Luigi Biancone

Exosomes/microvesicles as a mechanism of cell-to-cell communication

Microvesicles (MVs) are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes of most cell types. An increasing body of evidence indicates that they play a pivotal role in cell-to-cell communication. Indeed, they may directly stimulate target cells by receptor-mediated interactions or may transfer from the cell of origin to various bioactive molecules including membrane receptors, proteins, mRNAs, microRNAs, and organelles. In this review we discuss the pleiotropic biologic effects of MVs that are relevant for communication among cells in physiological and pathological conditions. In particular, we discuss their potential involvement in inflammation, renal disease, and tumor progression, and the evidence supporting a bidirectional exchange of genetic information between stem and injured cells. The transfer of gene products from injured cells may explain stem cell functional and phenotypic changes without the need of transdifferentiation into tissue cells. On the other hand, transfer of gene products from stem cells may reprogram injured cells to repair damaged tissues.

Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

Several studies demonstrated that treatment with mesenchymal stem cells (MSCs) reduces cisplatin mortality in mice. Microvesicles (MVs) released from MSCs were previously shown to favor renal repair in non lethal toxic and ischemic acute renal injury (AKI). In the present study we investigated the effects of MSC-derived MVs in SCID mice survival in lethal cisplatin-induced AKI. Moreover, we evaluated in vitro the effect of MVs on cisplatin-induced apoptosis of human renal tubular epithelial cells and the molecular mechanisms involved. Two different regimens of MV injection were used. The single administration of MVs ameliorated renal function and morphology, and improved survival but did not prevent chronic tubular injury and persistent increase in BUN and creatinine. Multiple injections of MVs further decreased mortality and at day 21 surviving mice showed normal histology and renal function. The mechanism of protection was mainly ascribed to an anti-apoptotic effect of MVs. In vitro studies demonstrated that MVs up-regulated in cisplatin-treated human tubular epithelial cells anti-apoptotic genes, such as Bcl-xL, Bcl2 and BIRC8 and down-regulated genes that have a central role in the execution-phase of cell apoptosis such as Casp1, Casp8 and LTA. In conclusion, MVs released from MSCs were found to exert a pro-survival effect on renal cells in vitro and in vivo, suggesting that MVs may contribute to renal protection conferred by MSCs.

Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells

Endothelial progenitor cells are known to reverse acute kidney injury by paracrine mechanisms. We previously found that microvesicles released from these progenitor cells activate an angiogenic program in endothelial cells by horizontal mRNA transfer. Here, we tested whether these microvesicles prevent acute kidney injury in a rat model of ischemia-reperfusion injury. The RNA content of microvesicles was enriched in microRNAs (miRNAs) that modulate proliferation, angiogenesis, and apoptosis. After intravenous injection following ischemia-reperfusion, the microvesicles were localized within peritubular capillaries and tubular cells. This conferred functional and morphologic protection from acute kidney injury by enhanced tubular cell proliferation, reduced apoptosis, and leukocyte infiltration. Microvesicles also protected against progression of chronic kidney damage by inhibiting capillary rarefaction, glomerulosclerosis, and tubulointerstitial fibrosis. The renoprotective effect of microvesicles was lost after treatment with RNase, nonspecific miRNA depletion of microvesicles by Dicer knock-down in the progenitor cells, or depletion of pro-angiogenic miR-126 and miR-296 by transfection with specific miR-antagomirs. Thus, microvesicles derived from endothelial progenitor cells protect the kidney from ischemic acute injury by delivering their RNA content, the miRNA cargo of which contributes to reprogramming hypoxic resident renal cells to a regenerative program.

Nephrin Redistribution on Podocytes Is a Potential Mechanism for Proteinuria in Patients with Primary Acquired Nephrotic Syndrome

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

Therapeutic potential of mesenchymal stem cell-derived microvesicles

Several studies have demonstrated that mesenchymal stem cells have the capacity to reverse acute and chronic kidney injury in different experimental models by paracrine mechanisms. This paracrine action may be accounted for, at least in part, by microvesicles (MVs) released from mesenchymal stem cells, resulting in a horizontal transfer of mRNA, microRNA and proteins. MVs, released as exosomes from the endosomal compartment, or as shedding vesicles from the cell surface, are now recognized as being an integral component of the intercellular microenvironment. By acting as vehicles for information transfer, MVs play a pivotal role in cell-to-cell communication. This exchange of information between the injured cells and stem cells has the potential to be bi-directional. Thus, MVs may either transfer transcripts from injured cells to stem cells, resulting in reprogramming of their phenotype to acquire specific features of the tissue, or conversely, transcripts could be transferred from stem cells to injured cells, restraining tissue injury and inducing cell cycle re-entry of resident cells, leading to tissue self-repair. Upon administration with a therapeutic regimen, MVs mimic the effect of mesenchymal stem cells in various experimental models by inhibiting apoptosis and stimulating cell proliferation. In this review, we discuss whether MVs released from mesenchymal stem cells have the potential to be exploited in novel therapeutic approaches in regenerative medicine to repair damaged tissues, as an alternative to stem cell-based therapy.

OBESTATIN PROMOTES SURVIVAL OF PANCREATIC β-CELLS AND HUMAN ISLETS AND INDUCES EXPRESSION OF GENES INVOLVED IN THE REGULATION OF β-CELL MASS AND FUNCTION

OBJECTIVE—Obestatin is a newly discovered peptide encoded by the ghrelin gene whose biological functions are poorly understood. We investigated obestatin effect on survival of β-cells and human pancreatic islets and the underlying signaling pathways. RESEARCH DESIGN AND METHODS—β-Cells and human islets were used to assess obestatin effect on cell proliferation, survival, apoptosis, intracellular signaling, and gene expression. RESULTS—Obestatin showed specific binding on HIT-T15 and INS-1E β-cells, bound to glucagon-like peptide-1 receptor (GLP-1R), and recognized ghrelin binding sites. Obestatin exerted proliferative, survival, and antiapoptotic effects under serum-deprived conditions and interferon-γ/tumor necrosis factor-α/interleukin-1β treatment, particularly at pharmacological concentrations. Ghrelin receptor antagonist [D-Lys3]-growth hormone releasing peptide-6 and anti-ghrelin antibody prevented obestatin-induced survival in β-cells and human islets. β-Cells and islet cells released obestatin, and addition of anti-obestatin antibody reduced their viability. Obestatin increased β-cell cAMP and activated extracellular signal–related kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt; its antiapoptotic effect was blocked by inhibition of adenylyl cyclase/cAMP/protein kinase A (PKA), PI 3-kinase/Akt, and ERK1/2 signaling. Moreover, obestatin upregulated GLP-1R mRNA and insulin receptor substrate-2 (IRS-2) expression and phosphorylation. The GLP-1R antagonist exendin-(9-39) reduced obestatin effect on β-cell survival. In human islets, obestatin, whose immunoreactivity colocalized with that of ghrelin, promoted cell survival and blocked cytokine-induced apoptosis through cAMP increase and involvement of adenylyl cyclase/cAMP/PKA signaling. Moreover, obestatin 1) induced PI 3-kinase/Akt, ERK1/2, and also cAMP response element–binding protein phosphorylation; 2) stimulated insulin secretion and gene expression; and 3) upregulated GLP-1R, IRS-2, pancreatic and duodenal homeobox-1, and glucokinase mRNA. CONCLUSIONS—These results indicate that obestatin promotes β-cell and human islet cell survival and stimulates the expression of main regulatory β-cell genes, identifying a new role for this peptide within the endocrine pancreas.

Improved route for the visualization of stem cells labeled with a Gd‐/Eu‐Chelate as dual (MRI and fluorescence) agent

A simple labeling procedure of stem/progenitor cells based on the use of Gd‐HPDO3A and Eu‐HPDO3A, respectively, is described. The Gd‐chelate acts as T1‐agent for MRI visualization, whereas the corresponding Eu‐chelate acts as reporter in fluorescence microscopy. Owing to their substantial chemical equivalence, the two chelates are equally internalized in EPCs (endothelial progenitor cells), thus allowing their visualization by both techniques. The lanthanide chelates are entrapped in endosomic vesicles and the labeled cells retain biological activity with preservation of viability and pro‐angiogenesis capacity. Hyperintense spots in MR have been observed for Gd‐labeled EPCs injected under mice kidney capsule or grafted on a subcutaneous Matrigel plug up to 14 days after transplantation. Magn Reson Med 51:938–944, 2004.

HIV-1 kills renal tubular epithelial cells in vitro by triggering an apoptotic pathway involving caspase activation and Fas upregulation.

HIV-infected patients suffer several renal syndromes, which can progress rapidly from renal insufficiency to end-stage renal disease. Histologically, HIV-induced nephropathy is characterized by prominent tubulopathy with apoptosis of tubular cells. Clinical and experimental evidence suggests that renal injury may be directly related to virus infection. Although HIV-1 is a polytropic and not solely lymphotropic pathogen, the susceptibility of renal cells to HIV-1 remains to be determined. This paper demonstrates in vitro the permissiveness of proximal tubular epithelial cells (PTEC) to HIV-1 and describes the effects of PTEC infection to explain the pathogenesis of tubular damage in vivo. The results indicate that PTEC express HIV-specific receptor and coreceptors and sustain virus replication. We observed that HIV-1 infection causes the death of tubular cells by triggering an apoptotic pathway involving caspase activation. Fas upregulation but not Fas ligand expression was found in the infected PTEC. However, after HIV-1 infection, tubular cells became susceptible to apoptosis induced through Fas stimulation. Caspase inhibition prevented the death of the infected PTEC in spite of persistent viral replication. These findings may explain the prominent histopathology of HIV-associated nephropathy and demonstrate that the apoptosis of nonlymphoid cells can be directly induced by HIV-1.

Effect of the intracellular localization of a Gd-based imaging probe on the relaxation enhancement of water protons

Gd‐HPDO3A has been internalized into rat hepatocarcinoma cells in the cytoplasm (by electroporation) or in intracellular vesicles (by pinocytosis), respectively. In the former case, the observed relaxation rates are likely dependent upon the amount of internalized paramagnetic complex, whereas in the latter case the relaxation enhancement is “quenched” to a plateau value (about 3 s−1) when the entrapped amount of Gd‐chelate is higher than 1 × 1010 Gd/cell. The observed behavior has been accounted in terms of a theoretical treatment based on equations formally derived by Labadie et al. (J Magn Reson B 1994;105:99–102). On this basis, entrapment into intracellular vesicles has been treated as a three‐site water exchange (extracellular/cytoplasm/vesicle compartments), whereas the cell pellets containing the paramagnetic agent spread out in the cytoplasm can be analyzed by a two‐site exchange system. Magn Reson Med, 2006.

PAF Produced by Human Breast Cancer Cells Promotes Migration and Proliferation of Tumor Cells and Neo-Angiogenesis

Platelet-activating factor (PAF), a phospholipid mediator of inflammation, is present in breast cancer tissue and correlates with microvessel density. In the present study, we investigated the biological significance of PAF synthesized within breast cancer. In vitro, we observed the production of PAF by two estrogen-dependent (MCF7 and T-47D) and an estrogen-independent (MDA-MB231) breast cancer cell lines after stimulation with vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor, thrombin but not with estrogen, progesterone, and oxytocin. The sensitivity to agonist stimulation and the amount of PAF synthesized as cell-associated or released varied in different cell lines, being higher in MDA-MB231 cells, which are known to be highly invasive. We further demonstrate, by reverse transcriptase-polymerase chain reaction and cytofluorimetry, that all of the breast cancer cells express the PAF receptor and respond to PAF stimulation in terms of proliferation. Moreover, in MDA-MB231 cells PAF elicited cell motility. In vivo, two structurally different PAF receptor antagonists WEB 2170 and CV 3988 significantly reduced the formation of new vessels in a tumor induced by subcutaneous implantation of MDA-MB231 cells into SCID mice. In conclusion, these results suggest that PAF, produced and released by breast cancer cells, can contribute to tumor development by enhancing cell motility and proliferation and by stimulating the angiogenic response.