INTERNA TIONAL JOURNAL OF IMMUNOP A THOLOGY AND PHARMACOLOGY Vol. 25, no. I, 75-85 (2012)
ENDOTHELIAL PROGENITOR CELL-DERIVED MICROVESICLES IMPROVE NEOV ASCULARIZA TION IN A MURINE MODEL OF HINDLIMB ISCHEMIA
A. RANGHINO!, V. CANTALUPPP, C. GRANGE!, L. VITILLOl , F. FOP!, L. BIANCONEl , M.C. DEREGIBUSl , C. TETTA2, G.P. SEGOLONP and G. CAMUSSP
JDepartment o fInternal Medicine, Research Center for Experimental Medicine (CeRMS) and Centerfor Molecular Biotechnology, and University ofTurin, Turin, Italy; ‘Fresenius Medical Care, Bad Homburg, Germany
Received March 24, 2011 – Accepted November 15, 2011
Paracrine mediators released from endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis following ischemia. Recently, we demonstrated that microvesicles (MVs) derived from EPCs are able to activate an angiogenic program in quiescent endothelial cells by a horizontal transfer of RNA. In this study we aim to investigate whether EPC-derived MVs are able to induce neoangiogenesis and to enhance recovery in a murine model of hindlimb ischemia. Hindlimb ischemia was induced in severe combined immunodeficient (SCID) mice by ligation and resection of the left femoral artery and mice were treated with EPC-derived MVs (MVs), RNase-inactivated MVs (RnaseMVs), fibroblast- derived MVs or vehicle alone as control (CTL). Since MVs contained the angiogenic miR-126 and miR-296, we evaluated whether microRNAs may account for the angiogenic activities by treating mice with MVs obtained from DICER-knock-down EPC (DICER-MVs). The limb perfusion evaluated by laserdoppler analysis demonstrated that MVs significantly enhanced perfusion in respect to CTL (0.50±0.08 vs 0.39±0.03, p < 0.05). After 7 days, immunohistochemical analyses on the gastrocnemius muscle of the ischemic hindlimb showed that MVs but not fibroblast-MVs significantly increased the capillary density in respect to CTL (MVs vs CTL: 24.7±10.3 vs 13.5±6, p < 0.0001) and (fibroblast-MVs vs CTL: 10.2±3.4 vs 13.5±6, ns); RNaseMVs and DICER-MVs significantly reduced the effect of MVs (RNaseMVs vs CTL: 15.7±4.1 vs 13.5±6, ns) (MVs vs DICER-MVs 24.7±10.3 vs 18.1±5.8, p < 0.05), suggesting a role ofRNAs shuttled by MVs. Morphometric analysis confirmed that MVs enhanced limb perfusion and reduced injury. The results of the present study indicate that treatment with EPC-derived MVs improves neovascularization and favors regeneration in severe hindlimb ischemia induced in SCID mice. This suggests a possible use of EPCs-derived MVs for treatment of peripheral arterial disease.
Peripheral arterial disease, caused by atherosclerotic occlusion of the leg arteries, is an important manifestation of systemic atherosclerosis along with coronary heart disease and cerebro- vascular disease. The age-adjusted prevalence of
peripheral arterial disease is approximately 12%, and affects men and women equally. The typical clinical manifestation is claudication, nevertheless 5% ofpatients undergo an amputation within 5 years (1,2).
Mailing address: Dr. G. Camussi, Dipartimento di Medicina Intema, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14,
10126, Torino, Italy
Tel: +39 Oil 6336708 Fax: +39 011 6631184 e-mail: email@example.com
0394-6320 (2012) Copyright © by B10LIFE, s.a.s. This publication and/or article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO THIS ARTICLE.
Key words: microvesicles, angiogenesis, hindlimb ischemia
A. RANGHINO ET AL.
Neovascularization is an important event in rescuing tissues after ischemia. Several studies have shown that stem cells and progenitor cells such as endothelial progenitor cells (EPCs) contribute to neoangiogenesis during hindlimb ischemia (3-7).
The mechanisms by which stem cells or EPCs are able to induce recovery in damaged organs or tissues are not completely understood. Nevertheless, some studies have demonstrated that a transient localization of stem cells in injured tissues might be sufficient to induce functional and regenerative events suggesting the release of paracrine mediators (8-12). The mechanisms underlying cell-to-cell communication involve the secretion of cytokines, chemokines, growth factors, adhesion molecules, tunneling nanotubules and circular membrane fragments called microvesicles (MVs) (13-16).
MVs are released from the cell surface ofnormal or damaged cells and are able to transfer mRNAs and microRNAs from the cell origin to other cells in a defined microenviroment (17-20). The shedding of MVs from cell surface is a physiological phenomenon that may increase during cell activation, hypoxia or irradiation, oxidative injury and exposure to protein from an activated complement cascade (15, 21-26).
Recently, we demonstrated that MVs derived from EPCs are able to promote proliferation and reduce apoptosis in vitro of human microvascular endothelial cells and human umbilical vein endothelial cells. These effects require the incorporation of the MVs into endothelial cells with transfer of genetic information. In vivo, human umbilical vein endothelial cells pre-incubated with MVs derived from EPCs formed patent vessels in Matrigel when implanted subcutaneously in SCID mice (18).
In the present study we investigated whether EPC-derived MVs could induce neoangiogenesis in a murine model of hindlimb ischemia that mimics a peripheral arterial disease to evaluate whether the MVs may represent a potential therapeutic strategy.
MATERIALS AND METHODS
Human endothelial progenitor cell cultures
EPCs were isolated from PBMCs ofhealthy donors by density centrifugation, seeded on type I collagen-coated plates (27) and characterized as previously described (18). When homogeneous monolayers with typical cobblestone
morphology were obtained, phenotypic characterization and functional evaluation of angiogenic properties were carried out as previously described (18). EPCs from 3- 5 passages were used to avoid monocyte and platelet contamination. By FACS, EPCs resulted negative for CD2, CD3, CD4, CD5, CD8, CDl6, CD20, CD62E, VEGFR-I, CDl4, CD45 but positive for CD34, CD133, Tie-2, VEGFR-3, VEGFR-2, P-selectin and CD42b. EPCs were characterized by dual-staining for 1,1′-dioctadecyl- 3,3,3′ ,3′,~tetramethylindocarbocyanine-Iabeled acetylated low-density lipoprotein and Ulex europaeus agglutinin-l and lectin and by the expression of endothelial marker proteins VEGFR-2, VE-cadherin, eNOS, and vWF. Endothelial phenotype was further confirmed by Western blot analysis and RT-PCR by expression.of markers characteristic for endothelial cells such as Tie-2, VEGFR- 2, VEGFR-3, but not ofVEGFR-1 (18).
Isolation and characterization of MVs released from EPCs
MVs were obtained from supernatants of EPCs by ultracentrifugation (Optima L-90K, Beckman Coulter, Fullerton, CA) as previously described (18). Transmission and scanning electron microscopy showed the spheroid morphology ofMVs with a size ranging from 60-130 nm. By FACS analysis, MVs were detectable under the 1 urn beads and expressed the hematopoietic stem cell marker CD34 and molecules essential for leukocyte adhesion such as a4 and ~I integrins and L-selectin. MVs did not express HLA class I and class II and markers of platelets (P-selectin, CD42b) and monocytes (CDI4). RNA extraction from MVs was performed using the mirVana isolation kit (Ambion, Austin, TX). RNA was analyzed by Agilent 2100 bioanalyzer (Agilent Tech. Inc., Santa Clara, CA). miR-126 and miR-296 expression levels were analyzed by qRT-PCR in a 48- well StepOne” Real Time System (Ambion): 200 ng of RNA was reverse-transcribed and the cDNA was used to detect and quantity miR-126 and miR-296 by qRT-PCR using the miScript SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). In selected experiments, MVs were treated with 1Ulml RNase (Ambion) for 3 h at 37°C. After RNase treatment the reaction was stopped by addition of 10 UI ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation (18). The efficacy of RNase treatment was evaluated by MV-RNA analyses by Agilent 2100 bioanalyzer (Agilent) and by evaluation of miR-126 and miR-296 by qRT-PCR. As control, MVs were obtained from human fibroblast as previously described (21).
Knock-down ofDICER in EPCs
Knock-down of DICER in EPCs was performed by a specific siRNA according to the manufacturer’s
instructions (Santa Cruz Biotech, Santa Cruz, CA, USA). An irrelevant siRNA (vector-MVs) was used as control. Western blot for DICER expression was performed by using an anti-DICER polyclonal antibody (Abeam, Cambridge, UK). The maximum knock-down of DICER was observed four days after transfection. At that time MVs derived from DICER knock-down-EPCs (DICER- MVs) or vector-MVs were collected by supernatants of engineered cells and used for proliferation and angiogenesis assays.
Murine model ofhindlimb ischemia
All procedures were approved by the Ethics Committee of the University of Torino and conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. scm mice (Charles River Laboratories), aged 7 to 8 wks and weighing 18 to 22 g, were anesthetized with i.m. injection of zolazepam 80 mg/Kg. Postoperatively, the animals were closely monitored, and analgesia with Ketorolac (5mg/Kg) was administered if required.
Under sterile conditions, a small skin incision was made overlying the middle portion ofthe left hindlimb of each mouse. The proximal end of the left femoral artery and the distal portion ofthe saphenous artery were ligated and dissected free and excised. The overlying skin was closed using a sterilized 6-0 silk suture (28). Immediately before the surgery the mice were treated with 50 pg of proteins o f MVs, RNase-inactivated MVs (RNase-MVs), DICER-MVs or vector-MVs, fibroblast-MVs or vehicle alone (CTL) administered intravenously, and after surgery with I 00 ug o f proteins o f the same preparations. Mice were sacrificed on day 7 (T7) after hindlimb ischemia.
Monitoring ofhindlimb bloodflow
After anesthesia, hair was removed from both legs using a depilatory cream, following which the mice were placed on a heating plate at 37°C for 3 min to minimize temperature variations. Hindlimb blood flow was measured using a Laser Doppler Blood Flow (LDBF) analyzer (PeriScan PIM 3 System, Perimed, Stockholm, Sweden). Immediately before surgery, and on postoperative days 0, 3, 7, LDBF analysis was performed on hindlimbs and feet. Blood flow was displayed as changes in the laser frequency using different color pixels. After scanning, stored images were analyzed to quantify blood flow. To avoid data variations caused by ambient light and temperature, hindlimb blood flow was expressed as the ratio of left (ischemic) to right (non-ischemic) LDBF (28).
Evaluation ofcapillary density
Capillary density within gastrocnemius muscles was
quantified by immunofluorescence analysis. Muscle samples were embedded in OCT compound (Miles) and snap-frozen in liquid nitrogen. Tissue slices (5 urn thick) were prepared and capillary endothelial cells were identified by immunofluorescence using a monoclonal antibody against mouse CD31 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Fifteen randomly chosen microscopic fields from three different sections in each tissue block were examined for the count ofcapillary endothelial cells for each mouse specimen. Capillary density was expressed as the number of CD-31-positive features per high power field (x400).
Macroscopic evaluation ofischemic severity
One week after the operation, the ischemic limb was macroscopically evaluated by using an ischemia scale assessed to detect less severe levels of ischemia. Tissue grade 0 corresponds to autoamputation of the leg; grade 1 to leg necrosis; grade 2 to foot necrosis; grade 3 relates to two or more toe discoloration; grade 4 to one toe discoloration; grade 5 to two or more nail discoloration; grade 6 to one nail discoloration and grade 7 to the absence ofnecrosis (29).
The gastrocnemius muscle from ischemic and non- ischemic limb was dissected out at day 7 after surgery, immediately fixed with 4% paraformaldeide for 8 hours and then embedded in paraffin. Tissue slices were stained with hematoxylin and eosin. Slides were examined under light microscopy at x200 magnification. Images were acquired from all the injured area of the ischemic limb sections for the count of the total muscle fibers and of the fibers with small-diameter and with multiple central nuclei that are indicative of the regeneration process. Random images from the total non-ischemic limb section were considered as a control (30).
Statistical analysis was performed with SPSS (SPSS Inc. Chicago, IL, USA). Differences between groups were analyzed with Student’s t-test, ANOVA with the Newmann-Keuls multicomparison tests and Mann-Whitney or Kruskal-Wallis non-parametric tests when appropriate. A p value of < 0.05 for all tests was considered statistically significant.
Ischemic hindlimb perfusion
Immediately after left femoral artery resection, the ratio between the ischemic and non-ischemic
Int. J. Immunopathol. Pharmacol.
A. RANGHINO ET AL.
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Fig. 1. Effect ofMVs on bloodperfusion. A) Representative images oflaser doppler bloodflow obtained before surgery, immediately after surgery (day 0) and 7 days after surgery (day 7) in CTL, and in mice treated with IVfVs and RNase inactivated MVs. Hues indicating regions from lower to higher perfusion are asfollows: dark blue, blue, green, yellow, red, dark red. B) Quantitative analysis ofperfusion measured by laser doppler bloodflow (LDBF) ratio indicates that MVs significantly enhancedperfusion in respect to CTL andRNaseMVs-treatedgroups. Data are expressed as mean±SD; *p value < 0.05, (Mann-Whitney non-parametric test).
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Fig. 2. Increased capillary density if! MVs-treated mice after hindlimb ischemia. Representative confocal images of capillaries stained with mAbs anti-CD31 in CTL (A), MVs-treated (B) and RnaseMVs-treated (C) mice (Original magnification x400). D) Quantitative analysis of capillary density in the ischemic hindlimb. Data are expressed as mean±SD;**pvalue< 0.0001, *pvalue< 0.05,(analysisofvariance,ANOVA).E) RepresentativeqRT-PCRanalysisfor miR-126 (l and 3) and miR-296 (2 and 4) in MVs incubated with vehicle alone (l and 2) or with 1 U/ml RNase (3 and 4) to inactivate RNAs. F) Representative qRT-PCR analysis for miR-126 (l and 3) and miR-296 (2 and 4) in MVs derived
from EPCs transfected with VectorsiRNA as control (l and 2) or DICER siRNA (3 and 4).
hindlimb decreased to 0.32±0.06 in the CTL group, 0.35±0.06 in the MVs group and 0.37±0.05 in the RNase-MVs group, (n=lO/group) indicating that the severity of the induced ischemia was similar in the three experimental groups. Fig. lA shows representative LDBF images o f hindlimb blood flow before, immediately after (day 0), at 3 and 7 days after surgery in the three groups (CTL, MVs
and RNaseMVs). In the MV-treated group hindlimb perfusion ratio significantly increased 3 days after surgery compared with the CTL group (0.5~0.08 vs 0.39±0.03, p < 0.05) and the RNase-MVs treated group (0.50±0.08 vs 0.41±0.06, p < 0.05). This significant increase in the perfusion ratio observed in the MV-treated group compared to the CTL and the RNase-MVs treated groups still persisted at day
IX •• <l
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Fig. 3. Administration ofMVs reduces limb loss and increases limb salvage. A) Representative macroscopic photographs o f mice showing the positive outcome o f MV-treated mice (l, limb salvage) compared to CTL mice (2, foot necrosis, as indicated by white arrow). B) Quantitative analysis ofmorphological changes in the different experimental conditions evaluated with the ischemia score as described in Methods section. Continuous variables did not follow a normal distribution and are presented as median (min-max); *p value < 0.05. The difference between groups was analyzed with Mann-Whitney and Kruskal-Wallis non-parametric tests.
7 (MVs 0.52±0.08 vs CTL OAl±0.05, p < 0.05; MVs 0.52±0.08 vs RNaseMVs OA3±0.03, p < 0.05) (Fig.
Quantitative analysis of CD3l positive cells in muscle sections obtained from ischemic hindlimb (n= 10/group) revealed that the capillary density was significantly increased in the MY-treated group compared with the CTL group (24.7±10.3 vs 13.5±6,
p < 0.0001) (Fig. 2, A-D). Fibroblast-derived MVs did not increase the capillary density in the ischemic hindlimb compared with CTL group (10.2±3A vs 13.5±6, ns). Despite MVs protecting RNA from inactivation by physiological concentration o f RNase, a previous study demonstrated that treatment with a high concentration o f RNase inactivate RNAs shuttled by MVs and inhibited MV biological activities (18, 20). Mice treated with RNase inactivated MVs did not show a significant increase
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Fig. 4. MV treatment protects from ischemic muscle damage and promotes muscle regeneration. Representative images ofhematoxylin-eosin stained muscle sections before surgery (A), and 7 days after surgery ofCTL (B), MVs-treated (C) and RNaseMVs-treated (D) mice. E) Quantitative analysis ofthe total number ofmuscle fibers. F) Quantitative analysis o fregenerative muscle fibers in the different experimental conditions. Data are expressed as mean±SD; *p value < 0.05, (ANOVA with the Newmann-Keuls multicomparison tests).
in capillary density compared with the CTL group (l5.7±4.1 vs 13.5±6, ns), (Fig. 2 C,D), suggesting that the biological activity o f EPC-deriveQ MVs was mediated by MV-shuttled RNA.
Role ofmiRNAs in MV-induced angiogenesis
As shown by RT-PCR, MVs derived from EPC contained miR-126 and miR-296 that are known to be angiogenetic (Fig. 2E). RNase treatment significantly reduced the content of miR-126 and miR-296 (Fig. 2E). To study the role of miRNAs in MV-induced angiogenesis, we engineered EPCs to knock-down DICER, the intracellular enzyme essential for miRNA synthesis. For these experiments, we used MVs derived from EPCs. qRT-PCR analysis showed reduced levels ofmiR-126 and miR-296 in DICER-
MVs but not in Vector-MVs (Fig. 2F). We found that DICER-MVs but not Vector-MVs significantly decreased capillary density (24.7±1O.3 vs 18.1±5.8, P < 0.05) (Fig. 2D).
Severity o f ischemic changes
One week after the operation, the ischemic limb was macroscopically evaluated (CTL, MVs and RNase-MVs, n=lO/group and DICER-MVs, Vector-MVs, Fibroblast-MVs n=5/group) by using an ischemia scale assessed to detect the levels of ischemia, according to Westvik, et al. (29). Mice treated with EPC-derived MVs exhibited significantly less severe degrees of limb ischemia compared to the CTL group injected with vehicle alone (5.8±2 vs 4.1±1.8, P < 0.05) and to the RNase-
A. RANGHINO ET AL.
MVs treated group (5.8±2 vs 4.6±1.6, p < 0.05). Animals treated with RNase-MVs or DICER-MYs showed a quite similar degree o f ischemia compared to the CTL group (4.6±1.7 vs 4.1±1.8, n.s. and 5±1.5 vs 4.1±1.8, ns, respectively), (Fig. 3 A,B).
We examined muscle histology of the ischemic limb dissected out at day 7 after surgery (Fig. 4A-D). Control mice demonstrated a significant reduction of muscle fibers per area compared to MV-treated mice (95.7±37.8 vs 116.3±40.7, p < 0.05) (Fig. 4E). No significant differences were noted between CTL and RNaseMVs-treated mice. In addition, the number of muscle fibers characterized by a small-diameter and with multiple central nuclei that are hallmarks o f the muscle regeneration process was significantly increased in MV-treated mice compared with the CTL group (39±28 vs 11±9, p < 0.05) (Fig. 4F).
The results of the present study demonstrate that MYs derived from EPCs promote neovascularization in an animal model of experimentally induced limb ischemia as a model ofperipheral arterial disease.
Rapid revascularization after ischemia is essential to restore tissue function. Several studies suggested that neoangiogenesis after an ischemic injury may involve the recruitment of EPCs by chemokines released from the injured tissues (31). Transplantation of EPCs was shown to facilitate revascularization o f various tissues including limb ischemia and postmyocardial infarction (4, 32). It is unclear whether EPCs directly contribute to the formation o f neovessels by generation o f mature endothelial cells or just by producing angiogenic factors. It has been recently shown that transient localization of EPCs in the injured tissues may be sufficient to induce functional and regenerative events, suggesting a paracrine mechanisms (8-12). Furthermore, pro-angiogenic EPCs may release MVs capable of exerting effects on surrounding cells. Recent studies indicate that MVs play an important role in cell-to-cell communication by direct stimulation o f target cells or by transferring proteins or mRNAs and microRNAs (17-20). We recently demonstrated that MYs released from EPCs were
able to activate in vitro and in vivo an angiogenic program in endothelial cells via a horizontal transfer of mRNAs. A proof of translation of MV-shuttled mRNA by recipient cells was obtained using MVs carrying GFP mRNA (18). EPC-derived MVs were shown to be incorporated in endothelial cells by interaction with a4 and ~1 integrins expressed on the MV surface. In addition, MYs were shown to activate in the recipient endothelial cells phosphatidyl inositol-3 kinase (PI3K) and eNOS, known to be involved in angiogenic and antiapoptotic programs. mRNA MVs were shown to carry biologically active miRNAs (19, 20). We herein showed that MYs derived from EPC contained miRNA-126 and miRNA-296 that are known to be proangiogenic mainly by regulating response to VEGF in endothelial cells (33, 34). However, MVs might contain other miRNAs that could contribute to the neoangiogenetic effect observed. It has been suggested that the pro-angiogenic potential of EPC obtained from the circulation may depend on the contamination with platelet-derived products (35). To avoid contamination, we used MVs isolated from EPCs after the 3’d passage in culture. Moreover, the cells used and the derived MVs expressed markers o f stem cells (CD34, CD133) and o f endothelium (VEGFR2, CD31) but not o f monocytes (CD 14) and platelets (P-selectin, CD41, CD42b).
In the present study we found that i.v. administration of EPC-derived MVs improved the revascularization after ischemic damage in a mouse ischemic hindlimb model (Figs. 1 and 2). Animals treated with MVs had a significantly lower incidence o f the biological consequences o f ischemia such as foot and limb necrosis compared to the animals injected with vehicle alone (Fig. 3). The enhanced perfusion was associated with the reduced muscle damaged in ischemic limb o f mice treated with MVs, suggesting a protective effect. In addition, there were signs of muscle regeneration as inferred by a significant increase in the number of small rounded myofibers with central nuclei in the damaged muscle area of MV-treated mice (Fig. 4). The specificity of the biological effect ofMVs derived from EPC was indicated by the absence ofprotective effect ofMVs derived from fibroblasts.
The observation that the RNase inactivation of EPC-derived MVs resulted in reduction of their
protective effect, suggests an involvement of RNA delivery. When MVs derived from DICER knock- down-EPCs were used the proangiogenic effect was also significantly reduced suggesting that the proangiogenic effect is at least in part mediated by MV miRNAs cargo.
Peripheral arterial disease along with coronary heart disease and cerebrovascular disease represent a serious and growing health problem in Western countries. Peripheral arterial disease causes intractable ischemia, impaired mobility, compromised wound healing, ulceration, and amputations (1, 2). EPC transplantation has been suggested as an angiogenic strategy. EPCs are able to promote repair o f endothelium (reendothelization) and postnatal formation o f new capillaries (neovascularization) (4,31). Nevertheless, cell therapy for limb ischemia using EPCs imply autologous transplantation o f cells because of the expression of HLA antigens, and EPCs obtained from peripheral blood require ex vivo expansion. The need of both autologous cells and in vitro expansion of the EPCs isolated from peripheral blood limit this procedure because the amount of cells required for transplantation is achieved only after several days of culture (6, 7). Our findings demonstrate the ability ofEPC-derived MVs to induce a neoangiogenic program after ischemia. In contrast with EPCs, MVs do not express HLA antigens, therefore MVs could be produced in large amounts using EPCs collected from healthy volunteers and promptly infused in patients with severe ischemia irrespective of their HLA pattern.
In conclusion, the proangiogenic effect of EPCs is mimicked by EPC-derived MVs, suggesting a paracrine mechanism. Further studies are needed to investigate whether MYs might be a valid and safety therapeutic option for peripheral arterial diseases.
We thank Professor Emilio Hirsh and Professor Mara Brancaccio for technical advice.
This work was supported by Italian Government Miur PRIN project, ‘Regione Piemonte, Piattaforme Biotecnologiche PiSTEM project, and Converging Technologies NanoIGT project and Ricerca Finalizzata and Local University Grants (‘ex60%).
named in a related patent (publication number: WO/2009/050742). C.T. (Fresenius Medical Care) is employed by a commercial company and contributed to the study as a researcher.
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