Original Article Islet Microvasculature in Islet Hyperplasia and Failure in a Model of Type 2 Diabetes Xianquan Li,1 Lanjing Zhang,1 Sasha Meshinchi,2 Claudia Dias-Leme,1 Diane Raffin,1 Jeffery D. Johnson,3 Mary K. Treutelaar,1 and Charles F. Burant1,4

Gene expression profiling of islets from pre-diabetic male Zucker diabetic fatty (ZDF) rats showed increased expres- sion of hypoxia-related genes, prompting investigation of fundamental question in the understanding of the vascular integrity of the islets. The islet microvascula- the pathogenesis of type 2 diabetes is the ture was increased approximately twofold in young male mechanisms underlying the maintenance of in- ZDF rats by both morphometric analysis and quantifying A sulin secretion in the presence of insulin resis- mRNA levels of endothelial markers. ZDF rats at 12 weeks tance for insulin and the mechanisms that lead to the of age showed a significant reduction in the number of failure of this adaptive response. Expansion of ␤-cell mass endothelial cells, which was prevented by pretreatment in response to insulin resistance is found in many animal with pioglitazone. Light and electron microscopy of normo- models, and obese humans have an increased islet mass glycemic 7-week-old ZDF rats showed thickened endothe- compared with lean individuals (1,2), suggesting a similar lial cells with loss of endothelial fenestrations. By 12 weeks of age, there was disruption of the endothelium and adaptation. The signal for expansion of islet mass is not intra-islet hemorrhage. Islets from 7- and 12-week-old ZDF clear but likely involves a response to increased glucose rats showed an approximate three- and twofold increase in flux and may be dependent on intact insulin signaling vascular endothelial growth factor (VEGF)-A mRNA and pathways within the ␤-cell (3,4). Similarly, decreases in VEGF protein secretion, respectively, compared with lean islet mass likely underlies the islet failure, leading to the controls. Thrombospondin-1 mRNA increased in 7- and development of type 2 diabetes (1,5–9). In mice and in rats, 12-week-old rats by 2- and 10-fold, respectively, and was and likely in humans, islet failure has a genetic basis. reduced by 50% in 12-week-old rats pretreated with piogli- Perhaps the most studied model of ␤-cell proliferation and tazone. Islets from young male control rats induced migra- failure is the Zucker fatty (fa/fa) and the Zucker diabetic tion of endothelial cells in a collagen matrix only after fatty (ZDF) rat, a substrain of the fa/fa. Both fa/fa and ZDF pretreatment with matrix metalloproteinase (MMP)-9. Is- rats carry mutation in the leptin receptor gene, while the lets from 7-week-old ZDF rats showed a fivefold increase in ZDF rat has additional abnormalities that predispose to migration score compared with wild-type controls, even islet failure (10). In male ZDF rats, progressive increases in without MMP-9 treatment. Islets from 15-week-old ZDF blood glucose occur around 7 weeks of age, and after rats did not induce migration; rather, they caused a signif- about 12 weeks, serum glucose levels can be Ͼ500 mg/dl. icant rounding up of the duct-derived cells, suggesting a The male and female Zucker and female ZDF rats remain toxic effect. These data suggest that in the ZDF rat model normoglycemic. Insulin-sensitizing treatments are effec- of type 2 diabetes, an inability of the islet to maintain tive in preventing ␤-cell hyperplasia and subsequent islet vascular integrity may contribute to ␤-cell failure. Diabetes 55:2965–2973, 2006 failure in the ZDF rat (11,12), suggesting that the islet failure is not intrinsic to the islet but is in response to insulin resistance. It is evident that the vascular endothelium is important in the growth and differentiation in a number of other tissues (13–17). A number of pro- and antiangiogenic peptides are elaborated by the islet. Vascular endothelial growth factor (VEGF) is expressed in the endocrine cells, From the 1Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; the 2Department of Cell and Developmental Biology, Univer- and the VEGF receptors flk-1 and flt-1 are expressed on sity of Michigan, Ann Arbor, Michigan; the 3Biology Division, Metabolex, endothelial cells within the islet (18,19). Overexpression of Hayward, California; and the 4Department of Molecular and Integrative VEGF in the developing pancreas leads to ectopic insulin Physiology, University of Michigan, Ann Arbor, Michigan. Address correspondence and reprint requests to Charles Burant, MD, PhD, production and islet hyperplasia (20). Disruption of University of Michigan Medical Center, Box 0678, 1500 E. Medical Center Dr., VEGF-A early in pancreatic development showed that the Ann Arbor, MI 48109. E-mail: [email protected]. absence of VEGF results in significantly smaller pancreata Received for publication 28 May 2006 and accepted in revised form 8 August 2006. with islet mass only one-third the size of normal (21). The X.L. and L.Z. contributed equally to this work. islets were relatively hypovascular and contained ultra- MMP, matrix metalloproteinase; PCNA, proliferating cell nuclear antigen; structural abnormalities in the remaining endothelial cells, TEM, transmission electron microscopy; Tsp-1, thrombospondin-1; VEGF, including decreased fenestrations and increased number vascular endothelial growth factor. DOI: 10.2337/db06-0733 of caveoli (22). There was also a significant decrease in © 2006 by the American Diabetes Association. glucose tolerance in these mice. In contrast, disruption of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance VEGF-A, specifically in islets, results in hypovascular islets with 18 U.S.C. Section 1734 solely to indicate this fact. and a minor alteration in glucose tolerance (23). These

DIABETES, VOL. 55, NOVEMBER 2006 2965 VASCULAR INTEGRITY IN DIABETIC ISLETS data suggest that VEGF is important for the development and function of the endocrine pancreas, as well as islet endothelial cells. In performing gene profiling of rat islets during the development of diabetes, we noted that changes in expres- sion of a number of genes related to hypoxia and vascular function. In light of the importance of the vasculature for the growth and maintenance of the islet, we examined changes in the vasculature of the islet in the ZDF rat.

RESEARCH DESIGN AND METHODS All animal procedures were approved by the institutional animal care and use committee at the University of Michigan. Male and female fa/fa and ZDF rats (ZDF/Gmi-fa/fa; ZDF rats) or lean littermate controls were purchased from Charles Rivers (Indianapolis, IN). In these experiments, pancreata were harvested on 2 consecutive days between 4 and 5, 7 and 8, and 12 and 14 weeks of age and throughout this article will be referred to as 4-, 7-, and 12-week-old animals. Male rats were fed standard rat chow (Purina 5006) in a 12:12 light:dark cycle or a chow in which pioglitazone was added at 1.6 mg/g to give an average daily dose of pioglitazone of ϳ40 mg/kg. To induce diabetes in female ZDF (Zucker fatty female [ZFF]) rats, they were fed a semipurified high-fat diet (48% fat, 16% protein; diet no. 13004; Research Diets, New FIG. 1. Gene expression determined by Affymetrix GeneChip profiling. Brunswick, NJ) from 8 to 13 weeks of age. Whole blood glucose was Average relative expression of the indicated genes found in profiles of determined using a Hemocue (Angelholm, Sweden). male lean control and ZDF rats. Data are means ؎ SE of average Islet oligonucleotide arrays and RNA hybridization. The use of custom difference scores of four individual pools of islets. *P < 0.05, #P < 0.01 rat islet Affymetrix GeneChips (Metabolex Rat Islet Oligonucleotide Arrays) vs. lean at each age. used in these studies has been described previously (18). Immunohistochemistry. Sections of fixed tissues (4% paraformaldehyde) were stained for nestin (1:100; Pharmingen, San Diego, CA) and insulin data were represented as Ct values, where Ct was defined as the threshold (1:2,000; Linco Research, St. Charles, MO) using a Vectastain ABC-Peroxidase cycle of PCR when the amplified product was first detected. The PCR and ⌬ kit (Vector Laboratories, Burlingame, CA). Biotinylated secondary antibodies protocol for the 18S ribosome were the same as described above. Ct was the and horseradish peroxidase–conjugated antibiotin were used to detect stain- difference in the Ct values derived from the specific gene being assayed and ⌬⌬ ing according to the manufacture’s protocols. For immunofluorescence stain- the 18S control, while Ct represented the difference between the paired ⌬⌬ ϭ⌬ Ϫ ing, blocked slides were incubated with nestin (1:100), VEGF (1:200; Santa tissue samples, as calculated by the formula Ct Ct of wild-type tissue ⌬ Cruz), or proliferating cell nuclear antigen (PCNA) (1:100; Sigma) antisera and Ct of other tissue. The N-fold differential expression in a specific gene of a ⌬Ct were developed with appropriate fluorescent secondary antibodies (Molecular sample compared with the wild-type counterpart was expressed as 2 . Probes). Relative RNA equivalents for each sample were obtained by normalizing to Quantification of nestin؉ cell number in pancreas sections. Four to five 18S levels. Each of the three samples per group was run in duplicate to random sections from each animal were stained for nestin and nuclei determine sample reproducibility, and the average relative RNA equivalents counterstained with DAPI (4Ј6-diamino-2-phenylindole). Two observers per sample pair was used for further analysis. Statistical analysis was counted the total number of nuclei in all of the islets from each section and performed using ANOVA. also quantified the number of nestinϩ cells in each section that was associated Angiogenesis assay. Primary duct cultures from rats were grown in four-well with the nuclei. The scores from each observer were averaged (Ͻ5% variabil- plates for 5 days as previously described (24) and trypsinized for 5 min, after ity in the counts between the observers) and the nestinϩ cell-associated nuclei which the cells were resuspended in 500 ␮l vitrogen 100 (Collagen). Islets divided by the total number of nuclei. from 7-week-old lean control or 7- or 14-week-old ZDF rats were isolated on Electron microscopy. Rat pancreas was excised and fixed by emersion in ficoll after collagenase digestion of the pancreas (12) and incubated for 24 h 2.5% glutaraldehyde in 0.1 mol/l Sorensen’s buffer, pH 7.4, and postfixed in 1% with or without matrix metalloproteinase (MMP)-9 to activate “angiogenesis” osmium tetroxide followed by en bloc stained with aqueous 3% uranyl acetate (25). Individual islets were placed with the trypsinized duct–derived cultures for 1 h, dehydrated, and embedded in Epon epoxy resin. Semithin sections and after 3 days scored for starburst formation, by a blinded observer, as an were stained with toluidine blue for tissue orientation. Selected areas were indication of migration of the cells in culture. ultrathin sectioned in 70-nm thickness and stained with uranyl acetate and Statistical analysis. ANOVA for single and repeated-measures ANOVA was lead citrate. They were examined using a Philips CM100 electron microscope used, where appropriate, to test for differences between groups, and Duncan’s at 60 kV. Images were recorded digitally using a Kodak 1.6 Megaplus camera multiple range test and Tukey’s Studentized range test were used for post hoc system operated using AMT software (Advanced Microscopy Techniques, evaluations. Danvers, MA). Total RNA isolation and real-time RT-PCR. Islets were isolated from lean RESULTS control rats or male and female ZDF rats using collagenase digestion and ficoll purification as previously described (12). Total RNA was isolated from 100 to Hypoxia-related genes are upregulated in pre-dia- 150 handpicked islets or from cultures, purified on RNeasy Quick spin betic and diabetic male ZDF rats. In performing Af- columns (Qiagen, Valencia, CA), and treated by DNA-free DNase (Ambion, fymetrix-based gene expression profiles from male ZDF Houston, TX). Maloney murine leukemia virus reverse transcriptase (Pro- rats at 6 and 9 weeks of age (26), we noted an upregulation mega, Madison, WI) was used for the reverse-transcription reaction in of a number of transcripts for genes known to be activated accordance with the manufacturer’s instructions. cDNA was purified by Quick during hypoxia (Fig. 1) (27). In general, these genes were spin kit (Qiagen). Real-time PCR was performed on a DNA Engine Opticon PCR cycler (MJ Research, Waltham, MA). PCR protocol was one cycle of 15 increased in the normoglycemic, 6-week-old ZDF rats and min at 95°C, 45 cycles of 15 s at 95°C, 30 s at 56°C, 30 s at 72°C, and plate read, in some cases were further increased in the mildly hyper- and 1 cycle of 10 min at 72°C, and 1 cycle of melting curve from 65 to 95°C. glycemic 9-week-old rats when compared with age- The following synthetic oligo-nucleotide primers were used in real-time PCR: matched lean controls. This prompted experiments to rat nestin forward 5Ј-AGGTGGGTGCTCTAAGGTT-3Ј, reverse 5Ј-AG assess the endocrine cell/vascular relationship in the islets Ј Ј GATCTCACCTCCCTTGCT-3 ; Rat Flk-1 (188 bp) forward 5 ctctacacttgtcgtgt of ZDF rats before, during, and after the induction of gaag-3Ј, reverse 5Јtaaccatacgacttctggcga-3Ј; rat VEGF forward (182 bp) 5Ј- CCAGGCTGCACCCACGAC-3Ј, reverse 5Ј-AGCCCGCACACCGCATTAG-3Ј; diabetes. and universal 18S forward (121 bp) 5Ј-ACTCAACACGGGAAACCTCACC-3Ј, Vascular endothelial cell in islets of ZDF rats. We (24) reverse 5Ј-CCAGACAAATCGCTCCACCAAC-3Ј. Results of the real-time PCR and others (28–30) have previously found that nestin, an

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TABLE 1 Characteristics of study animals Blood Age Weight glucose Cells Rat (weeks) (g) (mg/dl) per islet Lean (n) 4 5 ND 121 Ϯ 6 188 Ϯ 30.0 4 7 ND 112 Ϯ 4 200.3 Ϯ 25.4 4 12 ND 119 Ϯ 9 252.4 Ϯ 30.4 4 18 ND 124 Ϯ 10 249.7 Ϯ 28.6 fa/fa (n) 4 5 285 Ϯ 5 104 Ϯ 7 326 Ϯ 98.3 4 12 492 Ϯ 6 136 Ϯ 19 281 Ϯ 42.8 ZDF (n) 4 5 278 Ϯ 5 110 Ϯ 19 334 Ϯ 55.9 4 7 361 Ϯ 4 177 Ϯ 45 369 Ϯ 64.0 4 12 434 Ϯ 22 386 Ϯ 34 291 Ϯ 70.1 3 18 499 Ϯ 22 420 Ϯ 15 342 Ϯ 53.1

(؉ Female ZDF (n FIG. 2. Nestin endothelial cells in vivo and in vitro. A–C: Costaining of nestin (A) and the VEFG receptor Flk-1 (B) in pancreas of 7-week-old 3 (low-fat diet) 14 398 Ϯ 31 157 Ϯ 22 ND male lean rats. C: Merged image. The arrow indicates nestin and Flk-1, 3 (high-fat diet) 15 407 Ϯ 24 252 Ϯ 28 ND which costain in the muscularis area of the artery (asterisk). Note costaining of nestin and Flk-1 within in the islets (outlined by dashed Data are means Ϯ SD. ND, not determined. line). D: Costaining of nestin and the VEFG receptor Flk-1 in primary duct–derived cultures from lean male rats. Nestin is stained green and Flk-1 is stained red. ported (7), the average number of cells within islets increased in the fa/fa and ZDF animals compared with intermediate filament protein, is a marker of endothelial lean controls, with a 70–90% increase in cells/islets in cells in the islets of mice and humans. To confirm that we these populations compared with lean controls of the could use this easily detected antigen as an endothelial same age (Table 1). Serpentine nestinϩ cells were present marker in rat islet, we performed confocal microscopy on throughout the islet of all animals (Fig. 3A; fa/fa not pancreatic sections of control rats costained with mono- shown). In general, the nuclei associated with the endo- clonal anti-nestin antibodies and the VEGF receptor Flk-1 thelial cells were smaller and more elongated than the (VEGF-R2). As shown in Fig. 2A–C, we found that there nuclei in the endocrine cells. The relative nestinϩ endo- was 100% colocalization of Flk-1 with nestin within the thelial population was nearly doubled in the 4-week-old islet. Flk-1ϩ cells in large vascular structures cells did not fa/fa and ZDF rat islets compared with age-matched lean stain for nestin (Fig. 2A–C, asterisk), but some periductal controls. The increase in endothelial cell number was also and perivascular cells did show costaining (Fig. 2A–C, seen in the 7-week-old ZDF rat (Fig. 3D) before the onset arrows). In a previous publication, we demonstrated that of overt hyperglycemia. At 12 weeks, the fa/fa islet- primary duct cultures could give rise to nestinϩ cells. associated nestinϩ cell number continued to be signifi- About 30% of primary duct–derived cells were nestinϩ and cantly greater than the number in lean controls, while the also costained for Flk-1 (Fig. 2D). Thus, nestin staining can number of nestinϩ cells in the 12-week-old ZDF rats fell be used as a convenient marker for endothelial cells of the and continued to fall at 18 weeks of age and paralleled the islet and in cultured cells. islet degeneration (Fig. 3A and E). In addition to hyper- Islet microvasculature in insulin-resistant and dia- plasia, the endothelial cells in the 4- and 7-week-old ZDF betic rats. To determine if alterations in the microvascu- rats appeared hypertrophic compared with lean control lature of the islet occurs in a model of type 2 diabetes, we fa/fa rats (Fig. 3A; 7-week group). In male ZDF rats treated prospectively assessed potential changes in the microvas- with pioglitazone, the relative increase in the proportion of culature in the islets of male and female ZDF rats during nestinϩ endothelial cells was maintained at levels seen in islet expansion and islet failure and compared them with the 4- and 7-week-old ZDF animals (Fig. 3B and E), and the age-matched lean control and nondiabetic fa/fa rats. In morphology of the cells was not different from that of these studies, blood glucose levels rose with time in the control animals (Fig. 3B). male ZDF rats compared with lean littermates and were To independently confirm age-related changes in endo- frankly diabetic at 12 weeks of age (Table 1). Administra- thelial cell populations in the ZDF rat, islets were isolated tion of a diet containing the thiazolidinedione pioglitazone from 4- and 12-week-old rats, and the levels of nestin and prevented the onset of diabetes in the male rat. Lean Flk-1 mRNA expression were determined. We also exam- control male rats and fa/fa rats remain euglycemic ined nestin and Flk-1 mRNA expression in 12-week-old throughout the study. ZFF rats were significantly hyper- low-fat–fed (nondiabetic) and high-fat–fed (diabetic) ZFF glycemic only after administration of a proprietary high-fat rats. Nestin and Flk-1 mRNA levels were significantly diet for 4 weeks (31) (Table 1). lower in 12-week-old male ZDF rats compared with The proportion of nestinϩ endothelial cells in the islets 4-week-old animals and compared with age-matched lean of the experimental animals was determined by quantify- animals (Fig. 3F). As with the nestinϩ endothelial cell ing between 1,100 and 2,500 nuclei from histologically counts, pioglitazone prevented a significant fall in the identified islets in four to five random, nonconsecutive nestin mRNA levels in parallel to preventing the onset of sections from each of four different animals in each group diabetes, although the mRNA levels of Flk-1 tended to be and by determining the association of nuclei with nestinϩ lower than those seen in the pre-diabetic ZDF rats (Fig. cells determined by immunostaining. As previously re- 3F). Similar decreases in nestinϩ cell number were noted

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FIG. 3. Endothelial cells in the islets of male lean fa/fa and ZDF rats. A: Immunohistochemical staining of nestin in the islets of the different :strains of rats at the indicated ages. Magnification is 20؋ original. Arrows indicate nestin؉ cells within the islet and in the periductal area. Insets 7-week-old animals demonstrate endothelial cell hypertrophy. B: Nestin؉ cells in islets of 12-week-old male ZDF rats following 5 weeks of treatment with pioglitazone. C and D: Replicating cells in the islets of 14-week-old female rats fed a normal chow diet (C) or a diabetogenic high-fat diet (D) detected by costaining for nestin (red) and PCNA (green). A number of PCNA؉ cells are nestin؉ (C, arrows). Bright yellow cells are autofluorescent erythrocytes. E: Quantification of nestin؉ cell number in islets of individual rat strains. Each bar represents counts from 10 to 15 sections from four to five animals in each group. Significance levels determined by ANOVA are indicated. F: Nestin or VEGF receptor (Flk-1) mRNA levels in islets isolated from male lean or ZDF rats or female ZDF rats on a normal or diabetogenic high-fat diet. Significance values by .for each bar 5–4 ؍ ANOVA or Student’s t test are indicated. n in the islets of 14-week-old female ZDF rats after 6 weeks Early disruption of islet endothelium in ZDF rats. To of feeding a high-fat diet (not shown), which is in parallel better understand the loss of endothelial cells in this to the development of hyperglycemia, and this was paral- model of type 2 diabetes, we examined the ultrastructure leled by a decrease in nestin and Flk-1 mRNA levels in of lean control and ZDF islets by transmission electron islets isolated from the female rats (Fig. 3F). Interestingly, microscopy (TEM). The islets’ ultrastructure in 7-week-old confocal examination of islets of ZFF rat pancreata lean rats showed cells with multiple electron-dense gran- costained with nestin and the cellular proliferation marker ules within vesicles (Fig. 4A, arrows), which is indicative PCNA showed that a portion of the endothelial cells were of ␤-cells with intact epithelium. Higher-power images replicating in the control-diet–fed ZFF rats as shown by showed that the endothelium contained multiple fenestra- the presence of PCNA in nestinϩ cells. After the induction tions (Fig. 4B, arrows), which abut ␤-cells. In contrast, of diabetes, there was a paucity of nestinϩ endothelial TEM images from 7-week-old normoglycemic male ZDF cells and few double-positive cells (Fig. 3C and D, arrows). (Fig. 4C and D) rats showed several significant changes in There was evidence of endocrine cell replication, as the ␤-cells, including numerous vesicles without dense detected by PCNA staining, of typical islet endocrine cells granules (Fig. 4C and D, arrow heads), as well as evidence (Fig. 3C and D, asterisk). These results show that islets of swollen mitochondria (Fig. 4C and D, asterisk) and with active expansion in the nondiabetic state have pro- dilated endoplasmic reticulum multiple (Fig. 4C and D, liferating nestinϩ endothelial cells, while failing islets have double white arrow). The ultrastructure of endothelium a loss of these cells. was variable in 7-week-old ZDF rats, with some areas

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cells or endothelium in the exocrine portion of the pan- creas (data not shown). VEGF expression and secretion in islets. The progres- sive disruption of the endothelium in pre-diabetic ZDF rats may arise from decreases in production of endothelial survival factors or refractoriness of the endothelium to these factors either to intrinsic defects or due to the elaboration of antiangiogenic factors. VEGF-A, the most well-characterized proangiogenic factor, is expressed in the ␤-cells of islets (19,32,33), and disruption of islet VEGF-A results in abnormalities of islet endothelium and glucose intolerance (22). Immunohistochemistry of islets from different-aged lean control and ZDF rats showed that in all animals, VEGF-A was confined to the islets, and the number of cells that showed that VEGF staining in 14- week-old ZDF rats was decreased compared with those found in age-matched lean animals (Fig. 5A). However, the intensity of staining in the islet of the 15-week-old ZDF rat appeared greater than in the other groups of rats. Quanti- tative RT-PCR of VEGF-A mRNA from islets of pre- diabetic (7 weeks old) and diabetic (14 weeks old) ZDF rats showed that at both ages, VEFG-A mRNA levels were increased over that of 14-week-old lean control levels (Fig. 5B). At the same time, the insulin mRNA levels are decreased ϳ60% in the islets of 12-week-old male ZDF rats compared with 7-week-old lean controls, similar to that previously reported (34) (Fig. 5B). This result suggests that VEGF-A levels are increased per ␤-cell in the islets of the 7-week-old mZDF rat, and there is a relative preserva- tion of VEGF-A levels in the islets even as the islet fails. When compared with standard-diet–fed ZFF rats, ZFF fed the high-fat/high-carbohydrate diet showed a 30% increase in VEGF mRNA after 3 weeks of feeding (Fig. 5C). Pioglitazone treatment decreased the levels of VEGF-A mRNA to near control levels. We also measured the content of thrombospondin-1 (Tsp-1), a potent anti-VEGF FIG. 4. TEM of pancreatic islets. A: TEM of 7-week-old lean islet and peptide. Tsp-1 mRNA levels increased 2-fold in the 7-week- endothelium surrounding erythrocyte (RBC) showing normal electron- old pre-diabetic male ZDF rat and by 10-fold in the dense granules within vesicles (arrows). B: Fenestrations of endothe- 12-week-old diabetic animal. Pioglitazone blunted the rise lium indicated by arrows. C and D: TEM images from 7-week-old normoglycemic male ZDF rats showing vesicles in ␤-cells without dense in Tsp-1, but the levels were still fourfold greater than granules (arrow heads), swollen mitochondria (asterisk), and dilated control levels (Fig. 5B). The rise in Tsp-1 was most marked endoplasmic reticulum multiple (white arrow). Some endothelia in the ZFF rat, where the diabetogenic diet increased Tsp-1 showed areas showing normal appearing fenestrations (arrows), while most areas showed significantly thickened endothelium (black double expression nearly 20-fold (Fig. 5C). arrows). In D, the thickened endothelium demonstrates multiple intra- Following an 18-h incubation at 2 mmol/l glucose, the cytoplasmic vesicles (inset and arrowheads). E and F: TEM of islets islet content of VEGF-A was elevated twofold in islets from 14-week-old ZDF rats. The endothelium was severely disrupted (double black arrows). In F, autophagic vesicles containing degener- from 7-week-old ZDF rats compared with lean controls ating mitochondria and other intracellular organelles are seen (inset (Fig. 5D). Incubation in 20 mmol/l glucose increased the and arrows). VEGF-A content in islets from 7-week-old lean animals by 4.1-fold and in the ZDF animals by 5.8-fold in the ZDF showing normal-appearing fenestrations (Fig. 4C, arrows) islets. Even accounting for the increase in islet size, these and most areas showing significantly thickened endothe- values show an increase in islet content of VEGF-A in the lium (Fig. 4C and D, black double arrows), which likely ZDF islets. Unlike insulin, there was no acute release of corresponds to the hypertrophic endothelium seen in the VEGF-A from islets following exposure to 20 mmol/l pre-diabetic ZDF rat (Fig. 3A). In addition, there were glucose for 60 min (data not shown). Over a 24-h incuba- structures within the thickened epithelium of the pre- tion, secretion of VEGF-A into media was approximately diabetic animals that appeared as “target-like” structures. twofold higher in islets from ZDF rats incubated at both These vesicles contain electron-dense material, which may low and high glucose concentrations (Fig. 5D). The in- be endocytosed insulin (Fig. 4D, white arrowheads). In crease in VEGF-A secretion was dependent on de novo sections from 14-week-old ZDF rats, the endothelium was translation, as cyclohexamide prevented the rise following severely disrupted (Fig. 4E, arrows), with areas com- incubation in 20 mmol/l glucose (data not shown). These pletely denuded of endothelium (Fig. 4E and F, asterisk), data show that in the “failing” islets of the ZDF rat, there suggesting intra-islet hemorrhage. Autophagic vesicles is preserved and even enhanced synthesis and secretion of containing degenerating mitochondria and other intracel- VEGF-A. lular organelles were easily detected (Fig. 4F, arrows), Antiangiogenic factors in islets of diabetic rats. The suggesting a significant disruption in nutrient availability. degeneration of islet microvasculature in the ZDF rat with There was no difference in the appearance of the acinar intact VEGF-A secretion suggests an active inhibition of

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FIG. 5. VEGF in islets of lean and ZDF rats. A: Immunostaining for VEGF-A and insulin in islets from male lean and ZDF rats at the indicated ages. .for each bar. D: Islet VEGF-A content 7–5 ؍ B and C: Expression of insulin VEGF-A and TSP-1 mRNA in male (B) and female (C) ZDF rats. n Islets from 7-week-old lean control and ZDF rats were incubated for 24 h in the indicated glucose concentration and total islet–associated VEGF-A determined by enzyme-linked immunosorbent assay. Each bar represents three to four groups of five islets. P values are indicated. E: VEGF-A secretion from isolated islet from 7-week-old rats were incubated in increasing concentrations of glucose for 18 h. VEGF-A in the media assayed .islets determined in triplicate for each point. *Significantly different between groups, P < 0.05 5 ؍ was by enzyme-linked immunosorbent assay. n islet endothelial growth. To investigate this possibility, we the vitrogen gel matrix (Fig. 6A), independent of whether utilized a modified protocol of Folkman et al. (35) who MMP-9 was added to the cultures. The rounding up of cells showed that islets from normal mice became “angiogenic” was not seen in cocultures using 15-week-old islets from when treated with the protease MMP-9 via release of lean rats (data not show). These data suggest that a VEGF-A from the extracellular matrix. We isolated cells substance is secreted from the islet that has a detrimental from primary duct cultures that contained ϳ50% Flk-1ϩ effect on the duct-derived cells. cells (Fig. 2) and placed them in a vitrogen matrix (25). Islets from lean or ZDF rats were isolated and incubated for 24 h without or with MMP-9 to activate VEGF-A (35), DISCUSSION embedded in the endothelial cell–containing vitrogen ma- Expansion of the ␤-cell mass is a stereotypic response to trix, and scored for starburst formation, after 3 days, as an insulin resistance. The increase in mass has been attrib- indication of migration of the cells in culture. No apparent uted to both ␤-cell replication and neogenesis (36). Since growth of cells from the islets was noted when placed in islets are imbedded in the larger pancreas, an expansion of vitrogen without duct-derived cells (Fig. 6A) over the 25-h the endocrine cell mass would require a vasculature that period. Little migration of endothelial cells was seen in can grow in response to specific signals arising from the islets from lean control rats unless treated with MMP-9, as islet parenchyma or external signals. Studies in a number previously described (25). In contrast, there was enhanced of systems, including the endocrine pancreas, have shown migration of cells to the islets isolated from ZDF rats in the that cues for tissue growth and remodeling can arise from absence of MMP-9, with a smaller increase following the vasculature (21). The vasculature is also critical to MMP-9 treatment (Fig. 6A and B), suggesting endogenous maintaining the health of mature tissue by providing activation of VEGF-A in these cultures. We noted that the nutrients and oxygen, as well as additional growth signals. cultures containing 7-week-old ZDF islets also contained In the present study, we demonstrate morphological small rounded cells and elongated cells. Islets of 14-week- changes in the microvasculature of the pancreatic islet old ZDF mice induced the duct-derived cells to round up in before the onset of hyperglycemia with a continued par-

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observation that the endothelium of 7-week-old male ZDF rats show thickening and loss of endothelial fenestrations, similar to what has been described in the endothelium of mice following the inhibition of VEGF signaling by VEGF antibodies (40) or mice lacking VEGF-A in the pancreas (21). Interestingly, the latter mice demonstrate significant glucose intolerance, suggesting that changes in the endo- thelial morphology may contribute to abnormal insulin secretion. In the present study, the disruption of the vascular endothelium occurred before the elevation in glucose levels, suggesting that the degeneration may pre- cede the failure of the islet. Changes in capillary density in islets have been evalu- ated in previous studies. In db/db mice, there is capillary and pericyte hypertrophy (41). The apparent hypertrophy of endothelial cells is evident in both light microscopy and TEM of endothelial cells described in the present study. Similarly, in the Otsuka-Long-Evans-Tokushima fatty rat, islet degeneration leading to diabetes is associated with loss of capillary number within the islet (42). The phenom- ena of capillary hyperplasia followed by loss of capillary density in the male ZDF rat may not be limited to the islet. A recent study showed a similar phenomena occurring in the kidney of these rats. In this study, Gealekman et al. (43) observed hyperplasia of the endothelium of both the cortex and inner medulla in 8-week-old ZDF rats followed by a significant loss of capillary density at 22 weeks of age. Remarkably, they observed an identical increase in capil- lary growth from primary explants of the renal medulla from the younger rats and a marked diminution in growth from the 22-week-old ZDF rats, which is identical to the pattern we found in primary duct explants. The authors attributed part of these changes to oxidative stress and nitric oxide production changes, leading to reduced VEGF-A and Flk-1 receptor levels. The reason for altered capillary growth may be due to FIG. 6. Duct-derived cell migration induced by lean and ZDF islets. the paradoxical production of antiangiogenic factors in the Duct-derived cultures were established from mice, grown for 6 days, briefly trypsinized, and replated. The cells were resuspended in vitro- islets of the ZDF rat. In parallel to the disruption of gen as described with islets isolated from 7-week-old male lean (A and endothelial integrity in vivo, islets cultured in vivo show B)or7-(C–F) and 15-week-old (G and H) ZDF rats incubated for 24 h abnormal effects in cocultures, with endothelial cells with or without 20 ng/ml MMP-9 and placed in vitrogen alone or with providing evidence for the production of vascular growth the resuspended duct cells before gelling of the vitrogen solution. inhibitory factors. Tsp-1 has potent antiangiogenesis activ- ity (44,45), and mRNA for Tsp-1 is increased ϳ2-fold in allel degeneration of the microvasculature and ␤-cells pre-diabetic ZDF rats and ϳ5- to 10-fold in the islets of following the onset of hyperglycemia. Morphologically, it diabetic animals. The antiangiogenic effect of Tsp-1 is appears that nestin is a useful marker for the endothelium initiated by binding to the scavenger receptor CD36, which of the adult rat islet microvasculature, which confirms induces apoptosis via p38 and Jun NH2-terminal kinase. recent studies in other systems (30,37). The increase in the Tsp-1–null mice have enlarged hypervascular islets (46), relative number of nestinϩ cells in the islets of nondiabetic making them an attractive candidate for the antiangio- fa/fa and ZDF rats suggests that the expansion of the genic effect. However, crossing Tsp-1Ϫ/Ϫ mice with db/ vasculature is actively stimulated, perhaps via modulation dbleprϪ/Ϫ mice does not prevent diabetes (M.K.T., C.F.B., of signals arising from ␤-cells in response to the insulin- unpublished observations), suggesting that Tsp-1 does not resistant environment. An additional possibility is that the play a major role in the development of diabetes in increase in islet mass via ␤-cell replication and/or neogen- db/dbleprϪ/Ϫ mice, but we cannot rule out a role in the ZDF esis outstrips the vascular supply, causing a hypoxemic model. reaction. We recently reported that inhibition of MMPs can delay In both normal development (38) and during tumor the development of islet failure in the ZDF rat model (47). growth (39), hypoxia results in the development of addi- MMPs are a family of zinc-dependent enzymes responsible tional vasculature through the elaboration of vascular for matrix remodeling in several disease states (48). MMPs growth factors, notably VEGF-A. VEGF-A seems to only be have been shown to play a role in the degradation of basal produced in the ␤-cell of the islet. We found no evidence of lamina and disruption of the blood-brain barrier in animal acute release of VEGF-A with glucose stimulation but did models of intracerebral hemorrhage. MMP-9, an important find that glucose increases the synthesis and release of activator of VEGF in islets (25), has been found to be VEGF-A in cultured islets in response to glucose. VEGF-A associated with intracerebral hemorrhage (49), and inhi- mRNA and protein levels were elevated in the islets in bition of MMPs has been shown to be helpful in reducing 7-week-old male ZDF rats and is intriguing in light of the the edema associated with intracerebral hemorrhage

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(50,51). The finding that islets isolated from 7-week-old Training Center (DK-20572), and an Investigator Initiated rats induced endothelial cell migration without the need Award from Takeda Pharmaceutical North America. for MMP likely reflects the increased expression of MMPs, which we have previously documented in the ZDF rat (47). In addition to our proposed decrease in responsiveness of REFERENCES islet endothelial cells to VEGF, an increased MMP activity 1. Kloppel G, Lohr M, Habich K, Oberholzer M, Heitz PU: Islet pathology and could contribute to the intra-islet hemorrhages observed the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv in the present study. Synth Pathol Res 4:110–125, 1985 Recently, Johansson et al. (52) presented data suggest- 2. 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