Dottorato in Fisiopatologia Medica

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MICROVESICLES DERIVED FROM ENDOTHELIAL PROGENITOR CELLS ACCELERATE GLOMERULAR HEALING IN THY1.1 NEPHRITIS THROUGH THE RNA DEPENDENT INHIBITION OF COMPLEMENT-MEDIATED MESANGIAL INJURY AND TRIGGERING OF ANGIOGENESIS

Endothelial progenitor cells (EPCs) are bone marrow-derived stem cells that circulate in the peripheral blood and can localize within sites of endothelial injury, inducing a regenerative program. EPCs do not act via a direct trans-differentiation into mature endothelial cells but probably contribute to revascularization of damaged tissues favouring angiogenesis by secreting growth factors and other paracrine mediators of cell-to-cell communication.
Microvesicles (MVs) represent a heterogeneous population of micro-organoid bodies released by several cell types; they are enriched in proteins and different subsets of RNAs. For example, EPCs release MVs (EPC MVs), activating an angiogenic program in quiescent endothelial cells through horizontal mRNA transfer.
This study aimed to investigate whether EPC MVs may preserve damage and promote a regenerative program in an experimental model of anti-thy1.1 glomerulonephritis. This model is characterized by complement-mediated mesangiolysis and severe endothelial injury within the glomeruli.
We characterized EPC MVs at protein and RNA levels. We observed that MVs were enriched for microRNAs (miRNAs) involved in regulating processes such as proliferation, angiogenesis and apoptosis in target cells. In particular, we observed high levels of the pro-angiogenic miRNAs miR-126 and miR-296. Moreover, EPC MVs carried proteins and mRNAs coding for inhibitors of complement activation such as CD55, CD59 and Factor H.
Experiments of inactivation of MVs inferred the role of RNAs carried by MVs in the acceleration of glomerular healing by using RNase. Moreover, in selected experiments, we evaluated the role of microRNAs (miRNA) carried by MVs analyzing the effect of MVs derived from EPCs subjected to the knock-down of Dicer with a small interfering RNA (MV siRNA Dicer), the intracellular enzyme essential for maturation of miRNAs.
In vivo, we set up an anti-thy1.1 experimental nephritic model characterized by the presence of typical early lesions such as mesangiolysis, pre-ballooning, ballooning and loss of microvasculature with the formation of pseudoaneurysms and increase of glomerular size. We observed that animals treated with EPC MVs showed at all time points decreased levels of proteinuria and increased creatinine clearance in comparison to rats with anti-thy1.1 injury alone. However, no protective effect was observed in rats injected with EPC MVs pre-treated with RNAse. Furthermore, the protective effects of EPC MVs in antithy1.1 glomerular injury were not observed when pre-treated with RNase. These results suggest that the MV-mediated acceleration of glomerular injury may be due to an RNA dependent mechanism.
On day 4 after the induction of glomerulonephritis, EPC MVs accelerated glomerular healing with a significant decrease of histologic lesions and apoptotic cells; we observed lower endothelial injury and leukocyte infiltration into glomeruli in comparison to rats with anti-thy1.1 injury alone. On day 8, EPC MVs inhibited leukocyte infiltration, basal matrix accumulation and inhibited podocyte injury. We hypothesized that the protective effect of EPC MVs might be related to a dual mechanism: a) triggering of an angiogenic program b) preservation from complement cascade activation. Indeed, we observed that EPC MVs inhibited intraglomerular staining for the terminal fraction of complement C5b9, also known as membrane attack complex (MAC). All these effects were RNA dependent because pretreatment of EPC MVs with RNase abated these biological effects.
In selected experiments, we stained EPC MVs with the red fluorescent dye PKH26 to evaluate the localization in rats after intravenous injection. We observed that labelled-MVs localized 2h after injection within resident injured glomerular cells, particularly within endothelial glomerular cells and podocytes as demonstrated by containing with RECA and synaptopodin, respectively.
In vitro, we observed that PKH26 MVs were internalized in different glomerular cell lines mainly through an L-selectin-dependent mechanism. We set up an experimental model of anti-thy-1.1 complement-mediated lytic injury on rat mesangial cells (RMC). EPC MVs, but not EPC MVs pre-treated with RNase, increased proliferation and resistance to apoptosis and inhibited C5b-9 activation confirming the data observed in vivo. Furthermore, we found the transfer of mRNAs coding for inhibitors of complement activation CD55, CD59 and Factor H from EPC MVs to target RMCs treated 3h after incubation. After 24h, these mRNAs decreased, and we observed at protein level the increased production of CD55, CD59 and Factor H.
Then, we cultured human mesangial cells (HMC), glomerular endothelial cells (GEC) or podocytes (PODO) with pro-inflammatory cytokines (TNFα, IFNγ) to evaluate at different levels the biological effect of EPC MVs in an inflammatory microenvironment typical of glomerulonephritis.
MVs inhibited cytokine-induced adhesion of granulocytes and monocytes on GECs and HMCs. In addition, GECs treated with EPC MVs showed increased motility, proliferation, resistance to apoptosis and ability to form capillary-like structures on Matrigel modulating several genes involved in angiogenesis. However, these protective effects were significantly reduced, stimulating cells with EPC MVs pre-treated with RNAse or EPC MVs siRNA Dicer.
Moreover, to study the role of miRNAs carried by EPC MVs, we set up on GECs a knock-down model of key-miRNAs with specific antagomiRs directed to inhibit miRNAs involved in angiogenesis miR-126 and miR-296. The knockdown of these miRNAs significantly reduced proliferation and angiogenesis, but EPC MVs overcame the inhibitory effects of both antagomiRs.
Finally, PODOs treated with EPC MVs showed a preserved functional and structural integrity by maintaining cell polarity and resistance to apoptosis. In particular, we observed on PODOs that EPC MVs preserved the expression of the protein of slit diaphragm, nephrin. On the other hand, no protective effect was observed on cells cultured with EPC MVs pre-treated with RNAse.
In conclusion, EPC MVs exert a protective effect in the anti-thy1.1 experimental model of glomerulonephritis suggesting that they could be exploited as a new therapeutic approach for glomerulonephritis. These effects are mediated by the transfer of RNAs, particularly microRNAs, implicated in multiple pathways of glomerular kidney cells.
Some studies have suggested engraftment of stem cells by transdifferentiation or fusion in targeted organs. However, a growing number of evidence indicate that transient stem cell localization in the injured tissue may be sufficient to favour functional and regenerative events. MVs act with a switch on/off mechanism similar to a drug pharmacological mechanism. This characteristic makes EPC MVs a therapeutic option more suitable than EPC inoculation, resulting in maldifferentiation and tumorigenesis events.
EPC MVs as a therapeutic strategy could be to other diseases with the characteristics of endothelial injury and immune system activation such as ischemia/reperfusion damage following solid organ transplantation or antibody-mediated transplant rejection.

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