Dickkopf-1 (DKK1) drives growth and metastases in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is associated with a poor prognosis and remains an incurable fatal disease. Therefore, the identification of molecular markers involved in cancer progression is urgently needed to develop more-effective therapies. The present study investigated the role of the Wnt signaling modulator Dickkopf-1 (DKK1) in the growth and metastatic progression of mCRPC. DKK1 silencing through siRNA and deletion via CRISPR/Cas9 editing were performed in two different metastatic castration-resistant prostate cancer cell lines (PC3 and DU145). A xenograft tumor model was used to assess tumor growth and metastases. In in vitro experiments, both DKK1 silencing and deletion reduced cell growth and migration of both cell lines. DKK1 knockout clones (DKK1-KO) exhibited cell cycle arrest, tubulin reorganization, and modulation of tumor metastasis-associated genes. Furthermore, in DKK1-KO cells, E-cadherin re-expression and its membrane co-localization with ß-catenin were observed, contributing to reduced migration; Cadherin-11, known to increase during epithelial-mesenchymal transition, was down-regulated in DKK1-KO cells. In the xenograft mouse model, DKK1 deletion not only reduced tumor growth but also inhibited the formation of lung metastases. In conclusion, our findings support the key role of DKK1 in the growth and metastatic dissemination of mCRPC, both in vitro and in vivo.

Circulating extracellular vesicles derived from tumor endothelial cells hijack the local and systemic anti-tumor immune response: Role of mTOR/G-CSF pathway.

Circulating tumour-derived extracellular vesicles are supposed to contribute to the spreading of distant metastasis. In this study, we investigated the impact of circulating extracellular vesicles derived from tumour-endothelial cells (TEVs) in the expansion of the metastatic bulk. We focus on the role of immune cells in controlling this process using the 4T1 triple negative breast cancer (TNBC) syngeneic model. 4T1 cells were intravenously injected and exposed to circulating TEVs from day 7. The lung, spleen, and bone marrow (BM) were recovered and analysed. We demonstrated that circulating TEVs boost lung metastasis and angiogenesis. FACS and immunohistochemically analyses revealed a significant enrichment of Ly6G+/F4/80+/CD11b+ cells and Ly6G+/F4/80-/CD11b+ in the lung and in the spleen, while Ly6G+/F4/80-/CD11b+ in the BM, indicating the occurrence of a systemic and local immune suppression. TEV immune suppressive properties were further supported by the increased expression of PD-L1, PD-1, and iNOS in the tumour mass. In addition, in vitro experiments demonstrated an increase of CD11+ cells, PD-L1+ myeloid and cancer cells, upregulation of LAG3, CTLA4 and PD-1 in T-cells, release of ROS and NOS, and impaired T-cell-mediated cytotoxic effect in co-culture of TEVs-preconditioned PBMCs and cancer cells. Granulocyte-colony stimulating factor (G-CSF) level was increased in vivo, and was involved in reshaping the immune response. Mechanistically, we also found that mTOR enriched TEVs support G-CSF release and trigger the phosphorylation of the S6 (Ser235/236) mTOR downstream target. Overall, we provided evidence that circulating TEVs enriched in mTOR supported G-CSF release thereby granting tumour immune suppression and metastasis outgrowth.

Interleukin-3-Receptor-α in Triple-Negative Breast Cancer (TNBC): An Additional Novel Biomarker of TNBC Aggressiveness and a Therapeutic Target.

Tumour molecular annotation is mandatory for biomarker discovery and personalised approaches, particularly in triple-negative breast cancer (TNBC) lacking effective treatment options. In this study, the interleukin-3 receptor α (IL-3Rα) was investigated as a prognostic biomarker and therapeutic target in TNBC. IL-3Rα expression and patients' clinical and pathological features were retrospectively analysed in 421 TNBC patients. IL-3Rα was expressed in 69% human TNBC samples, and its expression was associated with nodal metastases (p = 0.026) and poor overall survival (hazard ratio = 1.50; 95% CI = 1.01-2.2; p = 0.04). The bioinformatics analysis on the Breast Invasive Carcinoma dataset of The Cancer Genome Atlas (TCGA) proved that IL-3Rα was highly expressed in TNBC compared with luminal breast cancers (p = 0.017, padj = 0.026). Functional studies demonstrated that IL-3Rα activation induced epithelial-to-endothelial and epithelial-to-mesenchymal transition, promoted large blood lacunae and lung metastasis formation, and increased programmed-cell death ligand-1 (PD-L1) in primary tumours and metastases. Based on the TCGA data, IL-3Rα, PD-L1, and EMT coding genes were proposed to discriminate against TNBC aggressiveness (AUC = 0.86 95% CI = 0.82-0.89). Overall, this study identified IL-3Rα as an additional novel biomarker of TNBC aggressiveness and provided the rationale to further investigate its relevance as a therapeutic target.

IL-3 signalling in the tumour microenvironment shapes the immune response via tumour endothelial cell-derived extracellular vesicles.

Antibody-based anti-cancer therapy is considered a successful approach to impair tumour progression. This study aimed to investigate the clinical impact of targeting the IL-3 signalling in the microenvironment of solid tumours. We intended to investigate whether the IL-3Rα blockade on tumour-derived endothelial cells (TEC) can modulate PD-L1 expression in tumour cells and peripheral blood mononuclear cells (PBMC) to reshape the anti-tumour immune response. Extracellular vesicles released by TEC after IL-3Rα blockade (aTEV) were used as the ultimate effectors of the antibody-based approach, while naive TEC-derived extracellular vesicles (nTEV) served as control. Firstly, we demonstrated that, either directly or indirectly via nTEV, IL-3 controls the expression of its receptor on TEC and PBMC respectively. Moreover, we found that nTEV, moulded by the autocrine secretion of IL-3, increased PD-L1 expression in myeloid cells both in vitro and in vivo. In addition, we found that nTEV-primed PBMC favour tumour cell growth (TEC and MDA-MB-231 cells), whereas PBMC-primed with aTEV still retain their anti-tumour properties. Isolated T-cells pre-conditioned with nTEV or aTEV and co-cultured with TEC or MDA-MB-231 cells have no effects, thereby sustaining the key role of myeloid cells in tumour immune editing. In vivo nTEV, but not aTEV, increased the expression of PD-L1 in primary tumours, lung and liver metastases. Finally, we demonstrated that the enrichment of miR-214 in aTEV impacts on PD-L1 expression in vivo. Overall, these data indicate that an approach based on IL-3Rα blockade in TEC rearranges EV cargo and may reshape the anti-tumour immune response.

Extracellular Vesicles Derived from Human Liver Stem Cells Attenuate Chronic Kidney Disease Development in an In Vivo Experimental Model of Renal Ischemia and Reperfusion Injury.

The potential therapeutic effect of extracellular vesicles (EVs) that are derived from human liver stem cells (HLSCs) has been tested in an in vivo model of renal ischemia and reperfusion injury (IRI), that induce the development of chronic kidney disease (CKD). EVs were administered intravenously immediately after the IRI and three days later, then their effect was tested at different time points to evaluate how EV-treatment might interfere with fibrosis development. In IRI-mice that were sacrificed two months after the injury, EV- treatment decreased the development of interstitial fibrosis at the histological and molecular levels. Furthermore, the expression levels of pro-inflammatory genes and of epithelial-mesenchymal transition (EMT) genes were significantly reverted by EV-treatment. In IRI-mice that were sacrificed at early time points (two and three days after the injury), functional and histological analyses showed that EV-treatment induced an amelioration of the acute kidney injury (AKI) that was induced by IRI. Interestingly, at the molecular level, a reduction of pro-fibrotic and EMT-genes in sacrificed IRI-mice was observed at days two and three after the injury. These data indicate that in renal IRI, treatment with HLSC-derived EVs improves AKI and interferes with the development of subsequent CKD by modulating the genes that are involved in fibrosis and EMT.

Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes.

Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.

Targeting IL-3Rα on tumor-derived endothelial cells blunts metastatic spread of triple-negative breast cancer via extracellular vesicle reprogramming.

The lack of approved targeted therapies highlights the need for new treatments for triple-negative breast cancer (TNBC) patients. Interleukin-3 (IL-3) acts as an autocrine factor for tumor-endothelial cells (TEC), and exerts pro-angiogenic paracrine action via extracellular vesicles (EVs). IL-3Rα blockade on TEC changes TEC-EV (anti-IL-3R-EV) microRNA (miR) content and promotes the regression of established vessels. As TEC is the doorway for "drug" entry into tumors, we aimed to assess whether IL-3R blockade on TEC impacts tumor progression via its unique EV cargo. First, the expression of IL-3Rα was evaluated in 27 human TNBC samples. It was noticed that, besides TEC and inflammatory cells, tumor cells from 55.5% of the human TNBC samples expressed IL-3Rα. Using human TNBC cell lines for in vitro studies, we found that, unlike native TEC-EVs (nEVs), anti-IL-3R-EVs increase apoptosis and reduced cell viability and migration. In vivo, anti-IL-3R-EV treatment induced vessel regression in established tumors formed of MDA-MB-231 cells, decreased Vimentin, ß-catenin, and TWIST1 expression, almost abolished liver and lung metastases from primary tumors, and reduced lung metastasis generated via the intravenous injection of MDA-MB-231 cells. nEVs depleted of miR-24-3p (antago-miR-24-3p-EVs) were effective as anti-IL-3R-EVs in downregulating TWIST1 and reducing metastatic lesions in vivo. Consistent with network analyses of miR-24-3p gene targeting, anti-IL-3R-EVs and antago-miR-24-3p-EVs upregulate SPRY2 in MDA-MB-231 cells. Finally, SPRY2 silencing prevented anti-IL-3R-EV and antago-miR-24-3p-EV-mediated apoptotic cues.Overall, these data provide the first evidence that IL-3Rα is highly expressed in TNBC cells, TEC, and inflammatory cells, and that IL-3Rα blockade on TEC impacts tumor progression.

Mesenchymal Stem Cell Derived Extracellular Vesicles Ameliorate Kidney Injury in Aristolochic Acid Nephropathy.

Limitations in the current therapeutic strategies for the prevention of progression of chronic kidney disease (CKD) to end stage renal disease has been a drawback to improving patient recovery. It is therefore imperative that a solution is found to alleviate this problem and improve the health and well-being of patients overall. Aristolochic acid (AA) induced nephropathy, a type of nephrotoxic CKD is characterised by cortical tubular injury, inflammation, leading to interstitial fibrosis. Extracellular vesicles derived from human bone marrow mesenchymal stem cells (MSC-EVs) display therapeutic properties in various disease models including kidney injury. In the current study, we intended to investigate the ability of MSC-EVs on ameliorating tubular injury and interstitial fibrosis in a mouse model of aristolochic acid nephropathy (AAN). The chronic model of AAN is comprised of an intraperitoneal injection of AA in NSG mice, followed by a three-day incubation period and then inoculation of MSC-EVs intravenously. This routine was performed on a weekly basis for four consecutive weeks, accompanied by the monitoring of body weight of all mice. Blood and tissue samples were collected post sacrifice. All animals administered with AA developed kidney injury and renal fibrosis. A gradual loss of body weight was observed, together with a deterioration in kidney function. Although no significant recovery was observed in weight loss following treatment with MSC-EVs, a significant reduction in: blood creatinine and blood urea nitrogen (BUN), tubular necrosis, and interstitial fibrosis was observed. In addition, infiltration of CD45 positive immune cells, fibroblasts, and pericytes which were elevated in the interstitium post AA induced injury, were also significantly reduced by MSC-EVs. Kidneys were also subjected to molecular analyses to evaluate the regulation of pro-fibrotic genes. MSC-EVs significantly reduced AA induction of the pro-fibrotic genes α-Sma, Tgfb1 and Col1a1. A downregulation in pro-fibrotic genes was also observed in fibroblasts activated by AA injured mTECs in vitro. Furthermore, meta-analyses of miRNAs downregulated by MSC-EVs, such as miR21, revealed the regulation of multiple pathways involved in kidney injury including fibrosis, inflammation, and apoptosis. These results therefore suggest that MSC-EVs could play a regenerative and anti-fibrotic role in AAN through the transfer of biologically active cargo that regulates the disease both at a protein and genetic level.

Extracellular vesicles from human liver stem cells inhibit renal cancer stem cell-derived tumor growth in vitro and in vivo.

Cancer stem cells (CSCs) are considered as responsible for initiation, maintenance and recurrence of solid tumors, thus representing the key for tumor eradication. The antitumor activity of extracellular vesicles (EVs) derived from different stem cell sources has been investigated with conflicting results. In our study, we evaluated, both in vitro and in vivo, the effect of EVs derived from human bone marrow mesenchymal stromal cells (MSCs) and from a population of human liver stem cells (HLSCs) of mesenchymal origin on renal CSCs. In vitro, both EV sources displayed pro-apoptotic, anti-proliferative and anti-invasive effects on renal CSCs, but not on differentiated tumor cells. Pre-treatment of renal CSCs with EVs, before subcutaneous injection in SCID mice, delayed tumor onset. We subsequently investigated the in vivo effect of MSC- and HLSC-EVs systemic administration on progression of CSC-generated renal tumors. Tumor bio-distribution analysis identified intravenous treatment as best route of administration. HLSC-EVs, but not MSC-EVs, significantly impaired subcutaneous tumor growth by reducing tumor vascularization and inducing tumor cell apoptosis. Moreover, intravenous treatment with HLSC-EVs improved metastasis-free survival. In EV treated tumor explants, we observed both the transfer and the induction of miR-145 and of miR-200 family members. In transfected CSCs, the same miRNAs affected cell growth, invasion and survival. In conclusion, our results showed a specific antitumor effect of HLSC-EVs on CSC-derived renal tumors in vivo, possibly ascribed to the transfer and induction of specific antitumor miRNAs. Our study provides further evidence for a possible clinical application of stem cell-EVs in tumor treatment.

miR-130a and Tgfß Content in Extracellular Vesicles Derived from the Serum of Subjects at High Cardiovascular Risk Predicts their In-Vivo Angiogenic Potential.

Serum-derived extracellular vesicles (sEV) from healthy donors display in-vivo pro-angiogenic properties. To identify patients that may benefit from autologous sEV administration for pro-angiogenic purposes, sEV angiogenic capability has been evaluated in type 2 diabetic (T2DM) subjects (D), in obese individuals with (OD) and without (O) T2DM, and in subjects with ischemic disease (IC) (9 patients/group). sEV display different angiogenic properties in such cluster of individuals. miRNomic profile and TGFß content in sEV were evaluated. We found that miR-130a and TGFß content correlates with sEV in-vitro and in-vivo angiogenic properties, particularly in T2DM patients. Ingenuity Pathway Analysis (IPA) identified a number of genes as among the most significant miR-130a interactors. Gain-of-function experiments recognized homeoboxA5 (HOXA5) as a miR-130a specific target. Finally, ROC curve analyses revealed that sEV ineffectiveness could be predicted (Likelihood Ratio+ (LH+) = 3.3 IC 95% from 2.6 to 3.9) by comparing miR-130a and TGFß content 'in Series'. We demonstrate that sEV from high cardiovascular risk patients have different angiogenic properties and that miR-130a and TGFß sEV content predicts 'true ineffective sEVs'. These results provide the rationale for the use of these assays to identify patients that may benefit from autologous sEV administration to boost the angiogenetic process.

HLSC-Derived Extracellular Vesicles Attenuate Liver Fibrosis and Inflammation in a Murine Model of Non-alcoholic Steatohepatitis.

Extracellular vesicles (EVs) are membrane vesicles released virtually by all cell types. Several studies have shown that stem cell-derived EVs may mimic both in vitro and in vivo the biological effects of the cells. We recently demonstrated that non-alcoholic steatohepatitis (NASH) is inhibited by treatment with human liver stem cells (HLSCs). The aim of the present study was to evaluate whether EVs released by HLSCs influence the progression of NASH, induced by a diet deprived of methionine and choline, in immunocompromised mice. EV treatment was initiated after 2 weeks of diet with a biweekly administration of three different doses. Bio-distribution evaluated by optical imaging showed a preferential accumulation in normal and, in particular, in fibrotic liver. EV treatment significantly improved liver function and reduced signs of liver fibrosis and inflammation at both morphological and molecular levels. In particular, we observed that, out of 29 fibrosis-associated genes upregulated in NASH liver, 28 were significantly downregulated by EV treatment. In conclusion, HLSC-derived EVs display anti-fibrotic and anti-inflammatory effects in a model of chronic liver disease, leading to an improvement of liver function.

Extracellular Vesicles From Adipose Stem Cells Prevent Muscle Damage and Inflammation in a Mouse Model of Hind Limb Ischemia: Role of Neuregulin-1.

OBJECTIVES: Critical hindlimb ischemia is a severe consequence of peripheral artery disease. Surgical treatment does not prevent skeletal muscle impairment or improve long-term patient outcomes. The present study investigates the protective/regenerative potential and the mechanism of action of adipose stem cell-derived extracellular vesicles (ASC-EVs) in a mouse model of hindlimb ischemia. Approach and Results: We demonstrated that ASC-EVs exert a protective effect on muscle damage by acting both on tissue microvessels and muscle cells. The genes involved in muscle regeneration were up-regulated in the ischemic muscles of ASC-EV-treated animals. MyoD expression has also been confirmed in satellite cells. This was followed by a reduction in muscle function impairment in vivo. ASC-EVs drive myoblast proliferation and differentiation in the in vitro ischemia/reoxygenation model. Moreover, ASC-EVs have shown an anti-apoptotic effect both in vitro and in vivo. Transcriptomic analyses have revealed that ASC-EVs carry a variety of pro-angiogenic mRNAs, while proteomic analyses have demonstrated an enrichment of NRG1 (neuregulin 1). A NRG1 blocking antibody used in vivo demonstrated that NRG1 is relevant to ASC-EV-induced muscle protection, vascular growth, and recruitment of inflammatory cells. Finally, bioinformatic analyses on 18 molecules that were commonly detected in ASC-EVs, including mRNAs and proteins, confirmed the enrichment of pathways involved in vascular growth and muscle regeneration/protection. CONCLUSIONS: This study demonstrates that ASC-EVs display pro-angiogenic and skeletal muscle protective properties that are associated with their NRG1/mRNA cargo. We, therefore, propose that ASC-EVs are a useful tool for therapeutic angiogenesis and muscle protection.

Human Liver-Derived Stem Cells Improve Fibrosis and Inflammation Associated with Nonalcoholic Steatohepatitis.

Cell therapy may be regarded as a feasible alternative to whole organ transplantation to treat end-stage liver diseases. Human liver stem cells (HLSCs) are a population of cells easily obtainable and expandable from a human adult liver biopsy. HLSCs share with mesenchymal stromal cells the same phenotype, gene expression profile, and differentiation capabilities. In addition, HLSCs show a specific commitment to the hepatic phenotype. Injection of HLSCs into immunodeficient mice fed with a methionine-choline-deficient diet to induce nonalcoholic steatohepatitis ameliorates liver function and morphology. In particular, HLSC treatment induced a reduction of liver fibrosis and inflammation at morphological and molecular levels. Moreover, HLSCs were able to persist for up to 3 weeks after the injection. In conclusion, HLSCs have healing effects in a model of chronic liver disease.

Stem cell-derived extracellular vesicles inhibit and revert fibrosis progression in a mouse model of diabetic nephropathy.

Extracellular vesicles (EVs) that are derived from mesenchymal stromal cells (MSCs) have been shown to reprogram injured cells by activating regenerative processes. We herein investigate the potential therapeutic effect of EVs, shed by human bone marrow MSCs and by human liver stem-like cells (HLSCs), on the progression and reversion of fibrosis in a mouse model of diabetic nephropathy, as induced by streptozotocin. After the development of nephropathy, stem cell-derived EVs were administered weekly to diabetic mice for four weeks. The stem cell-derived EV treatment, but not the fibroblast EV treatment that was used as a control, significantly ameliorated functional parameters, such as albumin/creatinine excretion, plasma creatinine and blood urea nitrogen, which are altered in diabetic mice. Moreover, the renal fibrosis that develops during diabetic nephropathy progression was significantly inhibited in stem cell EV-treated animals. A correlation was found between the down regulation of several pro-fibrotic genes in renal tissues and the anti-fibrotic effect of HLSC and MSC EVs. A comparative analysis of HLSC and MSC EV miRNA content highlighted some common and some specific patterns of miRNAs that target predicted pro-fibrotic genes. In conclusion, stem cell-derived EVs inhibit fibrosis and prevent its progression in a model of diabetes-induced chronic kidney injury.

PDGF enhances the protective effect of adipose stem cell-derived extracellular vesicles in a model of acute hindlimb ischemia.

We previously have shown that platelet-derived growth factor (PDGF) modulates the biological activity of extracellular vesicles released by adipose-derived mesenchymal stem cells (ASC-EVs). ASC-EVs may interact with blood and vessel cells by transferring proteins and nucleic acids and regulate their functions. In this study, we investigated immunomodulatory activity and protection from acute hindlimb ischemia of EVs released by PDGF-stimulated ASC (PDGF-EVs). PDGF treatment of ASC changed protein and RNA composition of released EVs by enhancing the expression of anti-inflammatory and immunomodulatory factors. In vitro, control EVs (cEVs) derived from non-stimulated ASC increased the secretion of both the IL-1b, IL-17, IFNγ, TNFα pro-inflammatory factors and the IL-10 anti-inflammatory factor, and enhanced the in vitro peripheral blood mononuclear cell (PBMC) adhesion on endothelium. In contrast, PDGF-EVs enhanced IL-10 secretion and induced TGF-ß1 secretion by PBMC. Moreover, PDGF-EVs stimulated the formation of T regulatory cells. In vivo, PDGF-EVs protected muscle tissue from acute ischemia, reduced infiltration of inflammatory cells and increased T regulatory cell infiltration in respect to cEVs. Our results suggest that PDGF-EVs are enriched in anti-inflammatory and immunomodulatory factors and induced in PBMC an enhanced production of IL-10 and TGF-ß1 resulting in protection of muscle from acute ischemia in vivo.

Human Liver Stem Cell-Derived Extracellular Vesicles Prevent Aristolochic Acid-Induced Kidney Fibrosis.

With limited therapeutic intervention in preventing the progression to end-stage renal disease, chronic kidney disease (CKD) remains a global health-care burden. Aristolochic acid (AA) induced nephropathy is a model of CKD characterised by inflammation, tubular injury, and interstitial fibrosis. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) have been reported to exhibit therapeutic properties in various disease models including acute kidney injury. In the present study, we aimed to investigate the effects of HLSC-EVs on tubular regeneration and interstitial fibrosis in an AA-induced mouse model of CKD. NSG mice were injected with HLSC-EVs 3 days after administering AA on a weekly basis for 4 weeks. Mice injected with AA significantly lost weight over the 4-week period. Deterioration in kidney function was also observed. Histology was performed to evaluate tubular necrosis, interstitial fibrosis, as well as infiltration of inflammatory cells/fibroblasts. Kidneys were also subjected to gene array analyses to evaluate regulation of microRNAs (miRNAs) and pro-fibrotic genes. The effect of HLSC-EVs was also tested in vitro to assess pro-fibrotic gene regulation in fibroblasts cocultured with AA pretreated tubular epithelial cells. Histological analyses showed that treatment with HLSC-EVs significantly reduced tubular necrosis, interstitial fibrosis, infiltration of CD45 cells and fibroblasts, which were all elevated during AA induced injury. At a molecular level, HLSC-EVs significantly inhibited the upregulation of the pro-fibrotic genes α-Sma, Tgfb1, and Col1a1 in vivo and in vitro. Fibrosis gene array analyses revealed an upregulation of 35 pro-fibrotic genes in AA injured mice. Treatment with HLSC-EVs downregulated 14 pro-fibrotic genes in total, out of which, 5 were upregulated in mice injured with AA. Analyses of the total mouse miRnome identified several miRNAs involved in the regulation of fibrotic pathways, which were found to be modulated post-treatment with HLSC-EVs. These results indicate that HLSC-EVs play a regenerative role in CKD possibly through the regulation of genes and miRNAs that are activated during the progression of the disease.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Serum-derived extracellular vesicles (EVs) impact on vascular remodeling and prevent muscle damage in acute hind limb ischemia.

Serum is an abundant and accessible source of circulating extracellular vesicles (EVs). Serum-EV (sEV) pro-angiogenic capability and mechanisms are herein analyzed using an in vitro assay which predicts sEV angiogenic potential in vivo. Effective sEVs (e-sEVs) also improved vascular remodeling and prevented muscle damage in a mouse model of acute hind limb ischemia. e-sEV angiogenic proteomic and transcriptomic analyses show a positive correlation with matrix-metalloproteinase activation and extracellular matrix organization, cytokine and chemokine signaling pathways, Insulin-like Growth Factor and platelet pathways, and Vascular Endothelial Growth Factor signaling. A discrete gene signature, which highlights differences in e-sEV and ineffective-EV biological activity, was identified using gene ontology (GO) functional analysis. An enrichment of genes associated with the Transforming Growth Factor beta 1 (TGFß1) signaling cascade is associated with e-sEV administration but not with ineffective-EVs. Chromatin immunoprecipitation analysis on the inhibitor of DNA binding I (ID1) promoter region, and the knock-down of small mother against decapentaplegic (SMAD)1-5 proteins confirmed GO functional analyses. This study demonstrates sEV pro-angiogenic activity, validates a simple, sEV pro-angiogenic assay which predicts their biological activity in vivo, and identifies the TGFß1 cascade as a relevant mediator. We propose serum as a readily available source of EVs for therapeutic purposes.