Publications by authors named "Benedetta Bussolati"

131 Publications

Novel Human Podocyte Cell Model Carrying G2/G2 APOL1 High-Risk Genotype.

Cells 2021 Jul 28;10(8). Epub 2021 Jul 28.

Department of Development and Regeneration, Katholieke Universiteit Leuven, 3000 Leuven, Belgium.

Apolipoprotein L1 () high-risk genotypes (HRG), G1 and G2, increase the risk of various non-diabetic kidney diseases in the African population. To date, the precise mechanisms by which risk variants induce injury on podocytes and other kidney cells remain unclear. Trying to unravel these mechanisms, most studies have used animal or cell models created by gene editing. We developed and characterised conditionally immortalised human podocyte cell lines derived from urine of a donor carrying HRG G2/G2. Following induction of APOL1 expression by polyinosinic-polycytidylic acid (poly(I:C)), we assessed functional features of APOL1-induced podocyte dysfunction. As control, APOL1 wild type (G0/G0) podocyte cell line previously generated from a Caucasian donor was used. Upon exposure to poly(I:C), G2/G2 and G0/G0 podocytes upregulated APOL1 expression resulting in podocytes detachment, decreased cells viability and increased apoptosis rate in a genotype-independent manner. Nevertheless, G2/G2 podocyte cell lines exhibited altered features, including upregulation of CD2AP, alteration of cytoskeleton, reduction of autophagic flux and increased permeability in an in vitro model under continuous perfusion. The human APOL1 G2/G2 podocyte cell model is a useful tool for unravelling the mechanisms of APOL1-induced podocyte injury and the cellular functions of APOL1.
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http://dx.doi.org/10.3390/cells10081914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8391400PMC
July 2021

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Pass through the Filtration Barrier and Protect Podocytes in a 3D Glomerular Model under Continuous Perfusion.

Tissue Eng Regen Med 2021 08 27;18(4):549-560. Epub 2021 Jul 27.

Department of Molecular Biotechnology and Health Sciences, University of Torino, via Nizza 52, 10126, Turin, Italy.

Background: Dynamic cultures, characterized by continuous fluid reperfusion, elicit physiological responses from cultured cells. Mesenchymal stem cell-derived EVs (MSC-EVs) has been proposed as a novel approach in treating several renal diseases, including acute glomerular damage, by using traditional two-dimensional cell cultures and in vivo models. We here aimed to use a fluidic three-dimensional (3D) glomerular model to study the EV dynamics within the glomerular structure under perfusion.

Methods: To this end, we set up a 3D glomerular model culturing human glomerular endothelial cells and podocytes inside a bioreactor on the opposite sides of a porous membrane coated with type IV collagen. The bioreactor was connected to a circuit that allowed fluid passage at the rate of 80 µl/min. To mimic glomerular damage, the system was subjected to doxorubicin administration in the presence of therapeutic MSC-EVs.

Results: The integrity of the glomerular basal membrane in the 3D glomerulus was assessed by a permeability assay, demonstrating that the co-culture could limit the passage of albumin through the filtration barrier. In dynamic conditions, serum EVs engineered with cel-miR-39 passed through the glomerular barrier and transferred the exogenous microRNA to podocyte cell lines. Doxorubicin treatment increased podocyte apoptosis, whereas MSC-EV within the endothelial circuit protected podocytes from damage, decreasing cell death and albumin permeability.

Conclusion: Using an innovative millifluidic model, able to mimic the human glomerular barrier, we were able to trace the EV passage and therapeutic effect in dynamic conditions.
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http://dx.doi.org/10.1007/s13770-021-00374-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325748PMC
August 2021

Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Human Corneal Endothelial Cells from Endoplasmic Reticulum Stress-Mediated Apoptosis.

Int J Mol Sci 2021 May 6;22(9). Epub 2021 May 6.

Department of Biotechnology and Health Sciences, University of Turin, 10126 Turin, Italy.

Corneal endothelial dystrophy is a relevant cause of vision loss and corneal transplantation worldwide. In the present study, we analyzed the effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in an in vitro model of corneal dystrophy, characterized by endoplasmic reticulum stress. The effects of MSC-EVs were compared with those of serum-derived EVs, reported to display a pro-angiogenic activity. MSC-EVs were able to induce a significant down-regulation of the large majority of endoplasmic reticulum stress-related genes in human corneal endothelial cells after exposure to serum deprivation and tunicamycin. In parallel, they upregulated the Akt pathway and limited caspase-3 activation and apoptosis. At variance, the effect of the serum EVs was mainly limited to Akt phosphorylation, with minimal or absent effects on endoplasmic reticulum stress modulation and apoptosis prevention. The effects of MSC-EVs were correlated to the transfer of numerous endoplasmic reticulum (ER)-stress targeting miRNAs to corneal endothelial cells. These data suggest a potential therapeutic effect of MSC-EVs for corneal endothelial endoplasmic reticulum stress, a major player in corneal endothelial dystrophy.
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http://dx.doi.org/10.3390/ijms22094930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125791PMC
May 2021

Angiogenic Properties of Placenta-Derived Extracellular Vesicles in Normal Pregnancy and in Preeclampsia.

Int J Mol Sci 2021 May 20;22(10). Epub 2021 May 20.

Department of Molecular Biotechnology and Health Sciences, University of Torino, 10124 Torino, Italy.

Angiogenesis is one of the main processes that coordinate the biological events leading to a successful pregnancy, and its imbalance characterizes several pregnancy-related diseases, including preeclampsia. Intracellular interactions via extracellular vesicles (EVs) contribute to pregnancy's physiology and pathophysiology, and to the fetal-maternal interaction. The present review outlines the implications of EV-mediated crosstalk in the angiogenic process in healthy pregnancy and its dysregulation in preeclampsia. In particular, the effect of EVs derived from gestational tissues in pro and anti-angiogenic processes in the physiological and pathological setting is described. Moreover, the application of EVs from placental stem cells in the clinical setting is reported.
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http://dx.doi.org/10.3390/ijms22105402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160914PMC
May 2021

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

J Extracell Vesicles 2021 May 21;10(7):e12093. Epub 2021 May 21.

Department of Cell and Molecular Biology QIMR Berghofer Medical Research Institute Brisbane Australia.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.
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http://dx.doi.org/10.1002/jev2.12093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138533PMC
May 2021

Effect of Stem Cell-Derived Extracellular Vesicles on Damaged Human Corneal Endothelial Cells.

Stem Cells Int 2021 16;2021:6644463. Epub 2021 Jan 16.

Department of Biotechnology and Health Sciences, University of Turin, Turin, Italy.

Purpose: Human corneal endothelial cells (HCECs) are essential to visual function; however, since they have limited proliferative capacity , they are prone to corneal endothelial dysfunction. At present, the only treatment is a corneal transplantation from donor cadavers. Also, due to a global shortage of donor corneas, it is important to find alternative strategies. Recent studies highlight that stem cell-derived extracellular vesicles (EVs) play a relevant role in stem cell-induced regeneration by reprogramming injured cells and inducing proregenerative pathways. The aim of this work is to evaluate whether EVs derived from mesenchymal stem cells (MSC-EVs) are able to promote regeneration of damaged HCECs.

Methods: We isolated HCECs from discarded corneas in patients undergoing corneal transplantation or enucleation ( = 23 patients). Bone marrow mesenchymal stem cells (MSCs) were obtained from Lonza, cultured, and characterized. MSC-EVs were obtained from supernatants of MSCs. In order to establish a valid damage model to test the regenerative potential of EVs on HCECs, we evaluated the proliferation rate and the apoptosis after exposing the cells to serum-deprived medium at different concentrations for 24 hours. We then evaluated the HCEC migration through a wound healing assay.

Results: In the selected serum deprivation damage conditions, the treatment with different doses of MSC-EVs resulted in a significantly higher proliferation rate of HCECs at all the tested concentrations of EVs (5-20 × 10 MSC-EV/cell). MSC-EVs/cell induced a significant decrease in number of total apoptotic cells after 24 hours of serum deprivation. Finally, the wound healing assay showed a significantly faster repair of the wound after HCEC treatment with MSC-EVs.

Conclusions: Results highlight the already well-known proregenerative potential of MSC-EVs in a totally new biological model, the endothelium of the cornea. MSC-EVs, indeed, induced proliferation and survival of HCECs, promoting the migration of HCECs .
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http://dx.doi.org/10.1155/2021/6644463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7834816PMC
January 2021

Coincubation as miR-Loading Strategy to Improve the Anti-Tumor Effect of Stem Cell-Derived EVs.

Pharmaceutics 2021 Jan 8;13(1). Epub 2021 Jan 8.

Department of Molecular Biotechnology and Health Sciences, University of Torino, 10126 Torino, Italy.

Extracellular vesicles are considered a novel therapeutic tool, due to their ability to transfer their cargoes to target cells. Different strategies to directly load extracellular vesicles with RNA species have been proposed. Electroporation has been used for the loading of non-active vesicles; however, the engineering of vesicles already carrying a therapeutically active cargo is still under investigation. Here, we set up a coincubation method to increase the anti-tumor effect of extracellular vesicles isolated from human liver stem cells (HLSC-EVs). Using the coincubation protocol, vesicles were loaded with the anti-tumor miRNA-145, and their effect was evaluated on renal cancer stem cell invasion. Loaded HLSC-EVs maintained their integrity and miR transfer ability. Loaded miR-145, but not miR-145 alone, was protected by RNAse digestion, possibly due to its binding to RNA-binding proteins on HLSC-EV surface, such as Annexin A2. Moreover, miR-145 coincubated HLSC-EVs were more effective in inhibiting the invasive properties of cancer stem cells, in comparison to naïve vesicles. The protocol reported here exploits a well described property of extracellular vesicles to bind nucleic acids on their surface and protect them from degradation, in order to obtain an effective miRNA loading, thus increasing the activity of therapeutically active naïve extracellular vesicles.
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http://dx.doi.org/10.3390/pharmaceutics13010076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826638PMC
January 2021

Extracellular Vesicles Released by Tumor Endothelial Cells Spread Immunosuppressive and Transforming Signals Through Various Recipient Cells.

Front Cell Dev Biol 2020 9;8:698. Epub 2020 Sep 9.

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, United States.

Head and neck squamous cell carcinoma (HNSCC) has a high recurrence and metastatic rate with an unknown mechanism of cancer spread. Tumor inflammation is the most critical processes of cancer onset, growth, and metastasis. We hypothesize that the release of extracellular vesicles (EVs) by tumor endothelial cells (TECs) induce reprogramming of immune cells as well as stromal cells to create an immunosuppressive microenvironment that favor tumor spread. We call this mechanism as non-metastatic contagious carcinogenesis. Extracellular vesicles were collected from primary HNSCC-derived endothelial cells (TEC-EV) and were used for stimulation of peripheral blood mononuclear cells (PBMCs) and primary adipose mesenchymal stem cells (ASCs). Regulation of ASC gene expression was investigated by RNA sequencing and protein array. PBMC, stimulated with TEC-EV, were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting. We validated the effects of TEC-EV on ASCs or PBMC by measuring invasion, adhesion, and proliferation. We found and confirmed that TEC-EV were able to change ASC inflammatory gene expression signature within 24-48 h. TEC-EV were also able to enhance the secretion of TGF-β1 and IL-10 by PBMC and to increase T regulatory cell (Treg) expansion. TEC-EV carry specific proteins and RNAs that are responsible for Treg differentiation and immune suppression. ASCs and PBMC, treated with TEC-EV, enhanced proliferation, adhesion of tumor cells, and their invasion. These data indicate that TEC-EV exhibit a mechanism of non-metastatic contagious carcinogenesis that regulates tumor microenvironment and reprograms immune cells to sustain tumor growth and progression.
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http://dx.doi.org/10.3389/fcell.2020.00698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509153PMC
September 2020

Intrinsic and Extrinsic Modulators of the Epithelial to Mesenchymal Transition: Driving the Fate of Tumor Microenvironment.

Front Oncol 2020 24;10:1122. Epub 2020 Jul 24.

Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

The epithelial to mesenchymal transition (EMT) is an evolutionarily conserved process. In cancer, EMT can activate biochemical changes in tumor cells that enable the destruction of the cellular polarity, leading to the acquisition of invasive capabilities. EMT regulation can be triggered by intrinsic and extrinsic signaling, allowing the tumor to adapt to the microenvironment demand in the different stages of tumor progression. In concomitance, tumor cells undergoing EMT actively interact with the surrounding tumor microenvironment (TME) constituted by cell components and extracellular matrix as well as cell secretome elements. As a result, the TME is in turn modulated by the EMT process toward an aggressive behavior. The current review presents the intrinsic and extrinsic modulators of EMT and their relationship with the TME, focusing on the non-cell-derived components, such as secreted metabolites, extracellular matrix, as well as extracellular vesicles. Moreover, we explore how these modulators can be suitable targets for anticancer therapy and personalized medicine.
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http://dx.doi.org/10.3389/fonc.2020.01122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393251PMC
July 2020

Molecular and functional characterization of urine-derived podocytes from patients with Alport syndrome.

J Pathol 2020 09;252(1):88-100

Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.

Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV α3, α4 or α5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non-invasive way. Here we isolated, immortalized, and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA-sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte-glomerular endothelial cell co-culture system, was established and AS podocyte co-cultures showed a significantly higher permeability of albumin compared to control podocyte co-cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..
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http://dx.doi.org/10.1002/path.5496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589231PMC
September 2020

International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19.

Cytotherapy 2020 09 16;22(9):482-485. Epub 2020 May 16.

Institute of Molecular and Cellular Biology, Agency for Science, Technology and Research, Singapore. Electronic address:

Statement: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.
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http://dx.doi.org/10.1016/j.jcyt.2020.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229942PMC
September 2020

Extracellular Vesicles Derived from Induced Pluripotent Stem Cells Promote Renoprotection in Acute Kidney Injury Model.

Cells 2020 02 17;9(2). Epub 2020 Feb 17.

Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil.

Induced pluripotent stem cells (iPSC) have been the focus of several studies due to their wide range of application, including in cellular therapy. The use of iPSC in regenerative medicine is limited by their tumorigenic potential. Extracellular vesicles (EV) derived from stem cells have been shown to support renal recovery after injury. However, no investigation has explored the potential of iPSC-EV in the treatment of kidney diseases. To evaluate this potential, we submitted renal tubule cells to hypoxia-reoxygenation injury, and we analyzed cell death rate and changes in functional mitochondria mass. An in vivo model of ischemia-reperfusion injury was used to evaluate morphological and functional alterations. Gene array profile was applied to investigate the mechanism involved in iPSC-EV effects. In addition, EV derived from adipose mesenchymal cells (ASC-EV) were also used to compare the potential of iPSC-EV in support of tissue recovery. The results showed that iPSC-EV were capable of reducing cell death and inflammatory response with similar efficacy than ASC-EV. Moreover, iPSC-EV protected functional mitochondria and regulated several genes associated with oxidative stress. Taken together, these results show that iPSC can be an alternative source of EV in the treatment of different aspects of kidney disease.
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http://dx.doi.org/10.3390/cells9020453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072760PMC
February 2020

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

Int J Cancer 2020 09 25;147(6):1694-1706. Epub 2020 Feb 25.

Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.

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.
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http://dx.doi.org/10.1002/ijc.32925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496472PMC
September 2020

Potential Applications of Extracellular Vesicles in Solid Organ Transplantation.

Cells 2020 02 5;9(2). Epub 2020 Feb 5.

Department of Medical Sciences, University of Turin, 10126 Turin, Italy.

Extracellular vesicles (EVs) play an important role in cell-to-cell communication by delivering coding and non-coding RNA species and proteins to target cells. Recently, the therapeutic potential of EVs has been shown to extend to the field of solid organ transplantations. Mesenchymal stromal cell-derived EVs (MSC-EVs) in particular have been proposed as a new tool to improve graft survival, thanks to the modulation of tolerance toward the graft, and to their anti-fibrotic and pro-angiogenic effects. Moreover, MSC-EVs may reduce ischemia reperfusion injury, improving the recovery from acute damage. In addition, EVs currently considered helpful tools for preserving donor organs when administered before transplant in the context of hypothermic or normothermic perfusion machines. The addition of EVs to the perfusion solution, recently proposed for kidney, lung, and liver grafts, resulted in the amelioration of donor organ viability and functionality. EVs may therefore be of therapeutic interest in different aspects of the transplantation process for increasing the number of available organs and improving their long-term survival.
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http://dx.doi.org/10.3390/cells9020369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072603PMC
February 2020

Adipose Mesenchymal Cells-Derived EVs Alleviate DOCA-Salt-Induced Hypertension by Promoting Cardio-Renal Protection.

Mol Ther Methods Clin Dev 2020 Mar 15;16:63-77. Epub 2019 Nov 15.

Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil.

Hypertension is a long-term condition that can increase organ susceptibility to insults and lead to severe complications such as chronic kidney disease (CKD). Extracellular vesicles (EVs) are cell-derived membrane structures that participate in cell-cell communication by exporting encapsulated molecules to target cells, regulating physiological and pathological processes. We here demonstrate that multiple administration of EVs from adipose-derived mesenchymal stromal cells (ASC-EVs) in deoxycorticosterone acetate (DOCA)-salt hypertensive model can protect renal tissue by maintaining its filtration capacity. Indeed, ASC-EVs downregulated the pro-inflammatory molecules monocyte chemoattracting protein-1 (MCP-1) and plasminogen activating inhibitor-1 (PAI1) and reduced recruitment of macrophages in the kidney. Moreover, ASC-EVs prevented cardiac tissue fibrosis and maintained blood pressure within normal levels, thus demonstrating their multiple favorable effects in different organs. By applying microRNA (miRNA) microarray profile of the kidney of DOCA-salt rats, we identified a selective miRNA signature associated with epithelial-mesenchymal transition (EMT). One of the key pathways found was the axis miR-200-TGF-β, that was significantly altered by EV administration, thereby affecting the EMT signaling and preventing renal inflammatory response and fibrosis development. Our results indicate that EVs can be a potent therapeutic tool for the treatment of hypertension-induced CKD in cardio-renal syndrome.
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http://dx.doi.org/10.1016/j.omtm.2019.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909095PMC
March 2020

Acute and chronic glomerular damage is associated with reduced CD133 expression in urinary extracellular vesicles.

Am J Physiol Renal Physiol 2020 02 23;318(2):F486-F495. Epub 2019 Dec 23.

Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

Extracellular vesicles released into urine (uEVs) can represent interesting biomarkers of renal cell damage. CD133, a stem/progenitor cell marker expressed by renal progenitor cells, is highly expressed in uEVs of healthy individuals. In the present study, we evaluated the level of CD133 in the uEVs of patients with acute and chronic glomerular damage by cytofluorimetric analysis. The level of CD133 uEVs was significantly decreased in pediatric patients with acute glomerulonephritis during the acute phase of renal damage, while it was restored after the subsequent recovery. A similar decrease was also observed in patients with chronic glomerulonephritis. Moreover, CD133 uEVs significantly declined in patients with type 2 diabetes, used as validation group, with the lowest levels in patients with albuminuria with diabetic nephropathy. Indeed, receiver-operating characteristic curve analysis indicates the ability of CD133 uEV values to discriminate the health condition from that of glomerular disease. In parallel, a significant decrease of CD133 in renal progenitor cells and in their derived EVs was observed in vitro after cell treatment with a combination of glucose and albumin overload, mimicking the diabetic condition. These data indicate that the level of CD133 uEVs may represent an easily accessible marker of renal normal physiology and could provide information on the "reservoir" of regenerating cells within tubules.
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http://dx.doi.org/10.1152/ajprenal.00404.2019DOI Listing
February 2020

Urinary Extracellular Vesicles Carrying Klotho Improve the Recovery of Renal Function in an Acute Tubular Injury Model.

Mol Ther 2020 02 18;28(2):490-502. Epub 2019 Nov 18.

Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy. Electronic address:

Acute kidney injury, defined by a rapid deterioration of renal function, is a common complication in hospitalized patients. Among the recent therapeutic options, the use of extracellular vesicles (EVs) is considered a promising strategy. Here we propose a possible therapeutic use of renal-derived EVs isolated from normal urine (urine-derived EVs [uEVs]) in a murine model of acute injury generated by glycerol injection. uEVs accelerated renal recovery, stimulating tubular cell proliferation, reducing the expression of inflammatory and injury markers, and restoring endogenous Klotho loss. When intravenously injected, labeled uEVs localized within injured kidneys and transferred their microRNA cargo. Moreover, uEVs contained the reno-protective Klotho molecule. Murine uEVs derived from Klotho null mice lost the reno-protective effect observed using murine EVs from wild-type mice. This was regained when Klotho-negative murine uEVs were reconstituted with recombinant Klotho. Similarly, ineffective fibroblast EVs acquired reno-protection when engineered with human recombinant Klotho. Our results reveal a novel potential use of uEVs as a new therapeutic strategy for acute kidney injury, highlighting the presence and role of the reno-protective factor Klotho.
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http://dx.doi.org/10.1016/j.ymthe.2019.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000999PMC
February 2020

Alternative Strategies to Inhibit Tumor Vascularization.

Int J Mol Sci 2019 Dec 7;20(24). Epub 2019 Dec 7.

Department of Molecular Biotechnology and Health Sciences; Universitty of Torino, 10126 Torino, Italy.

Endothelial cells present in tumors show different origin, phenotype, and genotype with respect to the normal counterpart. Various mechanisms of intra-tumor vasculogenesis sustain the complexity of tumor vasculature, which can be further modified by signals deriving from the tumor microenvironment. As a result, resistance to anti-VEGF therapy and activation of compensatory pathways remain a challenge in the treatment of cancer patients, revealing the need to explore alternative strategies to the classical anti-angiogenic drugs. In this review, we will describe some alternative strategies to inhibit tumor vascularization, including targeting of antigens and signaling pathways overexpressed by tumor endothelial cells, the development of endothelial vaccinations, and the use of extracellular vesicles. In addition, anti-angiogenic drugs with normalizing effects on tumor vessels will be discussed. Finally, we will present the concept of endothelial demesenchymalization as an alternative approach to restore normal endothelial cell phenotype.
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http://dx.doi.org/10.3390/ijms20246180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940973PMC
December 2019

Stem Cell-Derived Extracellular Vesicles and Kidney Regeneration.

Cells 2019 10 11;8(10). Epub 2019 Oct 11.

Department of Molecular Biotechnology and Health Sciences, University of Turin, via Nizza 52, 10126 Torino, Italy.

Extracellular vesicles (EVs) are membranous vesicles containing active proteins, lipids, and different types of genetic material such as miRNAs, mRNAs, and DNAs related to the characteristics of the originating cell. They possess a distinctive capacity to communicate over long distances. EVs have been involved in the modulation of several pathophysiological conditions and, more importantly, stem cell-derived EVs appear as a new promising therapeutic option. In fact, several reports provide convincing evidence of the regenerative potential of EVs released by stem cells and, in particular, mesenchymal stromal cells (MSCs) in different kidney injury models. Described mechanisms involve the reprogramming of injured cells, cell proliferation and angiogenesis, and inhibition of cell apoptosis and inflammation. Besides, the therapeutic use of MSC-EVs in clinical trials is under investigation. This review will focus on MSC-EV applications in preclinical models of acute and chronic renal damage including recent data on their use in kidney transplant conditioning. Moreover, ongoing clinical trials are described. Finally, new strategies to broaden and enhance EV therapeutic efficacy by engineering are discussed.
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http://dx.doi.org/10.3390/cells8101240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830104PMC
October 2019

Transient Receptor Potential Channel Expression Signatures in Tumor-Derived Endothelial Cells: Functional Roles in Prostate Cancer Angiogenesis.

Cancers (Basel) 2019 Jul 8;11(7). Epub 2019 Jul 8.

Univ. Lille, Inserm, U1003-PHYCEL-Physiologie Cellulaire, F-59000 Lille, France.

: Transient receptor potential (TRP) channels control multiple processes involved in cancer progression by modulating cell proliferation, survival, invasion and intravasation, as well as, endothelial cell (EC) biology and tumor angiogenesis. Nonetheless, a complete TRP expression signature in tumor vessels, including in prostate cancer (PCa), is still lacking. In the present study, we profiled by qPCR the expression of all TRP channels in human prostate tumor-derived ECs (TECs) in comparison with TECs from breast and renal tumors. We further functionally characterized the role of the '' channels in proliferation, sprout formation and elongation, directed motility guiding, as well as in vitro and in vivo morphogenesis and angiogenesis. : We identified three '' genes whose expression is upregulated in prostate TECs: TRPV2 as a positive modulator of TEC proliferation, TRPC3 as an endothelial PCa cell attraction factor and TRPA1 as a critical TEC angiogenic factor in vitro and in vivo. : We provide here the full TRP signature of PCa vascularization among which three play a profound effect on EC biology. These results contribute to explain the aggressive phenotype previously observed in PTEC and provide new putative therapeutic targets.
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http://dx.doi.org/10.3390/cancers11070956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678088PMC
July 2019

Purinergic Calcium Signals in Tumor-Derived Endothelium.

Cancers (Basel) 2019 Jun 1;11(6). Epub 2019 Jun 1.

Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy.

Tumor microenvironment is particularly enriched with extracellular ATP (eATP), but conflicting evidence has been provided on its functional effects on tumor growth and vascular remodeling. We have previously shown that high eATP concentrations exert a strong anti-migratory, antiangiogenic and normalizing activity on human tumor-derived endothelial cells (TECs). Since both metabotropic and ionotropic purinergic receptors trigger cytosolic calcium increase ([Ca]c), the present work investigated the properties of [Ca]c events elicited by high eATP in TECs and their role in anti-migratory activity. In particular, the quantitative and kinetic properties of purinergic-induced Ca release from intracellular stores and Ca entry from extracellular medium were investigated. The main conclusions are: (1) stimulation of TECs with high eATP triggers [Ca]c signals which include Ca mobilization from intracellular stores (mainly ER) and Ca entry through the plasma membrane; (2) the long-lasting Ca influx phase requires both store-operated Ca entry (SOCE) and non-SOCE components; (3) SOCE is not significantly involved in the antimigratory effect of high ATP stimulation; (4) ER is the main source for intracellular Ca release by eATP: it is required for the constitutive migratory potential of TECs but is not the only determinant for the inhibitory effect of high eATP; (5) a complex interplay occurs among ER, mitochondria and lysosomes upon purinergic stimulation; (6) high eUTP is unable to inhibit TEC migration and evokes [Ca]c signals very similar to those described for eATP. The potential role played by store-independent Ca entry and Ca-independent events in the regulation of TEC migration by high purinergic stimula deserves future investigation.
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http://dx.doi.org/10.3390/cancers11060766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627696PMC
June 2019

Effect of Bilastine on Diabetic Nephropathy in DBA2/J Mice.

Int J Mol Sci 2019 May 24;20(10). Epub 2019 May 24.

Department of Scienza e Tecnologia del Farmaco, University of Turin, Via P. Giuria 9, 10125 Turin, Italy.

Diabetic nephropathy is an unmet therapeutic need, and the search for new therapeutic strategies is warranted. Previous data point to histamine H receptor as a possible target for glomerular dysfunction associated with long term hyperglycaemia. Therefore, this study investigated the effects of the H receptor antagonist bilastine on renal morphology and function in a murine model of streptozotocin-induced diabetes. Diabetes was induced in DBA2/J male mice and, from diabetes onset (glycaemia ≥200 mg/dL), mice received bilastine (1-30 mg/kg/day) by oral gavage for 14 consecutive weeks. At the end of the experimental protocol, diabetic mice showed polyuria (+195.5%), increase in Albumin-to-Creatine Ratio (ACR, +284.7%), and a significant drop in creatinine clearance ( < 0.05). Bilastine prevented ACR increase and restored creatinine clearance in a dose-dependent manner, suggesting a positive effect on glomerular filtration. The ultrastructural analysis showed a preserved junctional integrity. Preservation of the basal nephrin, P-cadherin, and synaptopodin expression could explain this effect. In conclusion, the H receptor could contribute to the glomerular damage occurring in diabetic nephropathy. Bilastine preserved the glomerular junctional integrity, leading to the hypothesis of anti-H antihistamines as a possible add-on therapy for diabetic nephropathy.
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http://dx.doi.org/10.3390/ijms20102554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6566437PMC
May 2019

Extracellular Vesicles and Carried miRNAs in the Progression of Renal Cell Carcinoma.

Int J Mol Sci 2019 Apr 13;20(8). Epub 2019 Apr 13.

Molecular Biotechnology Centre, University of Turin, via Nizza 52, 10126 Turin, Italy.

The formation and maintenance of renal cell carcinomas (RCC) involve many cell types, such as cancer stem and differentiated cells, endothelial cells, fibroblasts and immune cells. These all contribute to the creation of a favorable tumor microenvironment to promote tumor growth and metastasis. Extracellular vesicles (EVs) are considered to be efficient messengers that facilitate the exchange of information within the different tumor cell types. Indeed, tumor EVs display features of their originating cells and force recipient cells towards a pro-tumorigenic phenotype. This review summarizes the recent knowledge related to the biological role of EVs, shed by renal tumor cells and renal cancer stem cells in different aspects of RCC progression, such as angiogenesis, immune escape and tumor growth. Moreover, a specific role for renal cancer stem cell derived EVs is described in the formation of the pre-metastatic niche. We also highlight the tumor EV cargo, especially the oncogenic miRNAs, which are involved in these processes. Finally, the circulating miRNAs appear to be a promising source of biomarkers in RCC.
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http://dx.doi.org/10.3390/ijms20081832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514717PMC
April 2019

Anti-tumor activity of stem cell-derived extracellular vesicles.

Oncotarget 2019 Mar 8;10(20):1872-1873. Epub 2019 Mar 8.

Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.

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http://dx.doi.org/10.18632/oncotarget.26759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443014PMC
March 2019

Improved Loading of Plasma-Derived Extracellular Vesicles to Encapsulate Antitumor miRNAs.

Mol Ther Methods Clin Dev 2019 Jun 9;13:133-144. Epub 2019 Jan 9.

Department of Medical Sciences, University of Turin, 10126 Turin, Italy.

Extracellular vesicles (EVs) carry various molecules involved in intercellular communication and have raised great interest as drug delivery systems. Several engineering methods have been investigated for vesicle loading. Here, we studied the electroporation of EVs isolated from plasma to load antitumor microRNAs (miRNAs). First, we optimized the transfection protocol using miRNA cel-39 by evaluating different parameters (voltage and pulse) for their effect on vesicle morphology, loading capacity, and miRNA transfer to target cells. When compared with direct incubation of EVs with miRNA, mild electroporation allowed more efficient loading and better protection of miRNA from RNase degradation. Moreover, electroporation preserved the naive vesicle cargo, including RNAs and proteins, and their ability to be taken up by target cells, supporting the absence of vesicle damage. EVs engineered with antitumor miRNAs (miR-31 and miR-451a) successfully promoted apoptosis of the HepG2 hepatocellular carcinoma cell line, silencing target genes involved in anti-apoptotic pathways. Our findings indicate an efficient and functional miRNA encapsulation in plasma-derived EVs following an electroporation protocol that preserves EV integrity.
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http://dx.doi.org/10.1016/j.omtm.2019.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370572PMC
June 2019

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

Human liver stem cell-derived extracellular vesicles enhance cancer stem cell sensitivity to tyrosine kinase inhibitors through Akt/mTOR/PTEN combined modulation.

Oncotarget 2018 Nov 16;9(90):36151-36165. Epub 2018 Nov 16.

Department of Molecular Biotechnology and Health Sciences, Torino, Italy.

It is well recognized that Cancer Stem Cells (CSCs) sustain the initiation, the maintenance and the recurrence of tumors. We previously reported that extracellular vesicles (EVs) derived from human liver stem cells (HLSCs) were able to limit tumor development. In this study, we evaluated whether EV derived from HLSCs could act in synergy with tyrosine kinase inhibitors (TKIs) on apoptosis of CSCs isolated from renal carcinomas. For this purpose, we administered to renal CSCs, HLSC-EVs and TKIs, as co-incubation or sequential administration. We found that HLSC-EVs in combination with Sunitinb or Sorafenib significantly increased renal CSCs apoptosis induced by low TKI dose. At variance, no synergistic effect was observed when bone marrow mesenchymal stem cell-derived EVs were used. In particular, renal CSCs chemosensitivity to TKIs was enhanced when HLSC-EVs were either co-administered with TKIs or added after, but not before. CSC apoptosis was also incremented at a percentage comparable to that of co-administration when TKIs were loaded in HLSC-EVs. By a mechanistic point of view, Akt/mTOR and Erk and Creb intracellular pathways, known to be pivotal in the induction of tumor growth and survival, appeared modulated as consequence of TKIs/HLSC-EVs co-administration. Together, our results indicate that the synergistic effect of HLSC-EVs with TKIs may increase the response to TKIs at low doses, providing a rational for their combined use in the treatment of renal carcinoma.
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http://dx.doi.org/10.18632/oncotarget.26319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281417PMC
November 2018

Pioneering updates in vascular biology.

Vascul Pharmacol 2019 01 7;112. Epub 2018 Dec 7.

Department of Biomedicine, University Hospital, University of Basel, DBM 407, Hebelstrasse 20, 4031 Basel, Switzerland.. Electronic address:

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http://dx.doi.org/10.1016/j.vph.2018.12.001DOI Listing
January 2019

Concise Reviews: Stem Cells and Kidney Regeneration: An Update.

Stem Cells Transl Med 2019 01 9;8(1):82-92. Epub 2018 Oct 9.

GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, Children's Hospital Los Angeles, Division of Urology, Saban Research Institute, University of Southern California, Los Angeles, California.

Significant progress has been made to advance stem cell products as potential therapies for kidney diseases: various kinds of stem cells can restore renal function in preclinical models of acute and chronic kidney injury. Nonetheless this literature contains contradictory results, and for this reason, we focus this review on reasons for apparent discrepancies in the literature, because they contribute to difficulty in translating renal regenerative therapies. Differences in methodologies used to derive and culture stem cells, even those from the same source, in addition to the lack of standardized renal disease animal models (both acute and chronic), are important considerations underlying contradictory results in the literature. We propose that harmonized rigorous protocols for characterization, handling, and delivery of stem cells in vivo could significantly advance the field, and present details of some suggested approaches to foster translation in the field of renal regeneration. Our goal is to encourage coordination of methodologies (standardization) and long-lasting collaborations to improve protocols and models to lead to reproducible, interpretable, high-quality preclinical data. This approach will certainly increase our chance to 1 day offer stem cell therapeutic options for patients with all-too-common renal diseases. Stem Cells Translational Medicine 2019;8:82-92.
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http://dx.doi.org/10.1002/sctm.18-0115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312445PMC
January 2019

Extracellular vesicles from human liver stem cells inhibit tumor angiogenesis.

Int J Cancer 2019 01 22;144(2):322-333. Epub 2018 Oct 22.

Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

Human liver stem-like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti-tumor activity. In our study, we investigated whether HLSC-derived EVs (HLSC-EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC-EVs). The results obtained indicated that HLSC-EVs, but not MSC-EVs, inhibited the angiogenic properties of tumor-derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC-EVs led to the down-regulation of pro-angiogenic genes. Since HLSC-EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC-EV specific miRNAs. We observed that four miRNAs, namely miR-15a, miR-181b, miR-320c and miR-874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC-EVs significantly enhanced expression of miR-15a, miR-181b, miR-320c and miR-874 associated with the down-regulation of FGF1 and PLAU. In summary, HLSC-EVs possess an anti-tumorigenic effect, based on their ability to inhibit tumor angiogenesis.
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http://dx.doi.org/10.1002/ijc.31796DOI Listing
January 2019
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