SEARCH

SEARCH BY CITATION

Keywords:

  • Endothelial cells;
  • engraftment;
  • rapamycin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Donor intra-islet endothelial cells contribute to neovascularization after transplantation. Several factors may interfere with this process and ultimately influence islet engraftment. Rapamycin, a central immunosuppressant in islet transplantation, is an mTOR inhibitor that has been shown to inhibit cancer angiogenesis. The aim of this study was to evaluate the effects of rapamycin on islet endothelium. Rapamycin inhibited the outgrowth of endothelial cells from freshly purified human islets and the formation of capillary-like structures in vitro and in vivo after subcutaneous injection within Matrigel plugs into SCID mice. Rapamycin decreased migration, proliferation and angiogenic properties of human and mouse islet-derived endothelial cell lines with appearance of apoptosis. The expression of angionesis-related factors VEGF, αVβ3 integrin and thrombospondin-1 on islet endothelium was altered in the presence of rapamycin. On the other hand, rapamycin decreased the surface expression of molecules involved in immune processes such as ICAM-1 and CD40 and reduced the adhesion of T cells to islet endothelium. Our results suggest that rapamycin exerts dual effects on islet endothelium inducing a simultaneous inhibition of angiogenesis and a down-regulation of receptors involved in lymphocyte adhesion and activation.


Abbreviations: 
EndoGF medium

endothelial growth factor-enriched medium

VEGF

vascular endothelial growth factor

bFGF

beta fibroblast growth factor

PDGF

platelet-derived growth factor

HGF

hepatocyte growth factor

TGFbeta

transforming growth factor beta

TSP-1

thrombospondin-1

mTOR

mammalian target of rapamycin

hIECs

human islet endothelial cells

mIECs

mouse islet endothelial cells

GFP

green fluorescent protein

vWF

von Willebrand factor

TNF-alpha

tumor necrosis factor-alpha

IFN-gamma

interferon-gamma

ICAM-1

intracellular adhesion molecule-1

OD

optical density

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Clinical islet transplantation has recently received a strong impulse from the results obtained by the introduction of a rapamycin-based glucocorticoid-free immunosuppressive regimen, leading to insulin independence at 1 year in 90% of the treated patients (1). However, several problems with the current procedure still need to be addressed to improve certain aspects, such as organ procurement and preservation, islet isolation and culture, modality of transplant and immunosuppression. Pancreata from multiple donors are still needed to guarantee a sufficient islet mass since a substantial number of transplanted islets fails to engraft into the liver or suffers from poor vascular engraftment. Indeed, native islets in vivo are richly vascularized and even though islets make up only 1% of the pancreatic mass, they receive about 10% of the blood flow (2). After transplantation, islets are revascularized most likely by both the host and the recipient endothelial cells that form a chimeric vascular tree (3). However, revascularization process is not immediate and transplanted islets show the first signs of angiogenesis (i.e. capillary sprout formation and protrusion) not earlier than 2 days after transplantation, and the entire process is completed after 10–14 days. In addition, the vascular density of vascularized transplanted islets is markedly reduced in comparison to native islets (4,5). Based on these considerations, the identification and consequent removal of factors that may impair the angiogenic processes after islet transplantation may likely increase the success of this procedure.

Rapamycin is widely used as central immunosuppressant for islet transplantation as part of the original Edmonton protocol (1). The immunosuppressive mechanism of rapamycin is based on the selective blockade of the mammalian target of rapamycin (mTOR) activation, a molecule known to play a pivotal role in cell cycle progression from late G1 into S phase in response to T-cell growth factor stimulation (6). Unfortunately, considering the ubiquitous expression of mTOR in different cell types, the effects of rapamycin are not restricted to the immune system but affect different physiopathological processes involved in cell survival and proliferation, inducing leucopenia (7), thrombocytopenia (7), delays in wound repair (8) and tubular regeneration after acute ischemic injury (9). Moreover, it has been recently shown that rapamycin inhibited metastatic tumor growth and angiogenesis in an in vivo mouse model (10). The dissection of this phenomenon revealed that rapamycin exerted antiangiogenic activities linked to a decrease in the production of vascular endothelial growth factor (VEGF) and to a markedly inhibited response of tumor endothelial cells to stimulation by VEGF itself (10).

The aim of this study was to evaluate the effects of rapamycin on islet endothelium in the early posttransplantation processes such as islet revascularization and endothelium–immune system interaction, events that may strongly affect intrahepatic engraftment of islets.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Reagents and antibodies

Dithizone, Hoechst 33258, propidium iodide (PI), TNF-alpha, IFN-gamma, mouse monoclonal anti-human thrombospondin 1 (TSP-1) antibody and rapamycin were from Sigma Chemical Company (St. Louis, MO). Rapamycin was solubilized in DMSO and stored at −20°C in the dark as previously described (11). In all experimental procedures, the same amount of solvent was used as control. Polyclonal rabbit anti-human insulin, anti-human CD31 (PECAM-1), anti-human tie-2 and anti-human VEGFR2 (KDR) antibodies were from Santa-Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-human VEGF was from US Biological (Swampscott, MA). Mouse monoclonal anti-human αVβ3 integrin was from Chemicon International (Temecula, CA). Rabbit polyclonal anti-human von Willebrand factor (vWF), Alexa Fluor-conjugated acetylated-LDL and Alexa Fluor-conjugated secondary antibodies were from Invitrogen (Carlsbad, CA). Mouse monoclonal anti-human CD40 and anti-human CD54 (ICAM-1) antibodies were from Serotec (Oxford, UK).

Cells

Four different preparations of freshly purified human islets discarded from transplant use for insufficient islet mass were prepared using the Ricordi method (12) and used in this study. Purified islets (>90% pure) were cultured in CMRL medium (Mediatech Inc., Herndon, VA) containing 5 mg/mL albumin (Kedrion Spa, Lucca, Italy) and 2 mM glutamine (GIBCO BRL, Gaithersburg, MD). Human (hIECs) and mouse (mIECs) pancreatic islet endothelial cell lines were generated as previously described (13). Briefly, cells outgrowing from islets were removed by trypsin/EDTA treatment and transfected with 4 μg pBR322 plasmid vector containing SV40-T large antigen gene at 250 mV and 960 μF in 4-mm electroporation cuvettes in an Invitrogen electroporator II (Invitrogen Corp., Carlsbad, CA). Clones were selected for 1 mg/mL G418 resistance and screened for immunofluorescence expression of vWF. Positive clones were further subcloned by limiting dilution method and cultured in RPMI (Sigma), containing 10% FCS (Hyclone, Logan, Utah) and 2 mM glutamine (GIBCO BRL). Where specified, cells were cultured in RPMI enriched with endothelial growth factors (named EndoGF medium and containing 10 ng/mL VEGF, 10 ng/mL bFGF, 10 ng/mL PDGF and 0.5 U/mL heparin from 10% FCS (Hyclone, Logan, Utah) and 2 mM glutamine (GIBCO BRL).

Assessment of islet viability and function

Viability assay of freshly purified islets was performed by UV light microscopy after double-staining the cells with 0.46 μM fluorescein diacetate (FDA, Sigma) and 14.34 μM PI (Sigma). Islet function was evaluated by ELISA-assay insulin secretory response (ALPCO Windham, NH) by preincubation for 1 h in 2.8 mM glucose medium followed by 2-h incubation in 25 mM glucose medium. The stimulation indices were calculated by dividing data of insulin secretion (mU/L/IEQ) in the presence of high glucose medium by mean basal insulin secretion levels using a spectrophotometric plate reader at 590-nm wave length.

Endothelial outgrowth from freshly purified islets

Freshly purified islets (500 IEQ) were plated on tissue culture dishes and incubated with EndoGF medium in the presence or absence of rapamycin. Endothelial outgrowth from islets was studied under a Nikon microscope system for living cell analysis (14). The same experimental procedures were performed on four different preparations of freshly purified islets.

Migration of hIECs and mIECs

HIECs and mIECs were plated and rested for 12 h with RPMI containing 1% FCS and then incubated with RPMI and different agonists. Cell migration was studied with a 10× phase-contrast objective under the above-mentioned Nikon system. The net migratory speed (velocity straight line) was calculated by the MicroImage software based on the straight line distance between the starting and ending points divided by the time of observation (14). Migration of at least 30 cells for each experimental point was analyzed.

In vitro angiogenesis assay of islets and hIECs

In vitro formation of vessel-like tubular structures was studied on 500 IEQ human islets or 5000 hIECs seeded on growth factor-reduced Matrigel (Becton Dickinson Labware, Bedford, MA) under an inverted microscope in a plexiglass Nikon NP-2 incubator at 37°C. After cells had attached, the medium was removed and 0.5 mL medium containing different stimuli was added. Image analysis was performed at 1-h intervals.

Generation of lentiviral vectors and islet endothelial cell lines infection

Viral production was performed as previously described (15,16). Briefly, 293T cells were transfected with a four-plasmid lentiviral system by the CaCl2 precipitation method. Supernatants were collected 48 and 72 h after transfection, filtered and concentrated by two successive ultracentrifugations. Viral preparation titers were determined by p24 ELISA (Alliance, PerkinElmer, Wellesley, MA), by GFP titer determination on 293T cells and by TaqMan real-time PCR determination of transduced proviral genomes. For gene marking, hIECs and mIECs were plated on 24-well tissue culture plates in EndoGF medium plus 10% FCS and then transduced overnight with a lentivector carrying a CMV-GFP expression cassette, at MOI about 20. HIECs and mIECs infected with lentiviral vectors were termed hIECs-GFP and mIECs-GFP, respectively.

Matrigel implants in mice

Subcutaneous (s.c.) implantation of islets or islet-derived endothelial cell lines in Matrigel plugs was performed to evaluate the antiangiogenic effects of rapamycin in vivo. Briefly, Matrigel was mantained at −20°C until use and thawed at 4°C O/N immediately before implant (17). Freshly purified islets (2000 IEQ), hIECs-GFP or mIECs-GFP (104 cells) were resuspended in 250 μL of fresh medium without FCS and mixed to 500 μL of Matrigel on ice using cooled pipette tips in the absence or presence of 10 ng/mL rapamycin and s.c. implanted into the scruff region of the neck of SCID mice (human islets and hIECs-GFP) or of C57Bl/6 mice (mIECs-GFP). After 2 weeks mice were sacrificed and Matrigel plugs were retrieved for histology and immunohistochemistry.

Immunofluorescence studies

Cells outgrown from islets or hIECs cultured in chamber slides were fixed with 1% paraformaldehyde, permeabilized with 0.1% Triton-X-100 (Sigma) and stained with antibodies directed to endothelial antigens. All samples were incubated with appropriate Alexa Fluor-conjugated secondary antibodies. Matrigel implants containing human islets were fixed in formaldehyde and embedded in paraffin prior to staining. All samples were counterstained with 1 μg/mL PI or with 0.5 μg/mL Hoechst, mounted with antifade mounting medium (Vector Laboratories, Burlingame, CA), and examined under a UV light microscope. The evaluation of intraislet revascularization was performed by confocal microscopy (Leica TCS SP2 Heidelberg, Germany) after costaining for insulin and the endothelial marker CD31. The MicroImage software was used to determine the number and the total area/section of CD31-positive cells in neoformed vessels within islets and in surrounding tissue.

HIECs and mIECs cytotoxicity assay

HIECs and mIECs were cultured on 24-well plates (Falcon Labware, Oxnard, CA) at a concentration of 5 × 104 cells/well, starved for 12 h without FCS and then incubated with different doses of rapamycin (0.1–100 ng/mL) in the presence or absence of endothelial growth factors in a medium without phenol red containing 250 μg/mL XTT (Sigma). The absorption values at 450 nm were measured. All experiments were done in triplicate.

Detection of apoptosis of hIECs and mIECs

HIECs and mIECs were subjected to TUNEL assay (terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling) (ApopTag, Oncor, Gaithersburg, MD) after starving for 12 h without FCS and incubation for 48 h with different doses of rapamycin (ranging from 0.1 ng/mL to 100 ng/mL) in the presence of EndoGF medium. After stimulation, both endothelial cell lines were fixed in 1% paraformaldehyde, postfixed in precooled ethanol-acetic acid 2:1, incubated with TdT enzyme in a humidified chamber at 37°C for 1 h and counterstained with antidigoxigenin-FITC antibody and with PI (1 μg/mL). Samples were analyzed under a UV light microscope with an appropriate mounting medium. Green-stained apoptotic cells were counted in different microscopic fields (magnification 100×).

Gene array technology

Human GEarray kit for the study of angiogenesis markers (SuperArray Inc., Bethesda, MD) was used to characterize the gene expression profiles of unstimulated or stimulated freshly purified islets with 10 ng/mL of rapamycin for 48 h. Hybridization was performed according to the manufacturer's instructions as previously described (18).

Adhesion of Jurkat to hIECs monolayers

Jurkat cells (ATCC, Rockville, MD) were labeled overnight with 10 μm Vybrant Cell Tracer kit (Invitrogen) according to manufacturer's in RPMI and 10% FBS (13). Labeled-Jurkat cells were counted, resuspended to 50 × 106/mL RPMI without FCS and added to confluent monolayer of hIECs cultured on six-well plates and previously incubated with vehicle alone or inflammatory cytokines (10 ng/mL TNF-alpha and 10 ng/mL IFN-gamma) in the presence or absence of 10 ng/mL rapamycin. Experiments were carried out in triplicate for 1 h at 37°C in conditions of slight agitation. At the end of incubation, plates were filled with medium and aspirated three times to remove unbound Jurkat cells. All samples were fixed with 1% paraformaldehyde and observed under a UV light microscope. Green fluorescent-Jurkat cells were counted on 10 different fields at 200× magnification.

FACS analysis

Unstimulated or stimulated hIECs were detached from tissue culture plates with EDTA and stained for 45 min at 4°C with appropriate antibodies. Cells were then fixed in 1% paraformaldehyde and subjected to FACS analysis (Becton Dickinson, Mountain View, CA).

Statistical analysis

All data of different experimental procedures are expressed as average ± SD. Statistical analysis was performed by Student's t-test or ANOVA with Newmann-Keuls multicomparison test where appropriate.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Effects of rapamycin on angiogenesis in freshly purified islet endothelium and inhIECs and mIECs endothelial cell lines

Endothelial cells outgrew from freshly purified human islets cultured in the presence of endothelial growth factors-rich medium (EndoGF medium) (Figure 1A). As shown in Figure 1B, the addition of rapamycin blunted this phenomenon. The antiangiogenic effect of rapamycin on freshly purified islet endothelium was confirmed by the marked decrease of capillary-like structures in vitro when islets were seeded on Matrigel-coated surfaces (Figure 1C,D). Cells outgrowing from islets were characterized as endothelial cells by staining with typical endothelial markers such as VEGFR2 (KDR, Figure 1E), vWF (Figure 1F), CD31 and tie-2 and by the uptake of acetylated-LDL (not shown). Notably, the dose of rapamycin used to obtain the inhibition of endothelial outgrowth (10 ng/mL) did not alter islet viability (comparable PI/FDA staining >90% in each sample) and function, evaluated as insulin response after high glucose challenge (stimulation indices 3.42 ± 0.85 for vehicle alone and 3.59 ± 0.97 for rapamycin-treated islets, respectively) after 5 days of culture.

image

Figure 1. Rapamycin inhibited the outgrowth of endothelial cells from freshly purified islets. (A and B) Representative images of dithizone-stained islets observed 5 days after plating on tissue culture dishes in EndoGF medium in the absence (A) or presence (B) of 10 ng/mL rapamycin (magnification 100×). (C and D) Representative images of endothelial cells forming tubular structures derived from islets 5 days after plating on Matrigel-coated surfaces and cultured in EndoGF medium in the absence (C) or presence (D) of 10 ng/mL rapamycin (magnification 100×). (E and F) Phenotypic characterization of cells outgrowing from islets in the presence of EndoGF medium. Endothelial phenotype was assessed by positive immunofluorescence staining with anti-VEGFR2 (KDR) (panel E, magnification 400×, nuclei counterstained by 0.5 μg/mL Hoechst) and with anti-vWF antibodies (panel F, magnification 400×, nuclei counterstained by 1 μg/mL propidium iodide).

Download figure to PowerPoint

To further investigate the inhibition of endothelial outgrowing from islets, we studied the effects of rapamycin on hIECs and mIECs motility by time-lapse recording microscopy. The baseline migration rate of hIECs and mIECs corresponding to the spontaneous motility of resting, unstimulated cells was first measured and found to remain steady for the whole period of observation never exceeding 5–6 μm/h. Rapamycin induced a significant decrease of spontaneous migration of both hIECs and mIECs (Figure 2A,B). As expected, the incubation with EndoGF medium induced a marked acceleration of spontaneous cell motility, peaking as early as 1 h after stimulation and remaining significantly higher compared to unstimulated cells throughout all the observation period (Figure 2A,B). The addition of rapamycin to EndoGF medium significantly reduced the migration of both endothelial cell lines (Figure 2A,B) in a dose-dependent manner (Figure 3A,B).

image

Figure 2. Inhibition of hIECs and mIECs motility induced by rapamycin. (A and B) Analysis of scattered hIECs (A) and mIECs (B) motility studied by time-lapse microscopy. Rapamycin induced a time-dependent inhibition of spontaneous and growth factor-induced migration of hIECs and mIECs. Migration of at least 30 cells for each experimental point was analyzed. All experimental points were conducted in triplicate. Data are expressed as speed averages (μm/h) ± SD. ANOVA with Newmann-Keuls multicomparison test was performed: rapamycin induced a significant decrease of both hIECs and mIECs motility in comparison to stimulation with normal or EndoGF medium at every period of time analyzed (p < 0.005 at 2, 6 and 12 h after incubation). Three independent experiments were performed with similar results.

Download figure to PowerPoint

image

Figure 3. Rapamycin-induced inhibition of migration of hIECs and mIECs was dose dependent. (A and B) Rapamycin decreases in a dose-dependent manner the EndoGF growth factor-induced motility of hIECs (A) and mIECs (B). Migration of at least 30 cells for each experimental point was analyzed. All experimental points were conducted in triplicate. Data are expressed as speed averages (μm/h) ± SD. ANOVA with Newmann-Keuls multicomparison test was performed: rapamycin induced a significant decrease of both hIECs and mIECs motility in comparison to stimulation with vehicle alone at the dose of 0.1 ng/mL, reaching a plateau at 50 ng/mL (*p < 0.005 EndoGF medium + different doses of rapamycin vs. EndoGF alone). Three independent experiments were performed with similar results.

Download figure to PowerPoint

We then studied the in vitro angiogenic ability of hIECs that form capillary-like structures when seeded on Matrigel-coated plates. The addition of EndoGF medium accelerated the angiogenic process, resulting in enhanced formation of a capillary network (Figure 4A,D) in comparison to vehicle alone (Figure 4A,B). Rapamycin significantly inhibited spontaneous (Figure 4A,C) and growth factor-induced (Figure 4A,E) formation of capillary-like structures.

image

Figure 4. Rapamycin inhibited in vitro hIECs angiogenesis. (A) Inhibitory effect of rapamycin on spontaneous and growth factor-induced formation of capillary vessel-like tubular structures by hIECs as shown by capillary connection counts per field. (B–E) Representative images of angiogenesis inhibition of hIECs induced by rapamycin challenge. When plated on Matrigel-coated wells, unstimulated hIECs show the formation of tubular structures within 12 h (B). EndoGF medium accelerates this angiogenic process (D), whereas 10 ng/mL rapamycin induced a decrease of capillary connections on both unstimulated (C) and growth factor-stimulated (E) hIECs. All experiments were conducted in triplicate and 10 fields/each were analyzed. Data are expressed as average number of capillary connections ± SD. ANOVA with Newmann-Keuls multicomparison test was performed: (*p < 0.005 rapamycin or EndoGF medium vs. vehicle alone); (#p < 0.005 EndoGF medium + rapamycin vs. rapamycin). Three independent experiments were performed with similar results.

Download figure to PowerPoint

The inhibition induced by rapamycin on islet angiogenesis was also evaluated in vivo by s.c. injection of freshly purified islets within Matrigel plugs into the scruff region of the neck of SCID mice. Matrigel s.c. implant in mice is a well-established model of angiogenesis (19) and it has been recently demonstrated as effective in transplanting islets (20). In the presence of rapamycin, implants showed a marked reduction of vascularization as detected by immunofluorescence staining for the endothelial marker tie-2 (Figure 5A,B) and by counts of number and total area of neoformed vessels within Matrigel (Figure 5C,D). In addition, rapamycin also induced a marked down-regulation of the vascular density within the islets (Figure 5E–H).

image

Figure 5. Reduced vascularization of freshly purified human islets after subcutaneous transplantation into SCID mice in the presence of rapamycin. (A and B) Representative images of immunofluorescence studies on freshly purified islets implanted into Matrigel plugs in the scruff region of the neck of SCID mice in the absence (A) or presence (B) of 10 ng/mL rapamycin. Rapamycin induced a marked decrease of endothelial cells (arrow heads) outgrowing from the transplanted islets (indicated by ‘I’) as detected by immunofluorescence analysis of tie-2 (blue staining in A and B), a marker of endothelial phenotype. All sections were counterstained by 1 μg/mL propidium iodide and evaluated at 100× magnification. (C and D) Rapamycin-induced significant inhibition of neovascularization of transplanted islets evaluated by counts of number (C) and total area (D, expressed in μm2/section) of neoformed vessels within Matrigel. Student's t-test was performed: (*p < 0.005 rapamycin vs. vehicle alone). (E and F) Confocal microscope analysis of costaining for insulin (red) and for the endothelial marker CD31 (green) showed a reduced microvascular density within islets treated with rapamycin (F) in comparison to vehicle (E). All sections were counterstained by 0.5 μg/mL Hoechst 33258 and evaluated at 200× magnification. (G and H) Rapamycin-induced significant inhibition of intraislet vascularization of transplanted islets evaluated by counts of number (G) and total area (H, expressed in μm2/section) of vessels. Student's t-test was performed: (*p < 0.005 rapamycin vs. vehicle alone). Six mice per group were used in this experiment.

Download figure to PowerPoint

We then performed xenografts of hIECs-GFP and isografts of mIECs-GFP by injection into the scruff region of the neck of SCID or C57Bl/6 mice, respectively. Consistently with the in vitro angiogenesis studies, in the presence of rapamycin hIECs showed a marked inhibition of their ability to proliferate and to form neovessels as detected by histologic (Figure 6A,B) and immunofluorescence studies (Figure 6C,D). Moreover, rapamycin induced a significant reduction of number and total area of vessels (Figure 6E,F). Immunofluorescence analysis also showed a marked alteration of normal structure of mIECs-GFP isografts (not shown).

image

Figure 6. Inhibition of in vivo hIEC angiogenesis induced by rapamycin. (A–D) Matrigel plugs implanted for 2 weeks into the neck scruff region of SCID mice and containing hIECs-GFP in the presence of vehicle alone (A and C) or 10 ng/mL rapamycin (B and D). Histologic analysis showed the abundant presence of neoformed capillaries in control Matrigel plugs (A) in comparison to their paucity in rapamycin-containing implants (B). By fluorescence analysis patent vessels were formed by GFP-positive cells in control Matrigel plugs (C), whereas in the presence of rapamycin mainly scattered hIECs-GFP were detectable (D). (E and F) Rapamycin induced a significant inhibition of in vivo hIECs angiogenesis evaluated by counts of number (E) and total area (F, expressed in μm2/section) of neoformed vessels in tissue sections. Student's t-test was performed: (*p < 0.005 rapamycin vs. vehicle alone). Six mice per group were used in this experiment.

Download figure to PowerPoint

We then studied the effect of rapamycin on islet endothelium growth. HIECs and mIECs were starved overnight without FCS and subsequently incubated with normal or EndoGF medium containing increasing doses of rapamycin (0.1–100 ng/mL). As expected, endothelial growth factors induced an increase of proliferation of endothelial cells in comparison to vehicle alone. Coincubation with rapamycin resulted in a significant inhibitory effect on spontaneous and growth factor-stimulated proliferation of both hIECs and mIECs (Figure 7A,B). This effect was detectable at doses of 0.1–1 ng/mL and reached a plateau at the dose of 50 ng/mL for both cell lines (Figure 7C,D).

image

Figure 7. Antiproliferative effect of rapamycin on hIECs and mIECs. (A and B) Time-course analysis of spontaneous and growth factor (EndoGF)-induced inhibition of proliferation exerted by rapamycin on hIECs (A) and mIECs (B), evaluated by XTT-based assay. A significant decrease of proliferation of both endothelial cell lines after 24 and 48 h of stimulation was observed (p < 0.005 rapamycin or EndoGF medium vs. vehicle alone and EndoGF + rapamycin vs. rapamycin). (C and D) Dose-dependent growth arrest induced by rapamycin challenge on hIECs (C) and mIECs (D) incubated with EndoGF. A significant decrease of proliferation of endothelial cell lines after 48 h of stimulation starting from a dose of rapamycin of 0.1 ng/mL for hIECs and 1 ng/mL for mIECs. This effect reached a peak with a rapamycin concentration of 50 ng/mL in both cell lines: (*p < 0.005 different doses of rapamycin vs. vehicle alone). Data are expressed as average OD intensity of three different experimental points ± SD. ANOVA with Newmann-Keuls multicomparison test was performed. Three independent experiments were performed with similar results.

Download figure to PowerPoint

In the transplantation setting, the efficiency of islet revascularization results from the net balance between opposing factors that may affect endothelial cell survival. For this reason, we evaluated the effect of rapamycin on endothelial growth factor-dependent rescue from apoptosis induced by serum deprivation. As shown in Figure 8, incubation with rapamycin worsened hIECs (Figure 8A) and mIECs (Figure 8B) apoptosis induced by serum deprivation while EndoGF medium significantly reduced cell death. In addition, rapamycin significantly inhibited the antiapoptotic effect of EndoGF-medium on hIECs (Figure 8A,C) and mIECs (Figure 8B,D) in a dose-dependent manner.

image

Figure 8. Rapamycin increased hIECs and mIECs cell death induced by serum deprivation and impaired growth factor-mediated rescue from apoptosis. (A and B) Evaluation of proapoptotic activity by TUNEL assay induced by rapamycin on hIECs (A) and mIECs (B) after 48 h incubation. Rapamycin significantly increased the number of apoptotic hIECs and mIECs in culture with or without coincubation with Endo GF medium. Data are expressed as average number of apoptotic cells/field ± SD (magnification 100×). ANOVA with Newmann-Keuls multicomparison test was performed: (*p < 0.005 rapamycin or EndoGF medium vs. vehicle alone); (#p < 0.005 Endo GF + rapamycin vs. rapamycin). (C and D) Dose-dependent proapoptotic effect of rapamycin on hIECs (C) and mIECs (D) after 48 h incubation in the presence of EndoGF medium. Data are expressed as average number of apoptotic cells/field ± SD. ANOVA with Newmann-Keuls multicomparison test was performed, showing a significant increase of apoptosis of both endothelial cell lines after 48 h of stimulation with a dose of rapamycin of 0.1 ng/mL. This effect reached a peak with a rapamycin concentration of 50 ng/mL: (*p < 0.005 different doses of rapamycin vs. vehicle alone). Three independent experiments were performed with similar results.

Download figure to PowerPoint

We then investigated the modulation of expression of selected molecules involved in angiogenesis after rapamycin challenge on cultured hIECs by immunofluorescence studies. Rapamycin-treated hIECs showed a decreased expression of VEGF and αVβ3 integrin and a marked increase of TSP-1 (Figure 9). Consistently with these results, we also observed a down-regulation of VEGF, PDGF, betaFGF, HGF, αVβ3 integrin genes, and up-regulation of the TSP-1 gene in human islets exposed to rapamycin (not shown).

image

Figure 9. Modulation of angiogenic factors induced by rapamycin on hIECs. Immunofluorescence analysis of hIECs cultured in the absence (control) or presence of 10 ng/mL rapamycin. Rapamycin induced a reduction of VEGF and αVβ3 integrin on hIECs. In addition, an overexpression of TSP-1, an angiogenesis inhibitor, was detected on rapamycin-treated hIECs (100× magnification, antibody staining in green and nuclear counterstaining in red with 1 μg/mL propidium iodide).

Download figure to PowerPoint

Effects of rapamycin on lymphocyte-islet endothelial interaction

Rapamycin has been shown to prevent up-regulation of immune cell surface molecules involved in cell adhesion and cross-talk during immune cell activation. To determine the role of rapamycin in intraislet lymphocyte recruitment, we conducted experiments aimed at evaluating the adhesion of Jurkat cells, a T-cell line, to hIECs monolayers. The addition of rapamycin inhibited the adhesion of Jurkat cells induced by 10 ng/mL TNF-alpha and 10 ng/mL IFN-gamma (Figure 10A). On cytokine-stimulated hIECs, the addition of rapamycin prevented the overexpression of the lymphocyte costimulatory molecule CD40 and of the adhesion receptor CD54 (ICAM-1) (Figure 10B).

image

Figure 10. Effects of rapamycin on T-cell adhesion on hIECs and on modulation of vascular surface receptors involved in lymphocyte-endothelium interaction. (A) In vitro adhesion assay of green fluorescent Jurkat T-cell line to hIEC monolayers. Labeled Jurkat cells were added to hIECs confluent monolayers incubated with vehicle alone or 10 ng/mL TNF-alpha plus 10 ng/mL IFN-gamma (cytokines) in the presence or absence of 10 ng/mL rapamycin. After extensive washing, all samples were fixed and analyzed under a UV light microscope. Fluorescent cells in 10 fields/well were counted at 200× magnification and data are representative of average number of adherent cells/field ± SD. Three independent experiments were performed with similar results. ANOVA with Newmann-Keuls multicomparison test was performed: (*p < 0.005 rapamycin or cytokines vs. vehicle alone); (#p < 0.005 cytokines + rapamycin vs. cytokines). (B) FACS analysis showed a slight basal expression of CD40 and ICAM-1 (bold lines) on hIECs in culture. Exposure of hIECs to cytokines resulted in a marked increase of both CD40 and ICAM-1 expression that was prevented by coincubation with 10 ng/mL rapamycin. Isotype-matched antibody controls are shown as dotted lines.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

A prominent role of islet vasculature conceivably takes place in the setting of islet transplantation. Recently, it was demonstrated that freshly purified islets retain viable native endothelial cells that may actively participate to the revascularization process by forming chimeric donor/recipient-derived vessels (3). This event may have direct consequences in facilitating islet engraftment and function (3) and may promote strategies aimed at preserving islet endothelium before transplantation by minimizing the pretransplant culture time (21) or by using endothelial growth factors such as VEGF (5,22). Indeed, several factors may positively or negatively influence islet endothelium viability and function in the setting of islet purification, culture and transplantation. In the past, immunosuppressive drug combinations have been indicated as a limiting factor for transplanted islets function due to the diabetogenic effect of steroids and to the direct toxic effect of calcineurin inhibitors (23). Clinical islet transplantation has recently received a strong impulse from the results obtained by the introduction of a rapamycin-based glucocorticoid-free immunosuppressive regimen leading to insulin independence at 1 year in 90% of the treated patients (1,24). However, an inhibitory effect of rapamycin on tumor angiogenesis has been recently described (10,25). In light of the evidences on the importance of neovascularization of the transplanted islets, we evaluated whether rapamycin may have a direct impact on the angiogenic ability of intraislet endothelial cells.

We observed that rapamycin markedly inhibited the outgrowth of endothelial cells from freshly purified islets in vitro and in vivo after s.c. injection into Matrigel plugs in mice. These results were paralleled by the inhibition of human and murine islet endothelial cell line migration, proliferation and angiogenesis induced by this immunosuppressant.

The antiangiogenic effect of rapamycin that we observed both on purified islets and on islet endothelial cell lines was exerted at rapamycin concentrations that correspond to the through blood rapamycin levels currently recommended for islet transplant recipients (1). Interestingly, the antiangiogenic effect was detectable at rapamycin doses that did not impair beta-cells viability or function in vitro. Moreover, it is conceivable that islet endothelium after transplantation may be exposed to even higher doses of rapamycin due to the peak rapamycin concentration in the portal circulation obtained after gastrointestinal absorption of this drug (26).

Furthermore, we analyzed the potential effect of rapamycin on apoptosis of islet endothelial cells. At supratherapeutic concentrations rapamycin was shown to have deleterious effects on the viability of rat and human islets (27). Other cell types such as monocytes (28), macrophages (28), BxPC3 and Panc-1 human pancreatic adenocarcinoma cell lines (29) were not susceptible to apoptosis after rapamycin challenge. In our study, rapamycin prevented the rescue effect of growth factors when hIECs and mIECs were exposed to detrimental culture conditions such as prolonged serum deprivation. This phenomenon may have relevance right after islet implant in the sinusoid vessels where islets are exposed to multiple mutual-conflicting signals that ultimately affect their survival and functional engraftment or their death.

The antiangiogenic effect of rapamycin has been partially elucidated and it is thought to depend on the inhibitory effect on VEGF-mediated activation of Akt, a serine/threonine protein kinase directly up-stream of mTOR in the rapamycin-sensitive signaling pathway and on decreased production of VEGF (10). It has been recently shown that rapamycin treatment inhibits tumor progression in renal transplant patients bearing Kaposi's sarcoma, whose lesions are characterized by abundant expression of VEGF and increased phosphorylation of Akt (25).

We performed experiments in the presence of endothelial growth factors that were shown to be expressed after islet implants within sinusoids (30–32), thus mimicking the proangiogenic milieau where islet neovascularization takes place. We showed that rapamycin challenge strongly inhibited VEGF production by islet endothelium and induced an altered expression of other molecules involved in tumor and normal tissue angiogenesis such as αVβ3 integrin and TSP-1. Overexpression of αVβ3 integrin on tumor vasculature has been associated with an aggressive phenotype of several solid tumor types (33) and we recently showed the up-regulation of this molecule on islet endothelium induced by platelet-activating factor (13). TSP-1 is a potent angiogenesis inhibitor that triggers apoptosis in activated endothelial cells (32,34). Interestingly, TSP-1−/− mice showed islet hyperplasia due to an increased density of blood vessels, suggesting a key role for this protein in vascularization of native and transplanted islets (35).

Taken together, our findings showed that rapamycin affects islet vascularization interfering with the production of different pro- and antiangiogenic factors within islet endothelium. Furthermore, the reduced release of these molecules by endothelial cells may affect beta cells surviving due to the deprivation of growth factors (36,37).

On the other hand, we provided data on the effect of rapamycin in modulating cell-to-cell receptor interaction among islet endothelial cells and lymphocytes that may potentially occur during allograft rejection. In addition, rapamycin also reduced the expression of CD40 and ICAM-1 in hIECs stimulated with TNF-alpha and IFN-gamma. In particular, endothelial CD40, a critical costimulatory receptor for lymphocyte activation, has been identified as a key molecule for the interaction with immune system in vascular processes involved in transplant rejection (38,39). Moreover, treatment with anti-CD40 ligand antibodies has been proven to be beneficial for the prevention of allograft rejection in nonhuman primate models of islet transplantation (40). ICAM-1 is a major surface molecule involved in leukocytes adhesion to endothelial cells and subsequent recruitment in sites of immune injury (41). These effects induced by rapamycin were previously observed also in antigen presenting cells such as dendritic cells (42).

Based on our results, a potential limitation of the current immunosuppressive protocols may be represented by the antiangiogenic activity of rapamycin, particularly detrimental in the early engraftment phase. However, its vascular immunomodulatory properties may be relevant in the following phases. In conclusion, new therapeutic sequential protocols that consider the whole activity profile of rapamycin may be highly advantageous to the field of islet transplantation.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This work was supported by Italian Ministry of University and Research (MIUR) FIRB project (RBNE01HRS5-001) to G.C. and COFIN 01 to L.B., G.C. and P.C.P., by Italian Ministry of Health (Ricerca Finalizzata 02) to G.C. and G.P.S. and by ‘Ricerca Finalizzata—Regione Piemonte’ to L.B.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
  • 1
    Shapiro AM, Lakey JR, Ryan EA et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343: 230238.
  • 2
    Carlsson PO, Mattsson G. Oxygen tension and blood flow in relation to revascularization in transplanted adult and fetal rat pancreatic islets. Cell Transplant 2002; 11: 813820.
  • 3
    Nyqvist D, Kohler M, Wahlstedt H, Berggren PO. Donor islet endothelial cells participate in formation of functional vessels within pancreatic islet grafts. Diabetes 2005; 54: 22872293.
  • 4
    Biancone L, Ricordi C. Pancreatic islet transplantation: An update. Cell Transplant 2002; 11: 309311.
  • 5
    Lai Y, Schneider D, Kidszun A et al. Vascular endothelial growth factor increases functional beta-cell mass by improvement of angiogenesis of isolated human and murine pancreatic islets. Transplantation 2005; 79: 15301536.
  • 6
    Kirken RA, Wang YL. Molecular actions of sirolimus: Sirolimus and mTor. Transplant Proc 2003; 35(3 Suppl): 227S230S.
  • 7
    Hering BJ, Wijkstrom M. Sirolimus and islet transplants. Transplant Proc 2003; 35(3 Suppl): 187S190S.
  • 8
    Valente JF, Hricik D, Weigel K et al. Comparison of sirolimus vs. mycophenolate mofetil on surgical complications and wound healing in adult kidney transplantation. Am J Transplant 2003; 3: 11281134.
  • 9
    Lieberthal W, Fuhro R, Andry CC et al. Rapamycin impairs recovery from acute renal failure: Role of cell-cycle arrest and apoptosis of tubular cells. Am J Physiol Renal Physiol 2001; 281: F693F706.
  • 10
    Guba M, Von Breitenbuch P, Steinbauer M et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: Involvement of vascular endothelial growth factor. Nat Med 2002; 8: 128135.
  • 11
    Butzal M, Loges S, Schweizer M et al. Rapamycin inhibits proliferation and differentiation of human endothelial progenitor cells in vitro. Exp Cell Res 2004; 300: 6571.
  • 12
    Ricordi C, Lacy PE, Scharp DW. Automated islet isolation from human pancreas. Diabetes 1989; 38(Suppl 1): 140142.
  • 13
    Biancone L, Cantaluppi V, Romanazzi GM et al. Platelet-activating factor synthesis and response on pancreatic islet endothelial cells: Relevance for islet transplantation. Transplantation 2006; 81: 511518.
  • 14
    Biancone L, Cantaluppi V, Boccellino M et al. Motility induced by human immunodeficiency virus-1 Tat on Kaposi's sarcoma cells requires platelet-activating factor synthesis. Am J Pathol 1999; 155: 17311739.
  • 15
    Dull T, Zufferey R, Kelly M et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72: 84638471.
  • 16
    Galimi F, Noll M, Kanazawa Y et al. Gene therapy of Fanconi anemia: Preclinical efficacy using lentiviral vectors. Blood 2002; 100: 27322736.
  • 17
    Montrucchio G, Lupia E, Battaglia E et al. Tumor necrosis factor alpha-induced angiogenesis depends on in situ platelet-activating factor biosynthesis. J Exp Med 1994; 180: 377382.
  • 18
    Biancone L, Cantaluppi V, Duò D, Deregibus MC, Torre C, Camussi G. Role of l-selectin in the vascular homing of peripheral blood-derived endothelial progenitor cells. J Immunol 2004; 173: 52685274.
  • 19
    Jain RK, Schlenger K, Hockel M, Yuan F. Quantitative angiogenesis assays: Progress and problems. Nat Med 1997; 3: 12031208.
  • 20
    Bharat A, Benshoff N, Olack B, Ramachandran S, Desai NM, Mohanakumar T. Novel in vivo murine model to study islet potency: Engraftment and function. Transplantation 2005; 79: 16271630.
  • 21
    Olsson R, Carlsson PO. Better vascular engraftment and function in pancreatic islets transplanted without prior culture. Diabetologia 2005; 48: 469476.
  • 22
    Zhang N, Richter A, Suriawinata J et al. Elevated vascular endothelial growth factor production in islets improves islet graft vascularization. Diabetes 2004; 53: 963970.
  • 23
    Pileggi A, Ricordi C, Alessiani M, Inverardi L. Factors influencing Islet of Langerhans graft function and monitoring. Clin Chim Acta 2001; 310: 316.
  • 24
    Ryan EA, Paty BW, Senior PA et al. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54: 20602069.
  • 25
    Stallone G, Schena A, Infante B et al. Sirolimus for Kaposi's sarcoma in renal-transplant recipients. N Engl J Med 2005; 352: 13171323.
  • 26
    Desai NM, Goss JA, Deng S et al. Elevated portal vein drug levels of sirolimus and tacrolimus in islet transplant recipients: Local immunosuppression or islet toxicity? Transplantation 2003; 76: 16231625.
  • 27
    Bell E, Cao X, Moibi JA et al. Rapamycin has a deleterious effect on MIN-6 cells and rat and human islets. Diabetes 2003; 52: 27312739.
  • 28
    Woltman AM, De Fijter JW, Kamerling SW et al. Rapamycin induces apoptosis in monocyte- and CD34-derived dendritic cells but not in monocytes and macrophages. Blood 2001; 98: 174180.
  • 29
    Shah SA, Potter MW, Ricciardi R, Perugini RA, Callery MP. FRAP-p70s6K signaling is required for pancreatic cancer cell proliferation. J Surg Res 2001; 97: 123130.
  • 30
    Vasir B, Jonas JC, Steil GM et al. Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets. Transplantation 2001; 71: 924935.
  • 31
    Linn T, Schneider K, Hammes HP et al. Angiogenic capacity of endothelial cells in islets of Langerhans. FASEB J 2003; 17: 881883.
  • 32
    Welsh M, Claesson-Welsh L, Hallberg A et al. Coexpression of the platelet-derived growth factor (PDGF) B chain and the PDGF beta receptor in isolated pancreatic islet cells stimulates DNA synthesis. Proc Natl Acad Sci U S A 1990; 87: 58075811.
  • 33
    Jin H, Varner J. Integrins: Roles in cancer development and as treatment targets. Br J Cancer 2004; 90: 561565.
  • 34
    Jimenez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med 2000; 6: 4148.
  • 35
    Johansson M, Mattsson G, Andersson A, Jansson L, Carlsson PO. Islet endothelial cells and pancreatic beta-cell proliferation: Studies in vitro and during pregnancy in adult rats. Endocrinology 2006; 147: 23152324.
  • 36
    Cheng K, Fraga D, Zhang C et al. Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets. Gene Ther 2004; 11: 11051116.
  • 37
    Garcia-Ocana A, Vasavada RC, Cebrian A et al. Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice. Diabetes 2001; 50: 27522762.
  • 38
    Kirk AD, Burkly LC, Batty DS et al. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat Med 1999; 5: 686693.
  • 39
    Biancone L, Cantaluppi V, Camussi G. CD40-CD154 interaction in experimental and human disease (review). Int J Mol Med 1999; 3: 343353.
  • 40
    Koulmanda M, Smith RN, Qipo A, Weir G, Auchincloss H, Strom TB. Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term anti-CD154-based therapy: Nonimmunologic graft failure? Am J Transplant 2006; 6: 687696.
  • 41
    Park SY, Kim HW, Moon KC, Hong HK, Lee HS. mRNA expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in acute renal allograft rejection. Transplantation 2000; 69: 25542560.
  • 42
    Monti P, Mercalli A, Leone BE, Valerio DC, Allavena P, Piemonti L. Rapamycin impairs antigen uptake of human dendritic cells. Transplantation 2003; 75: 137145.