Benedetta Bussolati

Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury

Administration of mesenchymal stem cells (MSCs) improves the recovery from acute kidney injury (AKI). The mechanism may involve paracrine factors promoting proliferation of surviving intrinsic epithelial cells, but these factors remain unknown. In the current study, we found that microvesicles derived from human bone marrow MSCs stimulated proliferation in vitro and conferred resistance of tubular epithelial cells to apoptosis. The biologic action of microvesicles required their CD44- and beta1-integrin-dependent incorporation into tubular cells. In vivo, microvesicles accelerated the morphologic and functional recovery of glycerol-induced AKI in SCID mice by inducing proliferation of tubular cells. The effect of microvesicles on the recovery of AKI was similar to the effect of human MSCs. RNase abolished the aforementioned effects of microvesicles in vitro and in vivo, suggesting RNA-dependent biologic effects. Microarray analysis and quantitative real time PCR of microvesicle-RNA extracts indicate that microvesicles shuttle a specific subset of cellular mRNA, such as mRNAs associated with the mesenchymal phenotype and with control of transcription, proliferation, and immunoregulation. These results suggest that microvesicles derived from MSCs may activate a proliferative program in surviving tubular cells after injury via a horizontal transfer of mRNA.

Isolation of Renal Progenitor Cells from Adult Human Kidney

We describe here isolation and characterization of CD133+ cells derived from normal adult human kidney. These cells lacked the expression of hematopoietic markers and expressed PAX-2, an embryonic renal marker, suggesting their renal origin. Renal tissue-derived CD133+ cells and clones of individual cells were capable of expansion and limited self-renewal and differentiated in vitro into epithelial or endothelial cells. On subcutaneous implantation in SCID mice, the undifferentiated cells formed tubular structures expressing renal epithelial markers. At variance, when differentiated in endothelial cells, these cells formed functional vessels. On intravenous injection in SCID mice with glycerol-induced tubulonecrosis, the in vitro expanded renal-derived CD133+ cells homed into the injured kidney and integrated in tubules. We propose that CD133+ cells from kidney represent a multipotent adult resident stem cell population that may contribute to the repair of renal injury.

Microvesicles Released from Human Renal Cancer Stem Cells Stimulate Angiogenesis and Formation of Lung Premetastatic Niche

Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer development. Within the tumor mass, all cell types may contribute to MV shedding, but specific contributions to tumor progression have yet to be established. Here we report that a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 in human renal cell carcinoma releases MVs that trigger angiogenesis and promote the formation of a premetastatic niche. MVs derived only from CD105-positive cancer stem cells conferred an activated angiogenic phenotype to normal human endothelial cells, stimulating their growth and vessel formation after in vivo implantation in immunocompromised severe combined immunodeficient (SCID) mice. Furthermore, treating SCID mice with MVs shed from CD105-positive cells greatly enhanced lung metastases induced by i.v. injection of renal carcinoma cells. Molecular characterization of CD105-positive MVs defines a set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastases. Our results define a specific source of cancer stem cell-derived MVs that contribute to triggering the angiogenic switch and coordinating metastatic diffusion during tumor progression.

Isolation and Characterization of a Stem Cell Population from Adult Human Liver

Several studies suggested the presence of stem cells in the adult normal human liver; however, a population with stem cell properties has not yet been isolated. The purpose of the present study was to identify and characterize progenitor cells in normal adult human liver. By stringent conditions of liver cell cultures, we isolated and characterized a population of human liver stem cells (HLSCs). HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, and CD90 but not the hematopoietic stem cell markers CD34, CD45, CD117, and CD133. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin‐19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed albumin, α‐fetoprotein, and in a small percentage of cells, cytokeratin‐8 and cytokeratin‐18, indicating a partial commitment to hepatic cells. HLSCs differentiated in mature hepatocytes when cultured in the presence of hepatocyte growth factor and fibroblast growth factor 4, as indicated by the expression of functional cytochrome P450, albumin, and urea production. Under this condition, HLSCs downregulated α‐fetoprotein and expressed cytokeratin‐8 and cytokeratin‐18. HLSCs were also able to undergo osteogenic and endothelial differentiation when cultured in the appropriated differentiation media, but they did not undergo lipogenic differentiation. Moreover, HLSCs differentiated in insulin‐producing islet‐like structures. In vivo, HLSCs contributed to regeneration of the liver parenchyma in severe‐combined immunodeficient mice. In conclusion, we here identified a pluripotent progenitor population in adult human liver that could provide a basis for cell therapy strategies.

Altered angiogenesis and survival in human tumor-derived endothelial cells

Knowledge on the functional properties of tumor‐derived endothelial cells (TEC) can be relevant for the development of antiangiogenic therapeutic strategies. In the present study, we obtained and characterized endothelial cell lines from human renal carcinomas. TEC did not undergo senescence and showed constant expression of markers of endothelial activation and angiogenesis. In vitro, TEC, in contrast to normal endothelial cells, were resistant to apoptosis, proadhesive for renal carcinoma cells, and able to grow and organize in the absence of serum in persistent capillary‐like structures. In vivo, TEC were able to grow in immunodeficient mice and to form vascular structures connected with the circulation. At a molecular level, gene array analysis showed an increased expression of genes involved in survival and cell adhesion compared with expression in normal microvascular endothelial cells. Moreover, expression of angiopoietin‐1 and vascular endothelial growth factor (VEGF)‐D and the Akt survival pathway were up‐regulated. Inhibition of interaction of VEGFR‐2 or VEGFR‐3 with VEGF‐D but not of Tie‐2‐angiopoietin‐1 interaction with soluble receptors abrogated Akt activation and survival of TEC. These results indicate that at least some of the TEC within a tumor display abnormal characteristics in terms of survival and angiogenic properties and also indicate the presence of a functional autocrine pathway related to VEGF‐D.

Identification of a tumor-initiating stem cell population in human renal carcinomas

The purpose of the present study was to search for the presence of a tumor‐initiating stem cell population in renal carcinomas. Based on the recent identification of mesenchymal stem cells in normal kidneys, we sorted cells expressing the mesenchymal stem cell marker CD105 from 5 human renal carcinomas. Because the CD105+ but not the CD105− population showed enhanced tumorigenicity when injected in severely compromised immunodeficient (SCID) mice, we cloned and characterized CD105+ cells and evaluated their stemness, differentiative ability, and serial tumor generation. Characterization of the phenotype of CD105+ clones revealed several stem cell properties: 1) clonogenic ability, 2) expression of nestin, Nanog, Oct4 stem cell markers, and lack of differentiative epithelial markers, 3) ability to grow in non‐adhesive spheroids, 4) in vitro differentiation into epithelial and endothelial cell types, and 5) generation in vivo of serially transplantable carcinomas containing an undifferentiated CD105+ tumorigenic and a differentiated CD105− nontumorigenic population. In addition, some vessels present in carcinomas generated from CD105+ clones were of human origin, suggesting the capability of tumor‐initiating stem cells to in vivo differentiate also in endothelial cells. In conclusion, we demonstrate that CD105+ cells and clones derived from renal carcinomas were enriched in tumor‐initiating cells with stem characteristics.—Bussolati, B., Bruno, S., Grange, C., Ferrando, U., Camussi, G. Identification of a tumor‐initiating stem cell population in human renal carcinomas. FASEB J. 22, 3696–3705 (2008)

Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized rats

Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MVs) derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MVs in the hepatocytes by an α4‐integrin‐dependent mechanism. However, MVs pre‐treated with RNase, even if internalized, were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA‐dependent effect. Microarray analysis and quantitative RT‐PCR demonstrated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MVs accelerated the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre‐treatment of MVs. Using human AGO2, as a reporter gene present in MVs, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MVs. These data suggested a translation of the MV shuttled mRNA into hepatocytes of treated rats. In conclusion, these results suggest that MVs derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets.

Preeclamptic sera induce nephrin shedding from podocytes through endothelin-1 release by endothelial glomerular cells

In preeclampsia (PE), proteinuria has been associated with a reduced expression of nephrin by podocytes. In the present study, we investigated in vitro on human cultured podocytes the mechanism responsible for nephrin loss in PE. Sera from patients with PE did not directly downregulate the expression of nephrin. In contrast, conditioned medium obtained from glomerular endothelial cells incubated with PE sera induced loss of nephrin and synaptopodin, but not of podocin, from podocytes. Nephrin loss was related to a rapid shedding of the protein from the cell surface due to cleavage of its extracellular domain by proteases and to cytoskeleton redistribution. The absence of nephrin mRNA downregulation together with nephrin reexpression within 24 h confirm that the loss of nephrin was not related to a reduced synthesis. Studies with an endothelin-1 (ET-1) receptor antagonist that abrogated the loss of nephrin triggered by glomerular endothelial conditioned medium of PE sera indicated that ET-1 was the main effector of nephrin loss. Indeed, ET-1 was synthesized and released from glomerular endothelial cells when incubated with PE sera, and recombinant ET-1 triggered nephrin shedding from podocytes. Moreover, VEGF blockade induced ET-1 release from endothelial cells, and in turn the conditioned medium obtained triggered nephrin loss. In conclusion, the present study identifies a potential mechanism of nephrin loss in PE that may link endothelial injury with enhanced glomerular permeability.

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.

Magnetic Resonance Visualization of Tumor Angiogenesis by Targeting Neural Cell Adhesion Molecules with the Highly Sensitive Gadolinium-Loaded Apoferritin Probe

Tumor vessel imaging could be useful in identifying angiogenic blood vessels as well as being a potential predictive marker of antiangiogenic treatment response. We recently reported the expression of the neural cell adhesion molecule (NCAM) in the immature and tumor endothelial cell (TEC) lining vessels of human carcinomas. Exploiting an in vivo model of human tumor angiogenesis obtained by implantation of TEC in Matrigel in severe combined immunodeficiency mice, we aimed to image angiogenesis by detecting the expression of NCAM with magnetic resonance imaging. The imaging procedure consisted of (a) targeting NCAMs with a biotinylated derivative of C3d peptide that is known to have high affinity for these epitopes and (b) delivery of a streptavidin/gadolinium (Gd)-loaded apoferritin 1:1 adduct at the biotinylated target sites. The remarkable relaxation enhancement ability of the Gd-loaded apoferritin system allowed the visualization of TEC both in vitro and in vivo when organized in microvessels connected to the mouse vasculature. Gd-loaded apoferritin displayed good in vivo stability and tolerability. The procedure reported herein may be easily extended to the magnetic resonance visualization of other epitopes suitably targeted by proper biotinylated vectors.