous addition of IGFBP2 and IGFBP4 proteins to primary human renal podocytes did not affect their growth. However, when human renal podocytes were cultured with the supernatant from THP-1 cells, the growth of the podocytes was affected. IGFBP2 and IGFBP4 interact to stimulate the activation of the complement pathway in macrophages, which induces podocyte apoptosis and subsequently promotes the development of DKD.

Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.

Pregnancy-associated plasma protein-A (PAPP-A) is a key regulator of insulin-like growth factor (IGF) bioactivity, by releasing the IGFs from their corresponding IGF-binding proteins (IGFBPs). The minor allele of the single nucleotide polymorphism (SNP), rs7020782 (serine < tyrosine), in PAPPA has previously been associated with recurrent pregnancy loss as well as with significant reduced levels of PAPP-A protein in human ovarian follicles. The aim of the present study was to reveal a possible functional effect of the rs7020782 SNP in PAPPA by comparing recombinant PAPP-A proteins from transfected human embryonic kidney 293 T cells. The proteolytic cleavage of IGFBP-4 was shown to be affected by the rs7020782 SNP in PAPPA , showing a significantly reduced cleavage rate for the serine variant compared to the tyrosine variant (p-value < 0.001). The serine variant also showed a trend towards reduced cleavage rates, that was not significant, towards IGFBP-2 and IGFBP-5 compared to the tyrosine variant. No differences were found when analysing cell surface binding, complex formation between PAPP-A and STC2 or proMBP, nor when analysing STC1 inhibition of PAPP-A-mediated IGFBP-4 cleavage. Regulation of IGF bioactivity in reproductive tissues is important and the rs7020782 SNP in PAPPA may disturb this regulation by altering the specific activity of PAPP-A.

Purpose We have previously identified insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) as essential proteins for tip cell maintenance and sprouting angiogenesis. In this study, we aim to identify other IGF family members involved in endothelial sprouting angiogenesis. Methods Effects on sprouting were analyzed in human umbilical vein endothelial cells (HUVECs) using the spheroid-based sprouting model, and were quantified as mean number of sprouts per spheroid and average sprout length. RNA silencing technology was used to knockdown gene expression. Recombinant forms of the ligands (IGF1 and IGF2, insulin) and the IGF-binding proteins (IGFBP) 3 and 4 were used to induce excess effects. Effects on the tip cell phenotype were analyzed by measuring the fraction of CD34 + tip cells using flow cytometry and immunohistochemistry in a 3D angiogenesis model. Experiments were performed in the presence and absence of serum. Results Knockdown of IGF2 inhibited sprouting in HUVECs, in particular when cultured in the absence of serum, suggesting that components in serum influence the signaling of IGF2 in angiogenesis in vitro. We then determined the effects of IGFBP3 and IGFBP4, which are both present in serum, on IGF2-IGF1R signaling in sprouting angiogenesis in the absence of serum: knockdown of IGFBP3 significantly reduced sprouting angiogenesis, whereas knockdown of IGFBP4 resulted in increased sprouting angiogenesis in both flow cytometry analysis and immunohistochemical analysis of the 3D angiogenesis model. Other IGF family members except INSR did not affect IGF2-IGF1R signaling. Conclusions Serum components and IGF binding proteins regulate IGF2 effects on sprouting angiogenesis. Whereas IGFBP3 acts as co-factor for IGF2-IGF1R binding, IGFBP4 inhibits IGF2 signaling.

Background Ruptured atherosclerotic plaques often precipitate severe ischemic events, such as stroke and myocardial infarction. Unraveling the intricate molecular mechanisms governing vascular smooth muscle cell (VSMC) behavior in plaque stabilization remains a formidable challenge. Methods In this study, we leveraged single-cell and transcriptomic datasets from atherosclerotic plaques retrieved from the gene expression omnibus (GEO) database. Employing a combination of single-cell population differential analysis, weighted gene co-expression network analysis (WGCNA), and transcriptome differential analysis techniques, we identified specific genes steering the transformation of VSMCs in atherosclerotic plaques. Diagnostic models were developed and validated through gene intersection, utilizing the least absolute shrinkage and selection operator (LASSO) and random forest (RF) methods. Nomograms for plaque assessment were constructed. Tissue localization and expression validation were performed on specimens from animal models, utilizing immunofluorescence co-localization, western blot, and reverse-transcription quantitative-polymerase chain reaction (RT-qPCR). Various online databases were harnessed to predict transcription factors (TFs) and their interacting compounds, with determination of the cell-specific localization of TF expression using single-cell data. Results Following rigorous quality control procedures, we obtained a total of 40,953 cells, with 6,261 representing VSMCs. The VSMC population was subsequently clustered into 5 distinct subpopulations. Analyzing inter-subpopulation cellular communication, we focused on the SMC2 and SMC5 subpopulations. Single-cell subpopulation and WGCNA analyses revealed significant module enrichments, notably in collagen-containing extracellular matrix and cell-substrate junctions. Insulin-like growth factor binding protein 4 ( IGFBP4 ), apolipoprotein E (APOE ), and cathepsin C ( CTSC ) were identified as potential diagnostic markers for early and advanced plaques. Notably, gene expression pattern analysis suggested that IGFBP4 might serve as a protective gene, a hypothesis validated through tissue localization and expression analysis. Finally, we predicted TFs capable of binding to IGFBP4 , with Krüppel-like family 15 ( KLF15 ) emerging as a prominent candidate showing relative specificity within smooth muscle cells. Predictions about compounds associated with affecting KLF15 expression were also made. Conclusion Our study established a plaque diagnostic and assessment model and analyzed the molecular interaction mechanisms of smooth muscle cells within plaques. Further analysis revealed that the transcription factor KLF15 may regulate the biological behaviors of smooth muscle cells through the KLF15/IGFBP4 axis, thereby influencing the stability of advanced plaques via modulation of the PI3K-AKT signaling pathway. This could potentially serve as a target for plaque stability assessment and therapy, thus driving advancements in the management and treatment of atherosclerotic plaques.

Senescent cells secrete several molecules, collectively named senescence-associated secretory phenotype (SASP). In the SASP of cells that became senescent following several in vitro chemical and physical stress, we identified the IGFBP-4 protein that can be considered a general stress mediator. This factor appeared to play a key role in senescence-paracrine signaling. We provided evidences showing that genotoxic injury, such as low dose irradiation, may promote an IGFBP-4 release in bloodstream both in mice irradiated with 100 mGy X-ray and in human subjects that received Computer Tomography. Increased level of circulating IGFBP-4 may be responsible of pro-aging effect. We found a significant increase of senescent cells in the lungs, heart, and kidneys of mice that were intraperitoneally injected with IGFBP-4 twice a week for two months. We then analyzed how genotoxic stressors may promote the release of IGFBP-4 and the molecular pathways associated with the induction of senescence by this protein.

Abstract Context Anorexia nervosa (AN) can cause severe undernutrition associated with alterations in the IGF axis. Pappalysins (PAPP-A, PAPP-A2) and stanniocalcins (STC-1, STC-2) modulate IGF binding-protein (IGFBP) cleavage and IGF bioavailability, but their implications in AN are unknown. Objective We determined serum levels of PAPP-As and STCs in relationship with classical IGF axis parameters in female adolescents with AN and their association with nutritional status and secondary amenorrhea. Methods Parameters of the IGF axis were determined in fasting serum samples of 68 female adolescents with AN at diagnosis and 62 sex- and age-matched controls. Standardized body mass index (BMI) and bone mineral density (BMD) were calculated. Results Patients with AN had lower concentrations of total and free IGF-I, total IGFBP-3, acid-labile subunit (ALS), insulin, PAPP-A2, STC-1, and STC-2 and higher levels of IGF-II and IGFBP-2. Their free/total IGF-I ratio was decreased and the intact/total IGFBP-3 and −4 ratios increased. BMI was directly related to total IGF-I and intact IGFBP-3 and inversely with IGFBP-2 and intact IGFBP-4. Weight loss was directly correlated with intact IGFBP-4 and negatively with intact IGFBP-3, ALS, STC-2, and PAPP-A2 concentrations. BMD was directly related to intact IGFBP-3 and inversely with intact IGFBP-4 and PAPP-A2 levels. Patients with amenorrhea had lower levels of total IGF-I and IGFBP-3 than those with menses. Conclusion The reduction of PAPP-A2 in patients with AN may be involved in a decline in IGFBP cleavage, which could underlie the decrease in IGF-I bioavailability that is influenced by nutritional status and amenorrhea.

Elevated levels of pregnancy-associated plasma protein-A (PAPP-A) have been implicated in the pathogenesis of various malignancies, including breast cancers. Breast cancer is one of the most frequent carcinomas and is the second most common cancer type detected in women of child-bearing age. Throughout pregnancy PAPP-A is produced and secreted by the placental syncytiotrophoblast cells; co-incidentally pregnancy-associated breast cancers often have an aggressive clinical course. The components of the PAPP-A/IGF axis was assessed in a panel of breast cancer cell lines. Using neutralising antibodies the impact of PAPP-A/IGF axis on cell motility was evaluated. PAPP-A was expressed in four of the twelve breast cancer cell lines tested. Blocking PAPP-A and IGFBP4 with neutralising antibodies significantly decreased motiliy of MDA-MB-231 cells. Upregulation of PAPP-A expression in breast tumours resulted in a trend towards worse overall survival. Notably, PAPP-A expression also positively correlated with epithelial-to-mesenchymal transition markers. In conclusion, these results indicate that PAPP-A plays an important role in breast cancer progression and it may be a promising therapeutic target in breast cancer.