Serum Derived Extracellular Vesicles Mediated Delivery of Synthetic miRNAs in Human Endothelial Cells.

Extracellular vesicles (EVs) have emerged in the last decades as a cell-to-cell communication mechanism. One of their mechanism of action is the direct delivery of their cargo, composed of bioactive molecules to target cells. Different methods (direct electroporation, cell transfection, chemical transfection) were developed to vehicle therapeutic molecules through EVs. However, most of these techniques presented some limitations such as EV disruption and aggregation. In the present study, we demonstrated that a direct temperature-controlled co-incubation of EVs with defined miRNAs is a stable method to deliver information to target cells without affecting EV constitutive content. We chose serum as an easy and abundant source of EVs applicable to autologous treatment after EV modification. Exogenous cel-miR-39 loaded on serum EVs (SEVs) was taken up by human endothelial cells, demonstrating an adequate miRNA loading efficacy based on the co-incubation method. Moreover, SEVs co-incubation with the angiomiRNA-126 (miR-126) enhanced their angiogenic properties in vitro and in vivo by increasing the capacity to induce capillary-like structure formation of human endothelial cells. MiR-126 loaded EVs were also shown to stimulate mouse endothelial cells to invade Matrigel plugs and create more vessels with respect to the EV naive counterpart. When SEVs were loaded with miR-19b, an anti-angiogenic miRNA, they were able to reduce Vascular endothelial growth factors (VEGF) pro-angiogenic capacity, supporting the selective biological effect mediated by the carried miRNA. Lastly, we identified Annexin A2 (ANXA2) as one of the molecules involved in the exogenous RNA binding to serum EV surface, favoring miRNA delivery to target endothelial cells for potential therapeutic application.

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

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.

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

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..

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.

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

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.

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.

Role of CD133 Molecule in Wnt Response and Renal Repair.

Renal repair after injury is dependent on clonal expansion of proliferation-competent cells. In the human kidney, the expression of CD133 characterizes a population of resident scattered cells with resistance to damage and ability to proliferate. However, the biological function of the CD133 molecule is unknown. By RNA sequencing, we found that cells undergoing cisplatin damage lost the CD133 signature and acquired metanephric mesenchymal and regenerative genes such as SNAIL1, KLF4, SOX9, and WNT3. CD133 was reacquired in the recovery phase. In CD133-Kd cells, lack of CD133 limited cell proliferation after injury and was specifically correlated with deregulation of Wnt signaling and E-cadherin pathway. By immunoprecipitation, CD133 appeared to form a complex with E-cadherin and ß-catenin. In parallel, CD133-Kd cells showed lower ß-catenin levels in basal condition and after Wnt pathway activation and reduced TCF/LEF promoter activation in respect to CD133+ cells. Finally, the lack of CD133 impaired generation of nephrospheres while favoring senescence. These data indicate that CD133 may act as a permissive factor for ß-catenin signaling, preventing its degradation in the cytoplasm. Therefore, CD133 itself appears to play a functional role in renal tubular repair through maintenance of proliferative response and control of senescence. Stem Cells Translational Medicine 2018;7:283-294.

Urine of Preterm Neonates as a Novel Source of Kidney Progenitor Cells.

In humans, nephrogenesis is completed prenatally, with nephrons formed until 34 weeks of gestational age. We hypothesized that urine of preterm neonates born before the completion of nephrogenesis is a noninvasive source of highly potent stem/progenitor cells. To test this hypothesis, we collected freshly voided urine at day 1 after birth from neonates born at 31-36 weeks of gestational age and characterized isolated cells using a single-cell RT-PCR strategy for gene expression analysis and flow cytometry and immunofluorescence for protein expression analysis. Neonatal stem/progenitor cells expressed markers of nephron progenitors but also, stromal progenitors, with many single cells coexpressing these markers. Furthermore, these cells presented mesenchymal stem cell features and protected cocultured tubule cells from cisplatin-induced apoptosis. Podocytes differentiated from the neonatal stem/progenitor cells showed upregulation of podocyte-specific genes and proteins, albumin endocytosis, and calcium influx via podocyte-specific transient receptor potential cation channel, subfamily C, member 6. Differentiated proximal tubule cells showed upregulation of specific genes and significantly elevated p-glycoprotein activity. We conclude that urine of preterm neonates is a novel noninvasive source of kidney progenitors that are capable of differentiation into mature kidney cells and have high potential for regenerative kidney repair.

Human Urine as a Noninvasive Source of Kidney Cells.

Urine represents an unlimited source of patient-specific kidney cells that can be harvested noninvasively. Urine derived podocytes and proximal tubule cells have been used to study disease mechanisms and to screen for novel drug therapies in a variety of human kidney disorders. The urinary kidney stem/progenitor cells and extracellular vesicles, instead, might be promising for therapeutic treatments of kidney injury. The greatest advantages of urine as a source of viable cells are the easy collection and less complicated ethical issues. However, extensive characterization and in vivo studies still have to be performed before the clinical use of urine-derived kidney progenitors.

Extracellular vesicles in the urine: markers and mediators of tissue damage and regeneration.

As in several body fluids, urine is a rich reservoir of extracellular vesicles (EVs) directly originating from cells facing the urinary lumen, including differentiated tubular cells, progenitor cells and infiltrating inflammatory cells. Several markers of glomerular and tubular damage, such as WT-1, ATF3 and NGAL, as well as of renal regeneration, such as CD133, have been identified representing an incredible source of information for diagnostic purposes. In addition, urinary extracellular vesicles (uEVs) appear to be involved in the cell-to-cell communication along the nephron, although this aspect needs further elucidation. Finally, uEVs emerge as potential amplifying or limiting factors in renal damage. Vesicles from injured cells may favour fibrosis and disease progression whereas those from cells with regenerative potential appear to promote cell survival. Here, we will discuss the most recent findings of the literature, on the light of the role of EVs in diagnosis and therapy for damage and repair of the renal tissue.

Tumor suppressor PTEN in breast cancer: heterozygosity, mutations and protein expression.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is one of the most frequently mutated human tumor suppressor genes, implicated in cell growth and survival and suppressing tumor formation. Loss of PTEN activity, either at the protein or genomic level, has been related to many primary and metastatic malignancies including breast cancer. The present study investigates the heterozygosity, mutation spectrum and protein expression of PTEN in 43 patients with breast cancer or precursor lesions of the breast and 10 healthy individuals. Microsatellite analysis at the PTEN locus using D10S215, D10S541 and D10S579 markers indicated that the observed heterozygosity (Ho) is lower than the expected heterozygosity (Hs) in benign and malignant breast disease. Mutational analysis in exons 1, 5, 7 and 9 of the PTEN gene revealed several mutations, most of which cause truncation of the PTEN protein and consequently loss of activity. Increased circulating levels of PTEN and phosphorylated PTEN protein were also observed by immunostaining in patients with breast cancer and precursor breast lesions. In support, increased PTEN protein expression was detected in corresponding tissue specimens. Our data suggest an association between breast cancer and PTEN mutations, resulting in the production of truncated forms of the corresponding protein, thus indicating that breast carcinogenesis is potentially related to PTEN loss of activity rather than loss of expression. Peripheral blood sampling may provide an advantageous application for the determination of PTEN gene mutations and its protein expression in human cancer.