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Endocrinology Vol. 147, No. 5 2315-2324
Copyright © 2006 by The Endocrine Society

Islet Endothelial Cells and Pancreatic ß-Cell Proliferation: Studies in Vitro and during Pregnancy in Adult Rats

Magnus Johansson, Göran Mattsson, Arne Andersson, Leif Jansson and Per-Ola Carlsson

Departments of Medical Cell Biology (M.J., G.M., A.A., L.J., P.-O.C.) and Medical Sciences (P.-O.C.), Uppsala University, SE-751 23 Uppsala, Sweden; and Biomedical Research Institute, Department of Biological Sciences, University of Warwick (G.M.), Warwick CV4 7AL, United Kingdom

Address all correspondence and requests for reprints to: Dr. Magnus Johansson, Department of Medical Cell Biology, Biomedical Center, Husargatan 3, Box 571, SE-751 23 Uppsala, Sweden. E-mail: magnus.johansson{at}medcellbiol.uu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The growth of both tumors and nonneoplastic tissues may be influenced by signals from the vascular endothelium. In the present investigation we show that purified proliferating endothelial cells from pancreatic islets can stimulate ß-cell proliferation through secretion of hepatocyte growth factor (HGF). This secretion could be induced by soluble signals from the islets, such as vascular endothelial growth factor-A (VEGF-A) and insulin. During pregnancy, the pancreatic ß-cells display a highly reproducible physiological proliferation. We show that islet endothelial cell proliferation precedes ß-cell proliferation in pregnant animals. Vascular growth was closely associated with endocrine cell proliferation, and prominent expression of HGF was observed in islet endothelium on d 15 of pregnancy, i.e. coinciding with the peak of ß-cell proliferation. In summary, our results suggest the existence of an endothelial-endocrine axis within adult pancreatic islets, which is of importance for adult ß-cell proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE REGULATION OF islet ß-cell mass is still incompletely understood (1, 2). To be able to increase the number of functional ß-cells would, however, be advantageous in several clinical settings. One such instance is large-scale clinical islet transplantation, where the limited availability of islets is a major obstacle. Another is type 2 diabetes, where such knowledge might provide new treatment regimens based on stimulation of endocrine cell growth. Recently, much attention has been paid to the importance of endothelium as a source of growth factors inducing proliferation of parenchymal cells. In testosterone-treated, castrated male rats, for example, a rapid increase in vascular mass precedes the growth of the prostate epithelium (3). An initial vascular growth preceding that of the parenchymal cells is also seen during liver organogenesis (4) and after liver injury in adults (5).

Pancreatic islets have a dense capillary network, which is of importance to provide nutrients, allow for accurate glucose sensing, and disperse hormones to the systemic circulation. Islet endothelial cells can glucose-dependently produce vasoactive mediators, such as nitric oxide, and a variety of cytokines (6, 7). It is likely that these substances have paracrine effects in view of the close proximity of endothelial and endocrine cells to one another. Observations that no insulin-positive cells are formed during early embryonic development without a close proximity to blood vessels are in line with these ideas (8). Furthermore, overexpression of endothelial cell mitogen vascular endothelial growth factor-A (VEGF-A) results not only in a hypervascularized pancreas, but also in hyperplasia of the pancreatic islets (8).

To what extent endothelium-derived substances affect islet endocrine function and growth in adults is unknown. In the present investigation we tested the hypothesis that there is an endothelial-endocrine interaction during ß-cell growth and islet angiogenesis. For this purpose, we developed an in vitro system in which potential interactions between purified proliferating islet endothelial cells and islet endocrine cells could be investigated. In this system, islet endothelial cells promoted ß-cell proliferation through the secretion of hepatocyte growth factor (HGF). This made us investigate also the relationship between endothelial and endocrine cell proliferation in vivo, and we chose for study the endocrine pancreas of pregnant rats, in which a highly reproducible physiological proliferation of pancreatic islet ß-cells occurs (9).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Twelve-week-old inbred male Wistar-Furth rats and control or pregnant female Wistar rats were purchased from B&K (Sollentuna, Sweden). Day 0 of pregnancy was defined as the day on which spermatozoa were found in the vaginal smear. The gestational length of Wistar rats is 21–22 d. The animals had free access to water and pelleted food throughout the course of the study. All experiments were approved by the animal ethics committee at Uppsala University.

Chemicals
All chemicals were purchased from Sigma-Aldrich Corp. (Irvine, UK) unless otherwise mentioned.

Islet isolation
Pancreatic islets from male Wistar-Furth rats were isolated as previously described (10). Groups of 150 islets were maintained free-floating at 37 C (air/CO2, 95:5) in islet culture medium [CM; RPMI 1640 supplemented with D-glucose (11.1 mmol/liter), L-glutamine (2 mmol/liter), benzylpenicillin (100 U/ml; Roche Diagnostics Scandinavia, Bromma, Sweden), streptomycin (0.1 mg/ml), and 10% (vol/vol) fetal calf serum].

Isolation of islet endothelial cells
Outgrowth of islet stromal cells on a collagen matrix was stimulated using a modification of a previously described protocol (6). Briefly, 20 hand-picked, apparently clean islets were transferred onto a collagen matrix (1.8 mg collagen type 1/ml; Collagen GmbH, Nutacon, Leimuden, The Netherlands) in a 24-well culture dish. Islets were cultured at 37 C (air/CO2, 95:5) in 1 ml endothelial cell culture medium [CM supplemented to 20% (vol/vol) fetal calf serum and with 100 µg endothelial cell growth supplement (Sigma-Aldrich)/ml; hereafter denoted as EcM]. Vascular sprouts growing out from the islets were removed before expanding cells reached confluence.

Cells were detached with 0.25% (wt/vol) trypsin (Invitrogen Life Technologies, Inc., Gaithersburg, MD) for less than 5 min at 37 C. The suspension was washed twice in CM. The endothelial cells were extracted from the cell suspension by a Dynabead (Dynal Biotech, Oslo, Norway) method previously described (7, 11). By the use of Bandeiraea (Griffonia) simplicifolia (BS-1)-coated Dynabeads, endothelial cells are separated from contaminating cells, and a purity of more than 90% is achieved (12).

As an additional control of endothelial cell purity, samples of endothelial cells were trypsinized, spun onto poly-L-lysine glass slides, fixed in 4% (vol/vol) paraformaldehyde for 2 h, and subsequently dehydrated. The slides were stained to detect endothelium with a monoclonal rat anti-CD31 antibody (clone MEC7.46; HyCult Biotechnology, Uden, The Netherlands) using the EnVision+ system (DakoCytomation, Glostrup, Denmark). Antigen retrieval was performed by boiling for 20 min with Target Retrieval Solution (pH 9.0; DakoCytomation) in a microwave oven (750 watts). The slides were counterstained with hematoxylin. After staining, the fraction of CD31-positive cells was counted in a light microscope (x400).

The detailed characterization of the endothelial cells has been described previously (7, 12), but included uptake of 1,1-dioctadecyl-3,3,3,3-tetramethyl indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and positive staining for endothelial nitric oxide synthase, angiotensin-converting enzyme, angiopoietin-2, and BS-1. The morphological appearance of purified cells was evaluated by scanning electron microscopy. We also verified that the endothelial cells migrate toward VEGF-A (data not shown).

Islet-conditioned CM (ICCM)
Islets were isolated, handpicked, and cultured in groups of 150 islets in 5 ml CM for 4 d, as described above, to minimize contamination with exocrine tissue and passenger leukocytes. Groups of 150 islets were thereafter cultured in 5 ml fresh CM for 24 h. This latter medium was centrifuged for 2 min at 2000 rpm to remove cells. The supernatant was collected and is hereafter denoted ICCM.

Endothelium-conditioned medium
Dynabead-purified endothelial cells were cultured for 4 d in EcM as described above. At this time point the endothelial cells were in exponential growth, but had not reached confluence. The wells were washed with CM to remove all EcM. Fresh CM was then added to some of the wells with endothelial cells, whereas ICCM was added to others. Additional wells without endothelial cells were used as controls. Some of the wells containing endothelial cells exposed to ICCM were supplemented with neutralizing VEGF-A antibody (1 µg/ml; NeoMarkers, Fremont, CA) or neutralizing HGF antibody (1 µg/ml; R&D Systems, Minneapolis, MN). Twelve hours later, the medium was collected after centrifugation for 2 min at 2000 rpm to remove cells. The samples were used for analysis of HGF concentration (enzyme immunoassay for rat HGF, Institute of Immunology, Tokyo, Japan) according to the manufacturer’s instructions or were used for the incubation experiments described below. Culture medium was also obtained from wells with endothelial cell-conditioned CM (ECCM) to which no VEGF or HGF antibody had been added, and from wells with islet and endothelial cell-conditioned CM (IECCM), with or without addition of VEGF antibody. A summary and overview of the conditioned media are given in Table 1Go.


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  		TABLE 1. Conditioning of culture media

 
Islet incubation in conditioned medium
One milliliter of CM, ICCM, ECCM, or IECCM was put into wells in 24-well plates. Neutralizing goat anti-HGF-antibody (1 µg/ml; R&D Systems) or rabbit antimouse antibody, as a control antibody (1 µg/ml; DakoCytomation), was added to wells with ECCM or IECCM. To separate wells with CM or IECCM, ovine prolactin (PRL; 500 ng/ml; ProSpec-Tany Technogene, Rehovot, Israel) was added. The concentration of PRL used was chosen to mimic physiological concentrations of the hormone during pregnancy (13). Twenty islets cultured for 5 d in CM were placed in each well. After 6 h of exposure to conditioned medium, [3H]thymidine (10 µCi/ml; Amersham Biosciences, Little Chalfont, UK) was added for an additional 60 min. The islets were then washed repeatedly in ice-cold PBS, fixed in 4% (vol/vol) paraformaldehyde for 2 h, and subsequently dehydrated. The islets were paraffin embedded, sectioned (5 µm), and stained with a monoclonal guinea-pig antiinsulin antibody (ICN Biomedicals, Aurora, OH).

Islet ß-cell proliferation in vitro
Wet slides with sections from isolated islets stained for insulin were dipped in 50% film emulsion (autoradiography emulsion, Eastman Kodak Co., Rochester, NY), in 75 mmol/liter ammonium acetate and kept in a light-proof chamber to dry overnight. The slides were then exposed for 3 wk at 4 C before being developed, fixed, and counterstained with hematoxylin. Tissue sections were evaluated in a light microscope (x400) by an observer (M.J.) unaware of the origin of the samples. All investigated islets were digitally photographed and stored as computer files. ß-Cells with 10 or more black silver grains over their nuclei were considered to be in the S phase of the cell cycle (14). Background thymidine incorporation was, in general, less than three grains per nucleus. The fraction of insulin-positive cells with [3H]thymidine incorporation in their nuclei was counted.

Islet stimulation with ovine PRL in vitro
Groups of 20 islets, freshly isolated from one rat, were transferred to a 24-well plate with 1 ml CM/well (well diameter, 16 mm). Recombinant ovine PRL (500 ng/ml; ProSpec-Tany Technogene) was added to some of the wells. Controls contained CM and islets only. The experiment was performed in duplicate on islets from five animals, and the mean value was calculated. The mean value for islets from one animal was then considered as one experiment in the subsequent statistical analysis. The medium was removed and frozen after 24 h of culture. VEGF-A contents in the medium were determined with ELISA (R&D Systems).

Islet endothelial cell stimulation with VEGF, insulin, and ovine PRL in vitro
Islet-derived endothelial cells were isolated and cultured in 24-well culture dishes as described above. The wells were washed with RPMI 1640 to remove all EcM. Islet-derived endothelial cells were thereafter exposed to serum-free medium [RPMI 1640 supplemented with D-glucose (11.1 mmol/liter), L-glutamine (2 mmol/liter), benzyl penicillin (100 U/ml), and streptomycin (0.1 mg/ml)] for 12 h. Some of the wells were supplemented with different combinations of VEGF-A (3–40 ng/ml; R&D Systems), insulin (1.0–100 µg/ml; Actrapid, Novo Nordisk, Malmo, Sweden), ovine PRL (250 or 500 ng/ml), and rapamycin (10 nmol/liter).

The ICCM that induced HGF expression contained 19.8 ± 4.0 ng VEGF-A/ml (n = 10) and 0.84 ± 0.07 µg insulin/ml (n = 9), as determined by ELISA [ELISAs for VEGF-A and insulin were obtained from R&D Systems and Mercodia (Uppsala, Sweden), respectively]. To mimic the composition of the conditioned medium with respect to insulin and VEGF-A, some of the wells were supplemented with 1.0 µg/ml insulin and 20 ng/ml VEGF-A. We have previously estimated the local insulin concentration within pancreatic islets to be in the range of 1–10 µg/ml (15), assuming a single islet blood flow of 10–15 nl/min (16, 17), that every ß-cell releases 5–15 granules/min (18) with a concentration of 74 mmol/liter in each granule (18), and that the vascular fraction of an islet is approximately 10% (19). Therefore, we also tested whether an insulin concentration of 1–10 µg/ml could induce HGF release in the presence of a low VEGF-A concentration (3 ng/ml). Moreover, the direct effects of ovine PRL (250 or 500 ng/ml), with or without 10 µg/ml insulin, on HGF secretion from islet endothelial cells were assessed.

Next we wanted to estimate the direct effects of ovine PRL on islet endothelial cell number. Islet endothelial cells were cultured in a 24-well plate at a density of 3220 ± 149 cells/well (n = 4). We exposed the cells to CM supplemented with ovine PRL (250 or 500 ng/ml) for 48 h. The cells were trypsinized and counted in a Burker chamber.

Studies in pregnant rats
Virgin female pregnant rats at d 5, 10, 15, 18, or 20 of gestation and d 2 or 7 postpartum were investigated. Blood obtained from the cut tip of the tail at 0800 h on the day of the experiment was used to measure blood glucose concentrations by test reagent strips (Medisense Sverige, Sollentuna, Sweden). The animals were then injected iv with 100 µl [3H]thymidine (0.5 µCi/g body weight; Amersham Biosciences) and were killed 2 h later. The pancreatic glands were carefully dissected free from surrounding tissues, removed, fixed in a 10% (vol/vol) formaldehyde solution, dehydrated, and embedded in paraffin. Sections, 5 µm thick and randomly chosen from all parts of the pancreas, were prepared, mounted on poly-L-lysine glass slides, and stained to detect endothelium with the lectin BS-1 or ß-cells with a monoclonal guinea-pig antiinsulin antibody (ICN Biomedicals), as previously described in detail (20). The staining specificity of BS-1 for endothelial cells was verified by staining pancreatic sections for CD31 as described above.

Islet endothelial, ß-cell, and total endocrine cell proliferation in pregnant rats
The proliferation of endothelial cells, ß-cells, and all endocrine cells was determined in the islets by autoradiography, as described above for the studies of islets in vitro. Also in this case, cells with 10 or more black silver grains over their nuclei were considered to be in the S phase of the cell cycle. An islet area of 0.391 ± 0.026 mm2 (44 ± 2.4 islets, corresponding to 3370 ± 223 ß-cells; n = 53) was investigated in each pancreas, using a computerized system for morphometry (Scion Image, Scion, Inc., Frederick, MD).

To analyze whether the proliferation of endothelial cells was associated with endocrine cell replication, we performed a linear regression analysis of the labeling index (LI) of endocrine and endothelial cells for each animal. Next we wanted to investigate whether endocrine LI was higher in islets in which [3H]thymidine-labeled endothelial cell nuclei were found. We therefore divided the islets from each animal into two groups. The first group consisted of islets in which at least one labeled endothelial cell nucleus was identified in the sections, whereas islets without detectable endothelial cell proliferation were referred to the second group. The means for the LI of endocrine cells in the two groups of islets were calculated for each animal and considered as one observation in the subsequent statistical analysis.

Measurements of islet mass, islet vascular density, and islet vascular mass
Total islet volume was measured by a direct point-counting technique in histological sections stained with hematoxylin (21). The number of intersections overlapping islets was estimated in a light microscope (x200). A total of 10 different fields were viewed in each pancreas (corresponding to ~1200 points). The islet mass was estimated by multiplying pancreatic weight by the islet volume fraction of the whole pancreas, assuming similar densities of the tissues. Indeed, the densities of the exocrine and endocrine pancreas differ by less than 1% (22).

The blood vessel density in the pancreatic islets was determined by a direct point-counting method (21). For this purpose, the number of intersections overlapping islet endothelial cells (stained with BS-1) was estimated (x400). A total of 657 ± 25 intersection points were counted in each pancreas. The blood vessel density was multiplied by the islet mass to obtain the blood vessel mass.

Double fluorescence staining for HGF and endothelium
Deparaffinized slides with 5-µm sections, randomly chosen from all parts of the pancreas, were incubated with neuroaminidase type X at 37 C for 2 h. The sections were washed in Tris-buffered saline (TBS) and thereafter incubated for 1 h with normal goat serum (DakoCytomation) diluted 1:20 with TBS containing 0.1% (wt/vol) BSA. Primary antibodies against HGF (1:20; clone Hyp-T6942, Institute of Immunology Institute of Immunology, Tokyo, Japan) and fluorescein isothiocyanate-labeled BS-1 (1:5) diluted in TBS were applied to the slides overnight at 4 C. The slides were washed (in TBS, three times, for 5 min each time) and incubated for 1 h with a Texas Red-conjugated secondary antibody (clone 111-075-144; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:50 in TBS. The slides were then washed again and mounted with a mounting medium containing 4',6-diamidino-2-phenylindole (Vectashield and DAPI, Vector Laboratories, Inc., Burlingame, CA).

Islet HGF expression
The islets exhibited a heterogeneous cytoplasmic staining pattern for HGF. To estimate the fraction of islets that stained positively for HGF, we used the direct point-counting method described above. The number of intersections overlapping areas that stained positively for HGF was estimated in a blinded fashion (x400). A total of 1310 ± 55 intersection points were counted in each pancreas (n = 5). Sections, stained for HGF and blood vessels, were investigated in a fluorescence microscope (x400) to evaluate the possible localization of HGF to blood vessels. The exocrine pancreas has previously been shown to express much HGF (23) and was used as a positive control.

Thrombospondin-1 (tsp-1) staining
Sections (5 µm), randomly chosen from all parts of the pancreas, were mounted on poly-L-lysine glass slides and stained for tsp-1 with the monoclonal mouse anti-tsp-1 antibody A6.1 (Lab Vision Corp., Fremont, CA), according to the manufacturer’s instructions. Antigen retrieval was performed by boiling for 20 min with citrate buffer (10 mmol/liter, pH 6.0; Lab Vision Corp.) in a microwave oven (750 watts). Negative control slides were incubated with normal rabbit serum instead of primary antibodies.

Tsp-1 expression in endocrine cells
Tsp-1-stained sections were examined in a blinded fashion with a light microscope (x400). The presence of tsp-1 staining was ranked by a dichotomic arbitrary scale: 0 indicated islets with very weak or unidentifiable tsp-1 staining in the cytoplasm of endocrine cells, whereas + denoted clearly tsp-1-positive islets. A total of 29.2 ± 2.5 islets were examined in each animal (three to seven in each group).

Statistical analysis
All values are given as the mean ± SEM. Multiple comparisons of parametric values with control values were performed by ANOVA with Dunnett’s post hoc test. Multiple comparisons of nonparametric values (such as fractions) were performed by nonparametric ANOVA and Dunn’s post hoc test. For comparisons of morphology score, or when only two groups were compared, Wilcoxon sign-rank test was used. Coefficients of correlation were obtained by simple linear regression, followed by evaluation of statistical significances of correlation by ANOVA (Sigmastat SSPS Science Software, Erfart, Germany). For all comparisons, a probability of chance differences of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purity of endothelial cell preparations
Staining for the endothelium-specific marker CD31 (Fig. 1AGo) was used to confirm the purity of the endothelial cells purified by BS-1-coated Dynabeads. Evaluation showed that our endothelial cell cultures contained 91 ± 4% CD31-positive cells (n = 4).


Figure 1
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  		FIG. 1. In vitro studies of endothelial cells and ß-cells. A, Micrograph of cells obtained from our endothelial cell cultures. The cells stain positively for CD31 (brown) and bind to BS-1-coated Dynabeads (arrow). Magnification, x1000. B, Effect of conditioned medium on the ß-cell LI. C, HGF release from purified islet-derived endothelial cells in vitro in response to different culture media. The detection limit of the assay (0.1 ng/ml) is indicated by the broken horizontal line. Values are expressed as the mean ± SEM for four to eight experiments. Multiple comparisons in B were performed with ANOVA on ranks and Dunn’s post hoc test. Multiple comparisons in C were performed with one-way ANOVA and Dunnett’s post hoc test. *, P < 0.05 vs. CM; #, P < 0.05 vs. IECCM.

 
Conditioned medium in vitro
IECCM, but not fresh CM, ICCM, or ECCM, enhanced ß-cell proliferation in rat islets in vitro (Fig. 1BGo). This proliferative response was prevented by the addition of a HGF-neutralizing antibody (Fig. 1BGo).

HGF was not possible to detect in CM or ECCM (data not shown), whereas ICCM contained low HGF concentrations (Fig. 1CGo). When purified endothelial cells were exposed to ICCM, thereby producing IECCM, they released HGF. This release was prevented when a neutralizing VEGF antibody was added to the ICCM before exposing endothelial cells to the medium (Fig. 1CGo). In contrast, when added to the IECCM after the endothelial cells had been exposed to ICCM, the VEGF antibody neither affected HGF secretion nor ß-cell proliferation (data not shown). Thus, the results were not due to direct effects by the neutralizing VEGF antibody. Neither the VEGF antibody nor the HGF antibody seemed to have any effects per se on isolated islets. The addition of either antibody to incubated islets did not alter basal ß-cell proliferation or insulin accumulation in the medium.

PRL, VEGF, and insulin supplementation in vitro
Addition of ovine PRL to CM increased ß-cell proliferation in rat islets in vitro approximately 2.5-fold (Fig. 2AGo). ß-Cell proliferation was further enhanced when ovine PRL was added to IECCM. In serum-free medium, i.e. RPMI 1640 (supplemented with L-glutamine and benzyl penicillin), neither VEGF-A (3–40 ng/ml) nor insulin (1–100 µg/ml) alone induced HGF expression in islet endothelial cells (Fig. 2BGo). Combinations of insulin and VEGF could, however, induce HGF expression in these endothelial cells (Fig. 2BGo). At higher insulin concentrations, a lower concentration of VEGF-A was needed to induce HGF expression. This stimulation was abrogated by the addition of rapamycin. Ovine PRL alone or in combination with insulin did not stimulate HGF production from islet endothelial cells. In vitro incubation of islets for 24 h with ovine PRL increased VEGF-A release to the culture medium by approximately 30% when paired comparisons were made (Fig. 2CGo), corresponding to absolute values of 7.4 ± 1.4 and 9.5 ± 1.2 ng/ml, respectively (n = 5). Ovine PRL also provided growth stimuli for the islet endothelial cell mass (Fig. 2DGo).


Figure 2
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  		FIG. 2. Effects of different growth factors on islets and islet endothelial cells. A, Effect of ovine PRL (oPRL) on the ß-cell LI in the absence or presence of IECCM. B, Effects of insulin, VEGF-A, oPRL, and rapamycin in RPMI 1640 supplemented with L-glutamine on islet endothelial secretion of HGF. C, Influence of oPRL on islet VEGF production. Freshly isolated islets were cultured in CM or CM supplemented with oPRL for 24 h. D, Effect of oPRL in CM on endothelial cell number. Values are expressed as the mean ± SEM for four to 10 experiments. Multiple comparisons in A and D were performed with ANOVA on ranks and Dunn’s post hoc test. Multiple comparisons in B were performed with one-way ANOVA and Dunnett’s post hoc test. The paired comparison in C was performed with Wilcoxon sign-rank test. * P < 0.05 vs. control.

 
Characteristics of pregnant animals
The animals allocated to the in vivo experiments during pregnancy weighed 228–336 g (Table 2Go). The pancreatic weights of all pregnant animals were similar and did not differ from those of virgin rats, but increased during lactation (Table 2Go). The blood glucose concentrations increased in early pregnancy (d 5), but decreased during late pregnancy (d 15–20; Table 2Go). After birth, the blood glucose concentrations were similar to those of nonpregnant rats.


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  		TABLE 2. Characteristics of the animals allocated to the in vivo studies

 
Endocrine cells in vivo
Both islet ß-cell proliferation (Fig. 3AGo) and total endocrine cell proliferation (data not shown) were significantly increased on d 15 of pregnancy. At all other time points, the ß-cell LI was similar to that in control animals (Fig. 3AGo). ß-Cells accounted for the majority of the proliferating endocrine cells on days 15 and 18 [82 ± 2% (n = 7) and 81 ± 3% (n = 6)]. In parallel, there was an increased islet mass on day 15 of pregnancy, which then normalized during late pregnancy and the early lactation period (Fig. 3BGo).


Figure 3
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  		FIG. 3. Growth of ß-cells during pregnancy and the first week postpartum. A, LI of ß-cells during pregnancy and postpartum. B, Measurement of total islet mass in pancreas. Values are expressed as the mean ± SEM for five to eight animals in each group. Multiple comparisons in A were performed with ANOVA on Ranks and Dunn’s post hoc test. In B, multiple comparisons were performed with one-way ANOVA and Dunnett’s post hoc test. *, P < 0.05 vs. d 0 of pregnancy. The broken vertical line denotes the separation between pre- and postpartum values.

 
Endothelial cells in vivo
Islet endothelial cell proliferation rates were increased on d 10, 15, 18, and 20 of pregnancy compared with those of virgin rats, but normalized immediately after delivery (Fig. 4AGo). Endothelial cell proliferation was associated with an increase in islet blood vessel density on d 15, 18 and 20 of pregnancy (Fig. 4BGo). The increased vascular densities, however, were only reflected in an increased blood vessel mass on d 15 and 18 of pregnancy (Fig. 4CGo).


Figure 4
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  		FIG. 4. Islet endothelial cells during pregnancy and the first week postpartum. A, LI of endothelial cells. B, Islet blood vessel density. C, Islet blood vessel mass. D, Linear regression analysis between endothelial and endocrine cell proliferation (r = 0.704; P < 0.001; n = 52 animals). The identification of individual points according to the day after conception is given in the right panel. E, Dichotomic analysis of whether demonstrable endothelial cell proliferation is coupled to endocrine cell proliferation. Values are expressed as the mean ± SEM for 36 animals. F, Microvascular endothelium, stained with BS-1 (red) in an islet on d 15 of pregnancy. Nuclei with film grains denote endothelial cell (arrowhead) and endocrine cell (arrow) that have incorporated [3H]thymidine. Multiple comparisons in A and B were performed with ANOVA on ranks and Dunn’s post hoc test. In C, multiple comparisons were performed with one-way ANOVA and Dunnett’s post hoc test. Data in D were evaluated by simple linear regression. In E, the comparison was performed with Wilcoxon’s sign-rank test. *, P < 0.05 vs. day 0 of pregnancy; #, P = 0.002 vs. LI of endocrine cells in islets without labeled endothelial cells in the section. Values in A–C are expressed as the mean ± SEM for five to eight animals in each group. The broken vertical line denotes the separation between pre- and postpartum values.

 
Correlation between endothelial cell and endocrine cell proliferation
Endothelial cell LI correlated to the degree of endocrine cell LI (Fig. 4DGo; y = 0.0889 + 0.151x; r = 0.704; P < 0.001; n = 52 animals). Furthermore, when individual islets were investigated, we found that islets with established endothelial cell proliferation had greater endocrine cell proliferation than other islets (Fig. 4Go, E and F). However, at late gestation (d 18 and 20), ß-cell proliferation decreased, whereas endothelial cell proliferation remained high.

Tsp-1
Tsp-1 was abundantly present in the cytoplasm of endocrine islet cells of virgin, pregnant, and lactating rats. In virgin rats, approximately 90% of the islets were tsp-1 positive (Fig. 5AGo). This percentage of islets transiently decreased to between 40–55% on d 10 and 15 of pregnancy (Fig. 5Go, A–C). On d 18 and 20 of pregnancy, both the exocrine and the endocrine pancreas exhibited very strong staining intensity for tsp-1, whereas the ductal structures remained negative (Fig. 5DGo).


Figure 5
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  		FIG. 5. Tsp-1 expression in islets during pregnancy. A, Percentage of tsp-1-positive islets during pregnancy. Values are expressed as the mean ± SEM for three to seven animals in each group. Multiple comparisons were performed with ANOVA on ranks and Dunn’s post hoc test. *, P < 0.05 vs. d 0 of pregnancy. The broken vertical line denotes birth. B, Histological section of pancreas on d 15 of pregnancy stained for the presence of tsp-1 (brown). Two tsp-1-negative islets are depicted (arrows; x400). C, Histological section of the same pancreas as that in B showing a tsp-1-positive islet (magnification, x400). D, Histological section of pancreas on d 18 of pregnancy stained for the presence of tsp-1 (magnification, x400).

 
HGF
In virgin rats and during early and late gestation (data not shown), islet HGF staining was absent or very weak. During midgestation, i.e. on d 15 of pregnancy, the intensity of staining for HGF peaked. We developed a fluorescence staining for islet microvasculature (Fig. 6AGo) and HGF (Fig. 6BGo) to evaluate whether endothelial cells expressed HGF during pregnancy. We found a colocalization of HGF and our endothelial cell marker BS-1 in a subpopulation of islets on d 15 of pregnancy (Fig. 6Go, C–F). HGF showed the strongest staining intensity in BS-1-positive cells. Also some BS-1-negative islet cells displayed weak cytoplasmic staining for HGF. In virgin rats, HGF rarely localized to BS-1-positive cells (data not shown).


Figure 6
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  		FIG. 6. Islet expression of HGF during pregnancy. A, Islet endothelium visualized with fluorescein isothiocyanate-conjugated BS-1 (green). B, Immunohistochemical visualization of the HGF staining pattern (red) using a Texas Red-conjugated secondary antibody. C, Double-fluorescence staining of blood vessel endothelium (green) and HGF (red). The yellow staining denotes colocalization of HGF and fluorescein isothiocyanate-conjugated BS-1. D, Same as C, with the addition of cell nuclei, visualized with 4',6-diamidino-2-phenylindole (blue). E, Islet endothelium visualized with FITC-BS-1 (green). F, Fluorescence staining of islet endothelium (green), HGF (red), and 4',6-diamidino-2-phenylindole (blue) similar to that in D, but shown at a higher magnification. The yellow staining in C, D, and F denotes colocalization of HGF and FITC-BS-1. The white line in A–D denotes the border between islet and acinar tissue. The exocrine pancreas is known to express HGF and was used as a positive control. Magnification: A–D, x400; E and F, x1000.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The endothelium has previously been considered to constitute a rather inert cell type. However, the importance of endothelial cells for vascular function has become more appreciated in the last decades. Recently, there has been an accumulating list of growth factors produced by endothelial cells that show tissue-specific characteristics. The present study tested the hypothesis that proliferating islet endothelial cells have the capacity to produce substances that can stimulate ß-cell proliferation. Indeed, isolated and purified proliferating islet endothelial cells exposed to medium from cultured islets were able to induce a marked increase in the ß-cell mitotic index.

In a recent study of livers, it was shown that endothelial cells can stimulate the growth of adjacent epithelial cells. In that study HGF expression was shown to be induced in the liver sinusoidal cells and cause proliferation of hepatocytes (5). Rat islets express both HGF and its receptor, c-met (24), and HGF is known to stimulate ß-cell proliferation and insulin content and to increase insulin release and islet mass in rodent tissues (25). Furthermore, transgenic mice expressing HGF under the insulin promoter have increased ß-cell proliferation and ß-cell mass (26). Recently, two independent groups investigated the effects of ß-cell specific ablation of c-met expression (27, 28). They confirmed that HGF has an essential role in normal glucose homeostasis. These combined observations led us to investigate whether HGF expression was induced in our in vitro system. We found that purified islet endothelial cells produced HGF in response to ICCM, but not in response to fresh CM, suggesting that factors released from islets participate in this stimulation. The concentrations of HGF in our in vitro study were similar to those previously shown to induce ß-cell proliferation in vivo (29), and we were able to prevent the ß-cell mitogenic response by the addition of neutralizing HGF antibodies to the conditioned culture medium.

Next we wanted to investigate the nature of the soluble islet-derived factors that induced HGF secretion from proliferating islet endothelial cells. In the liver, VEGF-A released by hepatocytes induces HGF expression in sinusoidal endothelial cells through paracrine effects (5). It should be noted that VEGF-A is constitutively expressed in pancreatic islet ß-cells (24, 30), and the islet microvasculature constitutively expresses the main receptors for VEGF-A, namely, VEGFR1 and VEGFR2 (31, 32, 33). Furthermore, VEGF-A stimulates the formation of new blood vessels in the islets during fetal development (19, 30), after transplantation of isolated pancreatic islets (34), and during islet carcinogenesis (30). In our experiments, immunoneutralization of VEGF inhibited endothelial cell HGF production induced by ICCM. Recombinant rat VEGF-A, however, did not induce HGF secretion from islet endothelial cells when given alone. This indicates that VEGF-A is a necessary component, but is not sufficient to stimulate HGF release from proliferating islet endothelium.

Insulin receptors and IGF-I receptors are expressed on endothelial cells and have been shown to potentiate the action of VEGF, at least in retinal endothelial cells (35, 36). We investigated whether insulin was involved in the stimulation of HGF secretion. Under serum-free conditions, insulin alone did not stimulate endothelial cell HGF production. A combination of VEGF-A and insulin, however, did stimulate islet endothelial cell HGF production. This response was eliminated by rapamycin, an inhibitor of mammalian target of rapamycin. This pathway has previously been shown to be involved in HGF synthesis (37, 38). We speculate that increasing local levels of insulin, in the presence of VEGF, stimulate the release of HGF from neighboring endothelial cells through either insulin or IGF-I receptors (Fig. 7Go). The increase in local insulin levels may be the result of increasing insulin resistance or a hormone-induced lowering of the insulin release threshold.


Figure 7
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  		FIG. 7. Schematic figure depicting the suggested paracrine interaction between islet endothelial cells and ß-cells during pregnancy. First, the pregnancy hormone PRL induces insulin and VEGF-A expression in ß-cells. This results in increased local levels of these growth factors, because islet blood perfusion is not increased during pregnancy. Second, endothelial cells react to these alterations in the microenvironment by secreting the ß-cell mitogen HGF.

 
Islet mass is relatively constant during adult life. It can, however, increase in response to an augmented demand, resulting from obesity, pregnancy or insulin resistance (2). The effects of pregnancy have been thoroughly investigated in rodents. The functional and morphological adaptations are highly reproducible, and this allows for comparisons between animals (9, 39, 40). It has previously been convincingly shown that placental lactogens and PRL mediate the increased islet mass and ß-cell proliferation seen during pregnancy (9, 41, 42), and that rat placental lactogen-1 and -2 and PRL mediate their effects solely through PRL receptors (43). However, PRL seems to directly stimulate not only ß-cell growth (44), but also an expansion of the islet endothelial cell mass. PRL increases insulin secretion in vitro and in vivo (45, 46), and we found increased VEGF production in vitro in response to PRL. VEGF and insulin can, as mentioned above, induce islet endothelial cell HGF production. However, PRL did not induce HGF release in isolated islet endothelial cells. Therefore, we believe the possible effects of PRL in HGF stimulation to be due to an increase in local insulin and VEGF, rather than a direct effect on the endothelial cells. It is noteworthy that IECCM containing HGF added to the direct stimulatory effects of PRL on ß-cell proliferation. We therefore decided to investigate possible interactions between endothelial and endocrine cells during pregnancy.

During pregnancy, we found that ß-cell and total endocrine cell proliferation were increased on d 15 and 18 of pregnancy. This is in line with previous reports (9). Islet endothelial cell proliferation, however, increased before that time point, viz. beginning on d 10. The blood vessel density of the pancreatic islets was increased on d 15, 18, and 20 of pregnancy. That is, blood vessel density was increased when islet mass increased. It can be argued that the expanding microvasculature paved the way for an increased islet blood perfusion to improve the oxygen and nutrient supply in the expanding endocrine cell mass. However, we have recently found that islet blood flow is not increased during pregnancy (47). Thus, it does not seem as if more blood vessels are associated with increased blood perfusion per se during these conditions. It is noteworthy that increased vascular density has been reported in several experimental models with islet hyperplasia, e.g. in insulin knockout fetuses (48) and models of islet carcinogenesis (49).

Despite an increased endothelial cell LI throughout the second half of pregnancy, islet vascular mass increased only between d 10 and 15 of pregnancy. This observation made us to investigate the possible involvement of tsp-1. Tsp-1 is a potent angiogenesis inhibitor that exerts its function in vivo by inducing apoptosis in activated endothelial cells rather than by inhibiting endothelial cell proliferation (50, 51). Furthermore, tsp-1–/– mice have increased islet vascular density and islet hyperplasia (52). We stained for tsp-1 on different days of pregnancy and found that the expression was decreased on d 10 and 15, i.e. at the time when islet blood vessel mass increased most markedly. On d 18 and 20, there was intense staining for tsp-1 in both endocrine and exocrine pancreas, providing a possible explanation for the increase in islet endothelial cell turnover during late gestation.

We found a strong correlation between the proliferation of endothelial and endocrine cells during pregnancy. We consistently found greater endocrine cell proliferation in islets with proliferating endothelial cells. Our in vitro findings suggested that proliferating islet endothelial cells can produce HGF. The pregnancy hormone PRL increases islet insulin and VEGF secretion. This, together with an unaltered blood perfusion (47), would increase the local levels of these growth factors and possibly induce HGF expression in islet endothelial cells. We therefore wanted to investigate whether islet endothelial cells produced HGF in vivo. In virgin rats, only occasional islet endothelial cells expressed HGF, and this was also true for pancreata obtained at the other studied time points during pregnancy. On d 15, in contrast, we observed a predominant expression of this factor in islet endothelium, i.e. coinciding with the peak of ß-cell proliferation. The reason for decreased ß-cell proliferation during late gestation despite continuous endothelial cell proliferation is obscure. HGF has previously been located to other islet cells, predominantly glucagon-producing cells, in both humans and rats (53). In accordance with this, we also noted some weak staining pattern in islet cells other than the endothelium. The nature of these cells was not elucidated in the present study.

In conclusion, purified proliferating islet endothelial cells were shown to secrete HGF in response to insulin and VEGF-A, thereby stimulating ß-cell proliferation. In pregnant rats, the proliferation of islet endothelial cells preceded and was closely associated with proliferating endocrine cells. The increase in blood vessel mass was associated with a decrease in islet tsp-1 expression, and the increase in islet mass coincided with islet endothelial cell expression of HGF. These findings suggest the existence of an endothelial-endocrine cell axis that is involved in cell proliferation within adult pancreatic islets.


   Acknowledgments
 
Birgitta Bodin, Ing-Marie Mörsare, Astrid Nordin, Lisbeth Sagulin, and Eva Törnelius are gratefully acknowledged for their skilled technical assistance.


   Footnotes
 
This work was supported by grants from the Swedish Research Council (17X-109 and 72XD-15043) and the Juvenile Diabetes Research Foundation, a European Foundation for the Study of Diabetes/Novo Nordisk Research Grant, the Swedish Diabetes Association, the Novo Nordic Fund, Svenska Barndiabetesfonden, Bergvalls Stiftelse, Thurings Stiftelse, Wibergs Stiftelse, Anérs Stiftelse, Clas Groschinskys Minnesfond, and the Familjen Ernfors Fond.

A.A., P.-O.C., L.J., M.J., and G.M. have nothing to declare.

First Published Online January 26, 2006

Abbreviations: BS-1, Bandeiraea (Griffonia) simplicifolia; CM, islet culture medium; ECCM, endothelial cell-conditioned islet culture medium; EcM, endothelial cell culture medium; FITC, fluorescein isothiocyanate; HGF, hepatocyte growth factor; ICCM, islet cell-conditioned culture medium; IECCM, islet and endothelial cell-conditioned CM; LI, labeling index; PRL, prolactin; TBS, Tris-buffered saline; tsp-1, thrombospondin-1; VEGF-A, vascular endothelial growth factor-A.

Received August 5, 2005.

Accepted for publication January 18, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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