Correspondence to: Daniel R. Salomon, Department of Molecular and Experimental Medicine MEM55, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. E-mail: dsalomon@scripps.edu
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Abstract |
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Cellcell and cellmatrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that vß3 and
vß5, two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of
vß3 and
vß5 integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins
vß3 and
vß5 and their ligands to morphogenetic events in the human endocrine pancreas.
Key Words:
pancreatic islets, endocrine progenitors, vß3, integrins, cell migration
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Introduction |
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Interactions of cells with their environment play a critical role in directing embryonic development and in maintaining tissue integrity in adult life. The structure/function paradigm dictates that the architectural elements that determine tissue structure also affect tissue function. In this context, the integrin family of adhesion receptors regulates many of the cellular interactions with the extracellular matrix (ECM),1 which provide critical elements of three-dimensional information for tissue organization. Integrins are remarkably multifunctional since they mediate not only adherence to specific ECM proteins, but also regulate the migration and spatial segregation of different cell types within tissues (
A distinctive feature of developing epithelia is the cell typespecific pattern of cell adhesion receptors whose expression and function is quantitatively and temporally regulated in a very precise manner. Integrins vß3 and
vß5 are among those receptors that contribute to the coordinated cell adhesion and movement of diverse cell types on various ECM components including vitronectin (VN), fibronectin (FN), and collagen IV (Coll-IV) (
vß3 that are downregulated upon cell differentiation (
vß5 has been found to be dynamically regulated in migrating versus stationary epithelial cells (
Cell growth and differentiation of endocrine progenitors in the pancreas provides a model of programmed epithelial morphogenesis. It is currently thought that pancreatic endocrine cells originate from a subpopulation of undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas (
Here we report the novel observation that pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium of the developing human pancreas are characterized by the expression of high levels of vß3 and
vß5 integrins. The expression and function of
vß3 and
vß5 integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of undifferentiated pancreatic epithelial cells. Using an in vivo model of human fetal islet development, we demonstrate that inhibiting the function of the
vß3 and
vß5 integrins results in a dramatic perturbation of islet cell emergence from the ductal epithelium. Thus, these data provide the first evidence for the contribution of these two integrins to morphogenetic events in the human pancreas.
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Materials and Methods |
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Tissues
Mid-gestation human fetal pancreata ranging from 18 to 20 wk of gestational age were obtained through a nonprofit organ procurement program (Advanced Bioscience Resources), which also obtained consent for tissue donation. Samples of human adult pancreas and pancreatic islets were prepared at The Diabetes Research Institute (University of Florida, Miami, FL) as previously described (
Immune Fluorescence, Confocal Microscopy, and Morphometric Analysis
Triple immune fluorescent labeling was performed on 8-µm-thick cryostat sections prepared from snap-frozen fetal pancreata (1820 wk of gestation), as previously described (vß3 (clone LM609; kind gift of Dr. David Cheresh, The Scripps Research Institute), mouse anti-
vß5 (clone 15F11; kind gift of Dr. Ingrid Stuiver, The Scripps Research Institute), and anti-ß1 (clone mAb 13; kind gift of Dr. Steve Akiyama, National Institute of Dental Research, Bethesda, MD). Anti-ECM antibodies were antiColl-IV, anti-VN, and anti-FN (all from GIBCO BRL). Goat antiplatelet endothelial cell adhesion molecule (PECAM-1; Santa Cruz Biotechnology, Inc.). All fluorophore-labeled secondary antibodies were used as F(ab)2 fractions and were from Jackson Immunoresearch Laboratories: lissamine rhodamine (LRSC)conjugated affinity-purified donkey antisheep IgGs (5 µg/ml; preadsorbed on chicken, guinea pig, hamster, horse, human, mouse, rabbit, and rat serum proteins); LRSC-conjugated affinity-purified donkey antigoat IgGs (5 µg/ml; preadsorbed on chicken, guinea pig, hamster, horse, human, mouse, rabbit, sheep, and rat serum proteins); FITC-conjugated affinity-purified donkey anti-rabbit IgGs (5 µg/ml; preadsorbed on bovine, chicken, goat, guinea pig, hamster, horse, human, mouse, rat and sheep serum proteins); indodicarbocyanine (Cy5)-conjugated affinity-purified donkey antimouse IgGs (5 µg/ml; preadsorbed on bovine, chicken, goat, guinea pig, hamster, horse, human, rabbit, rat and sheep serum proteins). In all experiments, separate sections were incubated with a mixture of normal sheep (or goat), rabbit, and mouse IgGs, followed by incubations with appropriate fluorophore-labeled secondary antibodies to be used as control reference for specificity of primary antibodies.
After extensive washes, sections were mounted in slow fade medium (Molecular Probes), and viewed on a Zeiss Axiovert 35M microscope equipped with a laser scanning confocal attachment (MRC-1024; Bio-Rad Laboratories), using an oil immersion x40 1.3 NA objective lens. Fluorescent images relative to each marker were collected by using the 488, 568, and 647 nm excitation lines from an argon/krypton mixed gas laser. Color composite images were generated using Adobe Photoshop 5.5 (Adobe Systems Inc.) running on a UMAX SuperMac S910/250 computer (UMAX Computer Corporation), and printed with a Fujix Pictography 3000 color printer (Fuji Photo Film U.S.A., Inc.).
Microscopic fields acquired from the fetal pancreatic sections (n = 50) immune stained for vß3 and
vß5 were analyzed for pixel intensity of
vß3- and
vß5-specific immune reactivity using the NIH Image software (National Institutes of Health, Bethesda, MD). Pixels' intensity units (0255) were recorded from a total of 250 domains of cellcell and/or cellmatrix contact for each cell type (ductal, endocrine, epithelial undifferentiated, and stromal).
Generation of Islet-like Cell Clusters and Cell Monolayers
Human fetal pancreata at 1820 wk of gestation were minced in small pieces and digested in a collagenase solution (6 mg/ml/HBSS; Collagenase-P; Boehringer) for 15 min at 37°C in a shaking water bath (150 cycles/min) as previously described (5% endocrine cells (mainly insulin- and glucagon-producing cells;
Adhesion Assay
We used a micro-adhesion assay method to optimize the use of limited tissue samples using 60-position micro templates. Each well has a volume of 20 µl and requires only 2,000 cells (
PCR
10 ng of total RNA from either fetal or adult human islets were reverse transcribed into cDNA. Then 2.5 and 5 µl of cDNA were used for each PCR reaction using either v-specific or cyclophilin-specific primes. Each of the cDNA samples was amplified 20 cycles with the same 3' primer (DS54, sense: atggcttttccgccgcggcgacggctg) and a 5' primer (DS60, anti-sense: gcactatcagcagtaaagccttgg) located upstream of the signal sequence (total amplified fragment, 3,259 bp). Nested PCR reactions were then performed with a series of
v-specific primer pairs amplifying sequences from 800 to 1,200 bp in length located in different regions of the
v sequence (DS57, sense: gccttcaacctagacgtggacagtc; DS58, anti-sense: tgcagccatctgctcgccagt); GenBank title: HUMVTNR; sequence data are available from EMBL/GenBank/DDBJ under accession no.
M14648. PCR conditions were as follows: 5 min at 94°C for hot start, followed by up to 35 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 4 min, with a final extension of 10 min at 72°C. The control was amplification of cyclophilin. To estimate the linear range of the nested reactions, we analyzed the PCR products at 10, 15, 20, 25, 30, and 35 cycles.
Transplantation Model
Explants of early gestational age human fetal pancreas are known to continue their differentiation program when transplanted under the kidney capsule of immune incompetent mice (vß3 and
vß5 integrins in the emergence and migration of endocrine cells from the pancreatic ductal epithelium. Fragments (35 mm) of 15-wk-old human fetal pancreas were transplanted under the kidney capsule of four NODlt/szSCID mice. 2 wk after transplantation (to allow time for the engraftment and revascularization of these tissue grafts), two of the mice were implanted subcutaneously with osmotic pumps loaded with the integrin-blocking "27O" arginine, glycine, aspartic acid (RGD) peptide analogue, and the other two mice were implanted with pumps loaded with a control peptide analogue, "39M". The RGD peptide analogue 27O [sequence: AcPen(OmeY) ARGDN(Tic)CNH2] and the control peptide 39M [sequence: GKGESP] were provided by Integra Life Sciences. Peptide 27O and the control peptide 39M were supplied in 50% DMSO/water at a concentration of 11.2 mM and 34.3 mM, respectively. Miniosmotic pumps (model 2001; Alza Pharmaceuticals; mean delivery rate 1 µl/h x 7 d) were loaded with 200 µl of peptides at a concentration of 11 µM. The osmotic pumps were changed weekly thereafter for 12 consecutive weeks. At the completion of the experiments the grafts were removed from the two groups of animals and analyzed by immune fluorescence staining for the four islet hormones. Pumps were checked at the time of explantation and consistently found empty by visual inspection.
Morphometric Analysis of Pancreatic Grafts
A total of 275 slides per graft were stained by immune fluorescence for the four islet hormones and analyzed at a confocal microscope for the identification of islet cell types, their architectural organization, and their relationship with ductal structures. Images acquired for each specific immune reactivity were then analyzed for measurements of each islet cell typespecific surface area using Image-Pro Plus software (Media Cybernetics). Islet cells were considered "ductal associated" when directly adjacent to the ductal epithelium, or clustered and contiguous with ducts within a range of 30 µm.
Statistics
Where applicable, data were analyzed using Stat View 4.01 software (Abacus Concepts, Inc.) for determination of mean, standard deviation, and parametric statistics (paired t test).
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Results |
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Ductal Cells in the Developing Human Pancreas Express High Levels of vß3 and
vß5 Integrin Receptors
In humans, the development of islets of Langerhans occurs during fetal life, whereas growth and expansion of the exocrine tissue is a perinatal event (
Little is known about the mechanisms that regulate emergence and migration of endocrine progenitor cells from the ductal epithelium into the surrounding mesenchyme. Based on the known roles of integrin receptors in the regulation of cell interactions with the extracellular environment during development, we performed a series of studies using confocal microscopy on human fetal pancreata (1620 wk of gestation), to identify the integrin receptors highlighting the ductal epithelium and/or endocrine cells emerging from it. We found that a unique pattern of expression characterizes both vß3 and
vß5 integrin receptors: high in ductal cells and decreased in developing islet cell clusters.
As shown in Fig 1, vß3-specific immune reactivity (green) highlights the pancreatic ductal epithelium and clusters of cells branching from the ducts (A; arrows). The expression pattern of
vß3 is restricted to regions of cellcell and cellmatrix contacts. Most cell clusters budding from the ductal epithelium are contiguous with (or adjacent to) developing islets as identified by the insulin- (red) and glucagon-specific (blue) immune reactivity (D; arrowheads). We consistently observed that the brightness of
vß3-specific immune reactivity (green) is decreased in these developing endocrine cells when compared with the levels of
vß3 expression in ductal cells, including ductal cells expressing insulin and/or glucagon. Consistent with the current view that endocrine cells arise from precursors present within the ductal epithelium (
vß3 during early events determining migration from the ductal epithelium into the surrounding mesenchyme. Our studies further indicate that, after differentiation and clustering of endocrine cells into the highly organized islets of Langerhans, the expression of
vß3 is downregulated. Consistent with this observation, we found that expression of the
v transcripts is decreased in adult islet cells as compared with fetal islet cells. Thus, PCR analysis of
v-specific transcripts detected in the linear range of amplification demonstrated that although barely detectable levels of
v transcripts are observed after 10 cycles of PCR on mRNA of human adult islets (Fig 2, lane 4), significantly higher levels of
v-specific transcripts can be readily amplified from samples of fetal islets (Fig 2, lane 5). This quantitative difference becomes more evident after 15 cycles of PCR (compare lanes 9 and 10). Cyclophilin-specific cDNA amplified from both fetal and adult islets mRNA samples served as an intra-assay control reference cDNA at all cycles (Fig 2, lanes 7, 8, 12, and 13). Flow cytometric studies validated the results of this transcriptional analysis by demonstrating that lower levels of both
vß3 and
vß5 integrin proteins are expressed at the cell surface of human adult islet cells as compared with fetal cells (data not shown). This pattern suggests that the expression of these two integrins is developmentally regulated during islet ontogeny.
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Previous studies on vß3 expression in models of angiogenesis occurring during wound healing or tumor formation demonstrated that this integrin is significantly upregulated in newly formed vessels (
vß3 is involved in various forms of angiogenesis. In contrast to this prediction, and despite the rich vascular network evident within and around islet clusters (see Fig 4 and Fig 5), we did not observe significant expression of
vß3 in endothelial cells colonizing fetal islets (data not shown). This result suggests that there may be cell-, tissue-, or developmental stage-specific differences in the mechanisms regulating
vß3 expression during blood vessel formation.
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Analysis of vß5 expression in similar preparations of human fetal pancreata revealed a pattern similar to that observed for
vß3, with the stronger immune reactivity in ductal cells, and in islet cells emerging from the ductal epithelium (Fig 3 D; arrowheads). Similar to
vß3, the intensity of
vß5-specific immune reactivity decreased in endocrine cells forming larger and more organized islet-like cell clusters.
Quantitative Analysis of vß3 and
vß5 Expression in the Fetal and Adult Human Pancreas
To quantitatively evaluate the levels of vß3 and
vß5 expression in specific cell types, confocal images from human fetal pancreata were studied by computer-aided morphometric analysis. Table 1 shows the pixel intensities of
vß3- and
vß5-specific fluorescence recorded in ductal, insulin- and glucagon-positive cells, as well as in undifferentiated epithelial cells (e.g., islet hormone-negative and amylase-negative). Ductal cells, including isolated insulin- and glucagon-positive cells within or immediately adjacent to ducts, displayed the highest mean pixel intensity of
vß3-specific immune reactivity (173.9 ± 25.1). In contrast, insulin-positive cells associated with developing islet-like cell clusters displayed a significantly lower pixel intensity (93.1 ± 20.5; P < 0.001). Conversely, glucagon-positive cells in these clusters showed pixel intensity closer to that measured in the ductal cells (140.2 ± 25.4). This observation suggests that glucagon cells, recently proposed to derive from a cell lineage independent from that of insulin-producing cells (
vß5 revealed that although ductal cells display the highest values of pixel intensities (i.e., 205.7 ± 33.7), both glucagon- and insulin-positive cells located in developing islets showed statistically significant lower values (100.1 ± 22.9 and 95.3 ± 18.2, respectively). The stromal cells located in the interstitium are essentially negative.
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Identification of ECM Proteins in the Developing Human Pancreas
FN, VN, and Coll-IV are important ECM proteins with known adhesive sites for the integrins vß3 and
vß5. It has also been reported that
vß3 expressed by endothelial cells can bind and present MMP-2, an enzyme capable of processing collagen (
Fig 5 shows representative microscopic fields acquired from fetal pancreatic tissue immune stained for (A and B) VN (green), insulin (red), and glucagon (blue), and for (C) VN (green), PECAM-1 (red), and insulin (blue). VN-specific immune fluorescence highlights groups of cells that are adjacent to, or emerging from, the ductal epithelium (A and C; arrows). Some of these VN-positive cells coexpress glucagon and/or insulin (A and B; arrowheads), and appear to cluster with developing pancreatic islets (B; arrowhead). It is interesting that VN-specific staining is largely cellular rather than in basal membranes. Moreover, larger islet-like cell clusters contain few or no VN-positive cells. These observations suggest that endocrine progenitors may arise from a larger pool of VN-positive cells.
Immune staining for Coll-IV (Fig 6) reveals a distinct pattern marking the basal membranes of the epithelial compartment, which includes both ducts and developing islets. Note the strong fluorescent signal defining the basal membranes of the ducts (Fig 6 A; arrowheads). A similar strong staining highlights the basal membranes of developing pancreatic islets (A; arrow). This pattern suggests that Coll-IV may be required as a pathfinder matrix for endocrine progenitor cells and as a structural template for newly developing islets.
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The immune localization of these ECMs in the basal membrane of ductal structures, together with the identification of their integrin receptors vß3 and
vß5 in ductal cells (see Fig 1 and Fig 3) demonstrate identical histological compartmentalization of these two integrins and their ligands, and suggests their functional involvement in the emergence of endocrine progenitor cells from the ductal epithelium.
Fetal and Adult Pancreatic Epithelial Cells Adhere to ECM Proteins
The coexpression of integrins and ECM proteins in the pancreas does not provide evidence of function. Therefore, to test the hypothesis that these putative integrin/ECM interactions are functional, we assessed the adhesive properties of fetal and adult islet cells with different purified ECM proteins. Fig 7 shows the results of adhesion assays performed on increasing concentrations of Coll-IV, FN, LN, and VN. Both fetal (Fig 7 A) and adult (Fig 7 B) islets demonstrate a ligand concentrationdependent adhesion to these purified ECM proteins. Adhesion to purified Coll-IV was similar for fetal and adult cells at the lower concentrations (1.55 µg/ml), but increased significantly for adult cells at higher substrate concentrations (1545 µg/ml). We consistently observed that adhesion to VN at higher ligand concentrations was increased for fetal as compared with adult islet cells, a result consistent with the increased expression levels of vß3 and
vß5 in fetal cells. In separate experiments, to assess the specificity of adhesion of fetal cells mediated by
vß3 and
vß5 we used function-blocking monoclonal antibodies LM609 (specific for
vß3) and 15F11 (specific for
vß5). We observed that blockade of
vß3 by the LM609 antibody (50 µg/ml) resulted in a 71.2% inhibition of adhesion to VN and a 69.3% inhibition of adhesion to FN. Combination of LM609 and 15F11 antibodies in these adhesion assays inhibited adhesion of fetal pancreatic cells to VN by 84.9%. Adhesion to Coll-IV, also known to contain some RGD binding sequences for
vß3, was inhibited by 50.2% in the presence of LM609. Conversely, the
vß5-specific monoclonal, 15F11, inhibited adhesion of fetal pancreatic cells to VN by 75%, but had no effect on adhesion to FN and Coll-IV.
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Subcellular Localization of vß3 and
vß5 Integrins: Preferential Targeting to Focal Contacts
In our model system, when pancreatic fetal islet-like cell clusters, mainly composed of undifferentiated putative islet cell progenitors (vß3 and
vß5 integrins with special attention to focal adhesion contacts, known as critical regulatory domains for cell adhesion, spreading, and migration on ECM (
Fig 8 depicts undifferentiated fetal pancreatic epithelial cells obtained after the plating of islet-like cell clusters on 804G matrix. Representative confocal microscopic fields are shown to demonstrate distinct patterns of co-localization for vß3 (Fig 8 A) and
vß5 (Fig 8 B; green) with F-actin filaments (red) in focal adhesion contacts.
vß3 is confined to discrete sites located at the very tips of the F-actin filaments in the focal adhesion contacts (A; arrows). In contrast,
vß5 is detected in the focal adhesion contacts but also extends back along the F-actin filaments covering a larger area of the cell/ECM contact surface (B). Similar results were obtained when cells were plated on purified FN and VN (data not shown). These results demonstrate that the spatial organization of these two integrins with respect to the focal contacts is distinct and suggest that these two integrins may have different functions in mediating cell migration and maintaining cell/matrix contact during the formation of a cell monolayer (see below).
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Primary Role of vß3 and
vß5 Integrins in the Attachment and Migration of Fetal Pancreatic Epithelial Cells on ECM Proteins
When islet-like cell clusters are plated on 804G matrixcoated surfaces, cells forming the three-dimensional islet-like cell clusters first adhere and then migrate out of the cluster to form a monolayer within 12 h. Therefore, we used this in vitro model system to test the effect of function-blocking antibodies for vß3 and
vß5 integrins during the initial process of adhesion and monolayer formation and at the later stage when the monolayers are already established. We reasoned that by providing ligands for multiple integrin adhesion receptors, this complex ECM would be a stringent test for assessing the functional significance of
vß3- and
vß5-mediated adhesion to a basal-like membrane.
To allow the identification of identical microscopic fields at specific time intervals, we used glass coverslips marked with an alpha-numerical grid and precoated with the 804G matrix. Islet-like cell clusters were plated in the presence of either a control, isotype-matched mouse IgG (MAR), or with function blocking antibodies specific for vß3 (LM609) and
vß5 (15F11). Fig 9 A shows representative light microscopic fields of cell clusters recorded serially after 1, 6, and 12 h. Most of the cell clusters adhered to the 804G substratum in all conditions after 15 min (data not shown). After 1 h, cell clusters remained attached with no significant evidence of cell spreading on the matrix-coated coverslips. However, after 6 h in culture the cell clusters plated in the presence of the control IgG started to migrate and spread onto the coverslips. This process is almost completely inhibited in conditions containing either the anti-
vß3 and/or the anti-
vß5 mAbs (25 µg/ml). After 12 h, while cells in the control condition have all migrated into a semiconfluent monolayer, only few cells in the presence of the anti-
vß3 and/or the anti-
vß5 mAbs have migrated, although the clusters remain attached. Thus, in the presence of anti-
vß3 or the anti-
vß5 mAbs most of the cells remain in three-dimensional clusters, suggesting that these two integrins are important mediators of cell migration. To quantify this phenomenon and integrate the results from multiple microscopic fields (n = 50/condition), we also measured the surface area of the cell monolayers in all conditions (Fig 9 B). This analysis reveals that the blocking antibodies produced >75% overall inhibition of monolayer development in these assays.
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In a separate set of experiments, we first established monolayers on the matrix-coated coverslips by overnight culture (18 h) and then tested whether the anti-vß3 and anti-
vß5 mAbs could disrupt cell adhesion over an additional 6 h. As shown in Fig 10, addition of anti-
vß3 and anti-
vß5 mAbs (25 µg/ml) disrupted the anchorage of cells to the ECM in the established monolayers. After 6 h with anti-
vß3 mAb, the leading edges of the cell monolayers detached and rolled back (Fig 10; arrows). In contrast, when anti-
vß5 mAb was added to the cell monolayers, most of the cells detached completely from the ECM and the integrity of the monolayer was lost. It is important to note that the concentrations of blocking antibodies were identical in the experiments described in Fig 9 and Fig 10. Thus, these data suggest that
vß3 and
vß5 cooperate to establish spreading and migration on matrix. However, the data also indicate that
vß5 plays a more critical role in maintaining cell anchorage in the established monolayer. We have already noted that the localization of these two integrins in focal contacts is such that
vß3 is concentrated at the tips of F-actin filaments in migrating cells, whereas
vß5 is found in the focal contacts and extending behind
vß3 (Fig 8). Thus, in light of the functional data presented here, we propose that the unique spatial distribution of these two integrins with respect to the focal adhesion contacts and F-actin filaments may reflect a distinct role for
vß3 mainly in islet progenitor cell migration, whereas
vß5, in addition to cell migration, may be also required to maintain cell anchorage to the ECM.
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Cyclic RGD Peptide Analogues Block the Emergence of Developing Endocrine Progenitors from the Ductal Epithelium
The primary RGD-dependent integrins expressed by the putative endocrine progenitors in the developing human pancreas are vß3 and
vß5, as demonstrated by flow cytometry, confocal microscopy, and the functional studies described above. Previous studies have shown that cyclic RGD peptide analogues can be effectively used to inhibit function of these integrins (
Fig 11 A shows two representative adjacent microscopic fields acquired from the pancreatic grafts from animals treated with the control 39M peptide. Several large, well-formed islet clusters are evident with the majority of insulin-positive cells (red) located in the center, surrounded by cells which are positive for glucagon (blue) and somatostatin/pancreatic polypeptide (green). In contrast, analysis of the pancreatic grafts harvested from mice treated with the RGD peptide inhibitor 27O revealed a profoundly perturbed organization and development of islet cell clusters. Thus, as can be observed in Fig 11 B, most islet cells appear closely associated with ductal structures (asterisk), often completely or nearly surrounding ductal elements. In addition, a dramatic reduction in insulin-positive cells (red) is observed and islet cells are grouped in small clusters containing mainly glucagon-positive cells (blue). These data were validated by morphometric analysis to identify the positional segregation of islet cell types from ductal structures and their architectural organization within developing islets, as well as to assess changes in the relative mass of insulin-, glucagon-, and pancreatic polypeptide/somatostatinproducing cells. To determine the relationship of islet cells with ductal structures, we defined as "ductal-associated" all islet cells which appeared directly adjacent to, and/or clustered with, ductal elements within a range of 30 µm from the pancreatic ductal epithelium. As shown in Fig 12 A, this analysis reveals that all four islet cell types are much more closely associated with ducts in the mice treated with the 27O blocking peptide than in control animals. Thus, a five to sevenfold increase in the ductal association of endocrine cells can be observed in grafts from mice treated with the 27O peptide analogue. These results strongly suggest that inhibition of vß3 and
vß5 integrins, the two RGD-dependent integrins we have demonstrated in the developing human pancreas, significantly reduces the emergence of islet cells from the ductal epithelium. However, we acknowledge that this approach does not exclude contributions from other RGD-dependent integrins that currently are unidentified. In this context, we note that both fetal and adult islet cells were negative for
5ß1 (data not shown). A surprising finding in grafts of mice treated with the RGD peptide analogue was the reduced number of insulin-positive cells associated with islet cell clusters. As shown in Fig 12 B, we quantified an
66% reduction in the number of insulin-positive cells as determined by measurements of cell surface areas. In contrast, the number of glucagon- and pancreatic polypeptide/somatostatinpositive cells is significantly increased. All together, these morphometric data show that despite the significant differences in relative representation of each islet cell type, the total endocrine cell mass is not decreased, but rather increased.
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Discussion |
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Little is known about the expression and function of integrins during pancreatic islet ontogeny. Thus, although it is axiomatic that the complex three-dimensional structure of the pancreatic islet is important to its endocrine function, the determinants of this structure are unknown. Moreover, the development of the architectural organization of islet clusters during fetal morphogenesis may involve mechanisms distinct from those required to maintain the structure of the mature islet during adult life. In this regard, integrin receptors and their ligands are likely candidates to participate in pancreatic islet morphogenesis. Thus, it has been shown that integrinECM interactions regulate a variety of cellular functions, encompassing cell adhesion to specific sequences within ECM components, migration and organization of cells within tissues, initiation and maintenance of cellular differentiation, and sustaining mature cellular functions. Here we describe the developmental expression and function of vß3 and
vß5 integrins in the adhesion and migration of undifferentiated human fetal pancreatic epithelial cells comprising putative progenitors of the endocrine lineage (
Our data show that vß3 and
vß5 integrins are expressed at the cell surface of cells obtained from fetal pancreatic islet-like cell clusters. These clusters are harvested from whole pancreata after short-term cultures of collagenase digests and are predominantly composed of undifferentiated epithelial cells as determined by previous morphometric studies (
vß3 and
vß5 expression, whereas levels of the ß1 family of integrins are essentially unchanged (data not shown). Interestingly, it was recently demonstrated that downregulation of
vß3 and signaling through
vß5 is critical for the differentiation of oligodendrocyte progenitor cells (
vß3 and
vß5 are important receptors regulating the emergence of islet cell progenitors from the pancreatic ductal epithelium during islet development.
Our morphological studies demonstrate that vß3 and
vß5 immune reactivity is highest on ductal cells and clusters of cells budding from the ductal epithelium. As endocrine differentiation and islet organization occurs,
vß3 and
vß5 expression in islet cell clusters decreases substantially, consistent with the hypothesis that these integrins are more important for early migration and organization events involved in recruiting endocrine progenitors from the ductal epithelium. Support for this interpretation is provided by evidence that VN and FN, two major ECM ligands for
vß3 and
vß5, are associated with cells emerging from, or adjacent to, the ductal epithelium. These cells often coexpress insulin and/or glucagon, a landmark of endocrine differentiation. In contrast, the distribution of Coll-IV and LN (data not shown) in discrete basal membrane structures in developing islet cell clusters as well as mature islets suggests that their receptors, including the ß1 family of integrins, are important for creating and maintaining islet architecture. The fact that expression levels of the ß1 integrins are similar in fetal and adult islet cells is consistent with the hypothesis that these integrins are important throughout development and in adult life in the maintenance of islet structure. In support of this view, it is known that
3ß1 is restricted to islet cells in the adult rat pancreas (
3ß1 and
6ß1 regulate not only islet cell adhesion but also insulin secretion (
To address the functional role of integrin adhesion receptors in the developing human pancreas, we performed a series of in vitro assays to assess integrin function, including adhesion and migration to both purified and cell-deposited matrix proteins. These studies demonstrate that both adult islet and fetal pancreatic cells adhere to purified basal membrane ECMs in a concentration-dependent fashion. We observed by electron microscopy that culture on cell-deposited matrix (i.e., 804G or HACAT) favored the formation of tight intercellular junctions and desmosomes not seen when the same cells are cultured on plastic (results not shown). Confocal microscopy of fetal pancreatic cells migrating on cell-deposited matrix demonstrated the colocalization of vß3 and
vß5 integrins in focal adhesion contacts. However, while
vß3 was confined to the very tips of the F-actin filaments in the focal contacts, the expression of
vß5 extended back along the filament at the lower surface of the cell. A specific spatial organization for
vß3 and
vß5 with respect to focal contacts in a migrating pancreatic cell suggests a functional cooperation of these two integrins. This interpretation is consistent with previous observations in a model of growth factordependent angiogenesis showing that different integrins are engaged in response to distinct sets of factors (
vß3 and
vß5, we developed an assay to determine the adhesion and migration of fetal islet-like cell clusters on cell-deposited matrix. First, we demonstrated that blocking antibodies for
vß3 and
vß5 inhibited the migration of cells out of intact islet-like cell clusters, and consequently prevented the establishment of cell monolayers. Second, we showed that the integrity of established cell monolayers developed on the same matrix was disrupted by these antibodies. Blocking
vß3 caused the edge of the intact monolayer to detach and roll back, consistent with the tensile forces exerted by growing monolayers on their leading edges (
vß5 resulted in the complete detachment of the monolayer from the matrix. These results demonstrate that both
v integrins are functional in mediating islet cell adhesion and migration onto matrix. They also provide evidence supporting our hypothesis that
vß3 and
vß5 play a role in mediating the recruitment of endocrine progenitors from the ductal epithelium during islet ontogeny.
Although there is evidence demonstrating that the ductal epithelium is a reservoir for endocrine progenitors during islet development, nothing is known about the molecular mechanisms regulating the emergence of these progenitors and the creation of islet cell clusters. Conceivably, islet ontogeny requires a series of events that includes early endocrine determination of progenitors, induction of cell migration from the duct into the adjacent interstitium, ECM production and modification, the initial clustering of islet cells, vasculogenesis, and ultimately the three-dimensional organization of the mature islet. In this context, our in vivo transplantation studies with human fetal pancreas and the RGD-blocking peptide 27O demonstrate that with inhibition of integrin function the migration of endocrine progenitors from the ductal epithelium is severely limited, resulting in many small endocrine cell clusters mostly located immediately adjacent to ducts. Secondly, the number of insulin-positive cells in these small clusters is also significantly reduced with a relative increase in glucagon- and somatostatin/pancreatic polypeptidepositive cells. These results support the conclusion that distinct integrin signals may be required for the migration, proliferation, and differentiation of each islet cell type. Thus, it is possible that vß3 and
vß5 signals regulate migration of all islet cell types and of their precursors, yet differentially affect the growth and differentiation of insulin and glucagon cell lineages. This view is consistent with the recent evidence that insulin- and glucagon-producing cells arise from two independent progenitor lineages (
In summary, our data provide the first evidence that vß3 and
vß5 integrins are expressed in the developing human pancreas and that undifferentiated putative endocrine progenitor cells can use these adhesion molecules to mediate adhesion and migration on ECM. The data also show that the ECM proteins, FN and Coll-IV, contribute to the complex architecture of the developing islet. In addition, the
vß3 and
vß5 ligands, FN and VN, colocalize with cell clusters comprising insulin- and/or glucagon-positive cells emerging from, or adjacent to, the ductal epithelium. Most importantly, functional inhibition of these integrins in an in vivo model of islet development significantly reduced the migration of putative progenitors of islet cells from the ducts and perturbed the architecture and size of developing islet clusters. We conclude that
vß3 and
vß5 are critical integrins in the ontogeny of pancreatic islets. We also suggest that integrin/ECM interactions are involved in maintaining the structural integrity of mature islets. To the extent that islet structure is critical to islet function, efforts toward the development of novel strategies to protect and enhance integrin/ECM interactions may prove useful to improve the success of islet transplantation in diabetes.
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Footnotes |
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1 Abbreviations used in this paper: Coll-IV, collagen IV; ECM, extracellular matrix; FN, fibronectin; LN, laminin; PECAM, platelet endothelial cell adhesion molecule; RGD, arginine, glycine, aspartic acid; VN, vitronectin.
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Acknowledgements |
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This paper is dedicated to the memory of George Klier, a trusted collaborator and friend.
We are grateful to Dr. L. Crisa (The Scripps Research Institute) for her insightful contribution to the design of the in vivo experiments, and Dr. Anthony Montgomery (University of California, San Diego) for his comments on the manuscript. We are grateful to Drs. R. Ingram and M. Pierschbacher (Integra Life Sciences) for their assistance in the design and conduct of the blocking RGD peptide experiments.
D.R. Salomon was supported by National Institutes of Health (NIH) grant AI42384; V. Quaranta by NIH grant GM46902; and V. Cirulli by NIH grant DK98183 as well as a Career Development Award (296112) and Research Grant (197009) from the Juvenile Diabetes Foundation International (JDFI). A. Hayek was supported by JDFI Research Grant (199952). We also acknowledge support for C. Ricordi by the JDFI Human Islet Distribution Program at the Diabetes Research Institute of the University of Miami. The National Center for Microscopy and Imaging Research is supported by NIH grants RR04050 and DK54441 to M.H. Ellisman. This is manuscript No. 12239-MEM from The Scripps Research Institute.
Submitted: 22 May 2000
Revised: 10 July 2000
Accepted: 19 July 2000
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