INTRODUCTION AND AIMS: EPCs are bone marrow-derived stem cells able to trigger tissue regeneration by paracrine mechanisms including the secretion of growth factors and MVs. MVs are small membrane fragments carrying genetic information that play a key role in cell-to-cell communication. MVs released from EPCs activate an angiogenic program in endothelial cells through the horizontal transfer of RNAs. The aim of this study was to evaluate whether EPC-derived MVs prevent IRI-associated AKI, identifying the role of microRNAs (miRNAs) shuttled by MVs in the reprogramming of renal resident cells. METHODS: EPCs were isolated from peripheral blood of healthy volunteers. MVs were isolated by ultracentrifugation and characterized for protein and RNA content. Wistar rats were treated as follows: 1) Sham; 2) IRI (renal pedicle clamping for 45 min); 3) IRI + 30 µg/ml EPC MVs; 4) IRI + 30 µg/ml EPC MVs pre-treated with 1U/ml RNase; 5) IRI + 30 µg/ml MVs from control fibroblasts. Renal function and histology were evaluated. In vitro, we studied the effects of MVs on isolated kidney endothelial, tubular epithelial and CD133+ progenitor cells. In selected experiments, MVs were isolated from EPCs engineered by siRNA to knock-down Dicer, the intracellular enzyme essential for miRNA production. RESULTS: MVs expressed markers of stem and endothelial cells and molecules of the integrin/selectin families essential for their internalization into target cells. MVs carried different mRNAs and 146 miRNAs such as miR-126 and miR-296 able to modulate proliferation, angiogenesis and apoptosis. After injection in rats, EPC MVs localized within peritubular capillaries and tubular cells and protected rats from IRI-associated AKI (decrease of serum creatinine/BUN and of histologic tubular injury) MVs significantly reduced apoptosis, sustained tubular proliferation and inhibited leukocyte infiltration. All these protective effects were not observed using fibroblast-derived MVs or when EPC MVs were pre-treated with RNase or produced by EPCs subjected to Dicer knock-down. In vitro, MVs inhibited hypoxia-induced apoptosis and functional alterations of kidney endothelial and epithelial cells by up-regulating genes involved in cell proliferation, differentiation, angiogenesis and inhibition of inflammation. In addition, MVs induced the differentiation of CD133+ renal progenitors towards an endothelial phenotype. CONCLUSIONS: EPC MVs induced a protective effect on IRI-associateed AKI. This effect may be ascribed to the reprogramming of renal resident cells induced by MV-shuttled miRNAs. Our results suggest that EPC-derived MVs could be exploited as a potential new therapeutic approach for AKI and other ischemic diseases without the potential risks of stem cell therapy such as maldifferentiation and tumorigenesis.
MICROVESICLES (MVs) DERIVED FROM ENDOTHELIAL PROGENITOR CELLS (EPCs) PROTECTS KIDNEYS FROM ISCHEMIA-REPERFUSION INJURY (IRI) THROUGH THE microRNA-DEPENDENT REPROGRAMMING OF RENAL RESIDENT CELLS
CANTALUPPI, Vincenzo;MEDICA, DAVIDE;FIGLIOLINI, Federico;BRUNO, Stefania;BIANCONE, Luigi;SEGOLONI, Giuseppe;CAMUSSI, Giovanni
2011-01-01
Abstract
INTRODUCTION AND AIMS: EPCs are bone marrow-derived stem cells able to trigger tissue regeneration by paracrine mechanisms including the secretion of growth factors and MVs. MVs are small membrane fragments carrying genetic information that play a key role in cell-to-cell communication. MVs released from EPCs activate an angiogenic program in endothelial cells through the horizontal transfer of RNAs. The aim of this study was to evaluate whether EPC-derived MVs prevent IRI-associated AKI, identifying the role of microRNAs (miRNAs) shuttled by MVs in the reprogramming of renal resident cells. METHODS: EPCs were isolated from peripheral blood of healthy volunteers. MVs were isolated by ultracentrifugation and characterized for protein and RNA content. Wistar rats were treated as follows: 1) Sham; 2) IRI (renal pedicle clamping for 45 min); 3) IRI + 30 µg/ml EPC MVs; 4) IRI + 30 µg/ml EPC MVs pre-treated with 1U/ml RNase; 5) IRI + 30 µg/ml MVs from control fibroblasts. Renal function and histology were evaluated. In vitro, we studied the effects of MVs on isolated kidney endothelial, tubular epithelial and CD133+ progenitor cells. In selected experiments, MVs were isolated from EPCs engineered by siRNA to knock-down Dicer, the intracellular enzyme essential for miRNA production. RESULTS: MVs expressed markers of stem and endothelial cells and molecules of the integrin/selectin families essential for their internalization into target cells. MVs carried different mRNAs and 146 miRNAs such as miR-126 and miR-296 able to modulate proliferation, angiogenesis and apoptosis. After injection in rats, EPC MVs localized within peritubular capillaries and tubular cells and protected rats from IRI-associated AKI (decrease of serum creatinine/BUN and of histologic tubular injury) MVs significantly reduced apoptosis, sustained tubular proliferation and inhibited leukocyte infiltration. All these protective effects were not observed using fibroblast-derived MVs or when EPC MVs were pre-treated with RNase or produced by EPCs subjected to Dicer knock-down. In vitro, MVs inhibited hypoxia-induced apoptosis and functional alterations of kidney endothelial and epithelial cells by up-regulating genes involved in cell proliferation, differentiation, angiogenesis and inhibition of inflammation. In addition, MVs induced the differentiation of CD133+ renal progenitors towards an endothelial phenotype. CONCLUSIONS: EPC MVs induced a protective effect on IRI-associateed AKI. This effect may be ascribed to the reprogramming of renal resident cells induced by MV-shuttled miRNAs. Our results suggest that EPC-derived MVs could be exploited as a potential new therapeutic approach for AKI and other ischemic diseases without the potential risks of stem cell therapy such as maldifferentiation and tumorigenesis.
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