Inhibition of Angiogenesis Is Associated With Reduced Islet Engraftment in Diabetic Recipient Mice

Inhibition of Angiogenesis Is Associated With Reduced Islet Engraftment in Diabetic Recipient Mice N. Zhang, S. Qu, J. Xu, J.S. Bromberg, and H.H. Dong ABSTRACT Rapid reestablishment of a functional microvasculature in transplanted islets is crucial for islet survival and function. To illustrate the importance of angiogenesis in islet engraftment, we took a loss-of-function approach to block angiogenesis in newly transplanted islets and determined the extent of islet engraftment in correlation with islet mass and glycemic control in diabetic recipient mice. Diabetic mice were transplanted with a marginal mass of 200 islets under the renal capsule, followed by once-daily oral administration of saline or 150 mg/kg of C-statin, a potent angiogenic inhibitor, for 14 days. Blood glucose profiles and the amplitude of glucose-stimulated insulin secretion in engrafted islets were determined. At 30 days posttransplant, islet grafts were retrieved for the determination of insulin content and vascular density by immunohistochemistry. When compared to sham-treated controls, diabetic recipient mice receiving a daily oral dose of C-statin exhibited significantly impaired blood glucose profiles and diminished glucose-stimulated insulin secretion in response to glucose challenge, correlating with significantly reduced intragraft insulin content and vascular density. Selective inhibition of angiogenesis was associated with reduced islet mass in diabetic mice. These data extend our view that rapid onset of angiogenesis is crucial for islet survival and engraftment and support the development of therapeutic strategies to stimulate angiogenesis in newly implanted islets for enhancing islet engraftment and improving the outcome of marginal islet transplantation.

I

SLET TRANSPLANTATION is considered a potentially curative treatment for type 1 diabetes,1 but its widespread clinical application is stymied by a number of factors, including the lack of sufficient islets and rejection by the host immune system. In addition, there is mounting evidence that the long-term survival and function of islet grafts is compromised by a third significant factor, namely, the rate and degree of islet engraftment posttransplantation.2– 6 Unlike whole organ transplantation, by which grafts are implanted as vascularized tissue, islets are transplanted as single islet or islet clusters that are considered avascular after collagenase digestion and isolation, such that the survival and function of newly transplanted islets depend on the reestablishment of new vessels within the grafts to derive blood for oxygen and nutrients from the host vascular system.2,7,8 There is evidence that engrafted islets, as opposed to native islets in the pancreas, are associated with significantly reduced vascularization. This impairment in islet engraftment appears to ensue regardless of the transplantation site; in the liver, spleen, or under the kidney capsule.3,6 Delayed and inadequate islet engraftment can

deprive islets of oxygen and nutrients, resulting in cellular apoptosis and death of islet cells, particularly within the core of engrafted islets. This accounts at least in part for the observation that only a fraction of transplanted islets (⬍30% of islet mass) becomes stably engrafted despite the infusion of a large quantity of islets (⬃10,000 islets/kg) per recipient.1,4,9 –11 To date, the molecular basis underlying islet engraftment remains undefined, but it is clear that microvascular perfusion to newly implanted islets does not resume readily after transplantation. Instead, it can take up to 2 to 3 weeks for the development of capillaries within engrafted islets and From the Children’s Hospital of Pittsburgh (S.Q., J.X., H.H.D.), Pittsburgh, Pennsylvania; and Department of Gene and Cell Medicine (N.Z., J.S.B.), Mount Sinai School of Medicine, New York, New York. Supported by the Juvenile Diabetes Research Foundation. Address reprint requests to H. Henry Dong, PhD, Children’s Hospital of Pittsburgh, Rangos Research Center, 3460 Fifth Avenue, Pittsburgh, PA 15213. E-mail: [email protected]

0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.10.097

© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

4452

Transplantation Proceedings, 37, 4452– 4457 (2005)

INHIBITION OF ANGIOGENESIS

reestablishment of intragraft blood flow. This delay in islet engraftment is attributable to attenuated angiogenic function in newly transplanted islets, presumably due to the destruction of intra-islet endothelium and damage of the extracellular matrix in islets following enzymatic digestion and isolation.4 Consistent with this notion, the expression of vascular endothelial growth factor (VEGF) is impaired in newly transplanted islets.2,12,13 Provision of VEGF in islet grafts either by vector-mediated gene expression in islets or collagen embedding of VEGF protein in encapsulated islets was associated with enhanced islet engraftment and improved blood glucose profiles in diabetic recipient mice.5,14,15 Thus, a gain of function of angiogenesis in newly transplanted islets contributes to augmented islet engraftment and better preservation of islet mass posttransplantation. In this study, we took a loss-of-function approach of using C-statin to block angiogenesis and address whether a selective inhibition of angiogenesis would impede islet engraftment. We employed a syngeneic mouse model for transplantation of a marginal islet mass in the presence and absence of C-statin. C-statin is a group of proteoglycan molecules extracted from the plant Convolvulus arvensis. Because of its potent anti-angiogenic function, C-statin has been used as an angiogenesis inhibitor for blocking angiogenesis and halting new vessel formation and growth for anti-angiogenesis therapy.16,17 We show that islet grafts in diabetic mice treated with C-statin during the initial posttransplant phase were associated with significantly reduced islet engraftment and functional islet mass, correlating with the lack of adequate glycemic control posttransplant. MATERIALS AND METHODS Animals Inbred BALB/c mice were purchased from the Jackson Laboratories (Bar Harbor, Me, USA) and housed in a pathogen-free barrier animal facility with a 12-hour light/dark cycle. Mice were fed standard rodent chow and water ad libitum. To induce diabetes, BALB/c mice (10 weeks old, body weight, 25 to 30 g) were injected intraperitoneally with streptozotocin (STZ, 180 mg/kg). Animals were considered diabetic when tail vein blood glucose levels were ⬎350 mg/dL for 2 consecutive days, as determined by glucometer (Bayer, Mishawaka, Ind, USA).

Islet Isolation and Transplantation Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), followed by intraductal infusion of 3 mL of cold Hank’s buffer containing 1.5 mg/mL of collagenase P (Roche Diagnostics, Indianapolis, Ind, USA). The pancreas was surgically procured and digested at 37°C for 20 minutes. Islets were purified on a discontinuous Ficoll gradient (Sigma, St. Louis, Mo, USA) as described.5 For marginal mass transplantation, 200 handpicked islets were implanted beneath the renal capsule of STZ-induced diabetic mice using the established procedure as described.5 Transplanted mice were randomly divided into two groups (n ⫽ 6). One group was treated by oral gavage daily with 400 ␮L of C-statin (prepared in saline, Ecomax Nutrition, Montreal, Canada) at the dose of 150 mg/kg per mouse and the other group was

4453 treated with 400 ␮L of saline daily as control starting from day 1 posttransplantation for 2 weeks. Islet graft function was defined as elevated blood glucose levels in diabetic recipient mice were reduced to less than 250 mg/dL on 2 consecutive days posttransplantation. The dose of C-statin was based on previous studies.16 When orally administrated to normal mice, this dose of C-statin did not result in detectable alterations in blood glucose levels after 2 weeks of treatment. All procedures were approved by the institutional animal care and usage committee of Mount Sinai School of Medicine (Protocol No 03-0031).

Glucose Tolerance Test Animals fasted for 5 hours and injected intraperitoneally with 50% dextrose solution (Abbott Laboratories, Chicago, Ill, USA) at 3 g/kg body weight. Blood glucose levels were determined before and after glucose infusion as described.5 Area under the curve (AUC) of blood glucose profiles during a glucose tolerance test (GTT) was calculated using the KaleidaGraph software (Synergy Software, Reading, Penn, USA). To determine glucose-stimulated insulin release, aliquots (25 ␮L) of tail vein blood were sampled before and 5 minutes after glucose infusion for the determination of plasma insulin levels by ELISA (Crystal Chem, Chicago, Ill, USA).

Immunohistochemistry and Morphometric Analysis Immunohistochemistry of islet grafts and morphometric analysis were performed as previously described.5 Mice were fasted for 16 hours and humanely killed by CO2 inhalation at 30 days posttransplantation. Islet grafts were retrieved from individual animals. After fixing in 10% phosphate-buffered formalin overnight, islet grafts were embedded in paraffin. Consecutive sections (8-␮m thick) of paraffin-embedded islet grafts were immunostained with guinea pig anti-insulin (1:200 dilution; DAKO, Carpentaria, Calif, USA) and rat anti-CD31 (1:50 dilution; Research Diagnostics Inc., Flanders, NJ, USA), as described.5 The immunoreactivity was detected using the multilink-HRP ultrasensitive system (BioGenex, San Ramon, Calif, USA). After immunostaining, slides were examined at ⫻200 magnification in a microscope that was linked to a computerized charge-coupled device camera. Microscopic views covering engrafted islets under the kidney capsule that were immunostained by insulin and CD31 antibodies were captured as digitized micrographic pictures using Adobe Photoshop software (Adobe Systems, San Jose, Calif, USA). Using the color range section option of Adobe Photoshop, insulin- or CD31-positively immunostained color (brown in each case) was selected for quantification of the relative intensity per islet graft by densitometry using NIH Image 1.62 software (National Institutes of Health, Bethesda, Md, USA). Using this procedure, the relative intensity of insulin, or CD31 from three sections, on average, per islet graft was evaluated for the determination of the mean values, which were subsequently compared between the C-statin and control groups, as described.5

Statistical Analysis Statistical analysis of data was performed by analysis of variance (ANOVA) using the StatView software (Abacus Concepts, Berkeley, Calif, USA). Unpaired ANOVA t-test was used to study the significance between two groups. Values are expressed as the mean values ⫾ SEM. P values ⬍ .05 are statistically significant.

4454

ZHANG, QU, XU ET AL

Fig 1. Blood glucose profiles. Blood glucose levels in C-statin (□) and control (‘) groups of diabetic mice were determined before and after islet transplantation. The Cstatin group of diabetic recipient mice received an oral dose of Cstatin (150 mg/kg in 400 ␮L) from day 1 to day 14 posttransplantation, whereas the control group received an equivalent volume of saline. P ⬍ .05 between C-statin and control groups.

RESULTS Effects of C-Statin Treatment on Glycemic Control

Effects of C-Statin Treatment on Glucose-Stimulated Insulin Release

To investigate the effect of angiogenesis on islet engraftment, we used C-statin, a potent angiogenic inhibitor, to selectively inhibit angiogenesis in newly transplanted islets in diabetic BALB/c mice. As shown in Fig 1, transplantation of a marginal islet mass significantly reduced the elevated blood glucose levels from 386 ⫾ 31 to 225 ⫾ 43 mg/dL in the control group of diabetic recipient mice. In contrast, diabetic mice transplanted with the same marginal islet mass in the C-statin treatment group maintained hyperglycemia (384 ⫾ 19 mg/dL) during the course of the study. These data suggest that C-statin treatment adversely affects the optimal performance of transplanted islets in glycemic control in diabetic recipient mice. To rule out the possibility that this lack of glycemic control in the C-statin group is due to potential side effects of C-statin, we treated normal BALB/c mice (n ⫽ 5) with either once-daily oral dose of C-statin (150 mg/kg, 400 ␮L in vol) or 400 ␮L saline for 2 weeks, followed by the determination of fasting blood glucose levels and blood glucose profiles in response to GTT (3 g/kg). The mean fasting blood glucose level in C-statin treated mice (89 ⫾ 9 mg/dL) was indistinguishable from that in control animals (86 ⫾ 7 mg/dL). Furthermore, no significant differences were detected in blood glucose levels at all time points during the GTT between C-statin and control groups. These results indicate that C-statin treatment did not result in detectable changes in blood glucose homeostasis in normal mice under the experimental condition.

To study the effect of C-statin treatment on glucosestimulated insulin secretion from transplanted islets, we performed GTT on diabetic recipient mice 2 and 3 weeks posttransplantation, followed by the determination of blood glucose profiles and plasma insulin levels under both basal and inducible conditions. As shown in Fig 2A and 2B, in response to glucose challenge, significantly better blood glucose profiles were detected in control diabetic recipient mice, when compared to the C-statin treatment group. These results were corroborated following the quantification of the area under the curve (AUC) of blood glucose profiles during GTT. In keeping with their impaired blood glucose profiles, diabetic recipient mice in the C-statin treatment group are associated with significantly higher AUC values, a reflection of relatively poor glycemic control, when compared to controls (Fig 2, C and D). Similar results were produced at 2 and 3 weeks posttransplantation. During GTT, aliquots of blood were taken from tail vein of diabetic recipient mice before and 5 minutes after glucose infusion for determination of the amplitude of glucose-stimulated insulin release. This parameter, which represents insulin secretory reservoir of islets, has been considered the best predictor of functional islet mass.11,18 As shown in Fig 2E and 2F, significantly lower levels of plasma insulin were detected in the C-statin group in response to glucose challenge, which correlated with their impaired ability to tolerate intraperitoneally infused glucose (Fig 2, A and B).

INHIBITION OF ANGIOGENESIS

4455

Fig 2. Glucose tolerance test. Two and 3 weeks after transplantation, diabetic recipient mice fasted for 5 hours, followed by injecting intraperitoneally 3 g/kg glucose solution. Blood glucose profiles (A, B) and the corresponding AUC values (C, D) in C-statin (□) and control ( ) groups of diabetic recipient mice were measured during the glucose tolerance test. Glucose-stimulated insulin release (E, F) was studied by determining the amount of plasma insulin after 5 min of glucose infusion. *P ⬍ .05 versus control.

‘

Effects of C-Statin Treatment on Insulin Content and Vascular Density

To correlate the physiology of glucose-stimulated insulin release with the intragraft insulin content and degree of islet engraftment in transplanted islets, diabetic recipient mice were sacrificed 1 month posttransplantation. Islet grafts were retrieved from individual recipients for anti-insulin and antiCD31 immunohistochemistry. Three nonconsecutive sections per islet graft in a total of six islet grafts per group were assessed for the determination of relative insulin and CD31 immunostaining intensities, as described.5 Islet grafts in the control group exhibited significant amounts of positive insulin

staining (Fig 3A), coinciding with their significantly higher levels of glucose-stimulated insulin release (Fig 2) and relatively greater intensities of anti-CD31 staining (Fig 3C). In contrast, islet grafts in the C-statin group were associated with much lesser extent of anti-insulin (Fig 3B) and anti-CD31 (Fig 3D) immunostaining. Quantification of the relative intensities of anti-insulin and anti-CD31 immunostaining reinforced this conclusion. As shown in Fig 4A and 4B, both insulin content and vascular density, defined by the relative intensity of anti-insulin and ant-CD31 staining in islet grafts, were significantly lower in the C-statin group, when compared to controls.

4456

ZHANG, QU, XU ET AL

Fig 3. Immunohistochemistry. Islet grafts were retrieved from recipient diabetic mice with mock (A, C) and C-statin treatment (B, D) 30 days posttransplantation. Paraffin-embedded islet grafts were sectioned and immunostained with anti-insulin (A, B) and anti-CD31 (C, D) antibodies, respectively. Islet grafts are marked by arrows in Panel A and B. Anti-CD31 positively stained vascular elements are indicated by arrows in Panel C and D. Bar, 100 ␮m.

DISCUSSION

We show that a selective inhibition of angiogenesis by C-statin in newly transplanted islets resulted in a significant reduction in islet engraftment, accompanied by significantly reduced functional islet mass. The rationale for C-statin administration for the first 14 days posttransplantation was based on the implicit assumption that rapid onset of angiogenesis in newly transplanted islets is crucial for islet engraftment and early inhibition of graft angiogenesis would be more detrimental to islet engraftment. Indeed, based on the time of onset of angiogenic gene expression and appearance of capillaries in engrafted islets, it has been suggested that islet engraftment initiates as early as on day 2 posttransplantation and concludes with the reestablishment of blood flow to islets grafts within 2 to 3 weeks in rodent models of type 1 diabetes.2,19 Consistent with this scenario, we show that discontinuation of C-statin administration at day 15 posttransplantation did not result in significant improvement in glycemic control, as hyperglycemia persisted in diabetic recipient mice in the C-statin group. These results underscore the critical importance of

angiogenesis in islet engraftment during the immediate posttransplantation period, which is line with previous observations that once implanted islets become stably engrafted, islet engraftment does not seem to improve over time.2,20 We would like point out that although C-statin has been clinically used for anti-angiogenesis therapy in humans,16,17 its mechanism of action remains elusive. This did not preclude us from harnessing the anti-angiogenic property of C-statin to address the functional importance of angiogenesis in islet engraftment. Our data are consistent with the idea that islet engraftment constitutes a distinctive factor that can compromise the viability of transplanted islets and limit the optimal functional performance of transplanted islets. Recently, we and others have showed that adenovirusmediated VEGF production in islets resulted in augmented islet engraftment, contributing to significantly increased islet mass and improved glycemic control in diabetic mice.5,14 Using a different strategy by which islets were immobilized in VEGF-containing collagen and trans-

INHIBITION OF ANGIOGENESIS

4457

REFERENCES

Fig 4. Intragraft insulin content and CD31 immunostaining intensity. The relative immunostaining intensity of insulin (A) and CD31 (B) in engrafted islets was compared between C-statin and control groups by morphometric analysis. *P ⬍ .05 versus control.

planted into the peritoneal cavity of diabetic mice, Sigrist et al15 show that VEGF-induced angiogenesis conferred a beneficial effect on the viability of engrafted islets and the quality of glycemic control in diabetic mice. These data validate the concept that a gain-of-function of angiogenesis by elevating VEGF levels locally in newly grafted islets accelerates islet engraftment and support the development of therapeutic angiogenesis for preserving islet mass and improving the success rate of islet transplantation.

1. Shapiro AM, Lakey JR, Ryan EA, et al: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med 343:230, 2000 2. Jansson L, Carlsson PO: Graft vascular function after transplantation of pancreatic islets. Diabetologia 45:749, 2002 3. Carlsson PO, Palm F, Mattsson G: Low revascularization of experimentally transplanted human pancreatic islets. J Clin Endocrinol Metab 87:5418, 2002 4. Zhang N, Anthony K, Shinozaki K, et al: Angiogenic gene therapy for improving islet graft vascularization. Gene Ther Mol Biol 7:153, 2003 5. Zhang N, Richter A, Suriawinata J, et al: Elevated vascular endothelial growth factor production in islets improves islet graft vascularization. Diabetes 53:963, 2004 6. Mattsson G, Jansson L, Nordin A, et al: Evidence of functional impairment of syngerneically transplanted mouse pancreatic islets retrieved from the liver. Diabetes 53:948, 2004 7. Menger MD, Yamauchi J-I, Vollmar B: Revascularization and microcirculation of freshly grafted islets of Langerhans. World J Surg 25:509, 2001 8. Konstantinova I, Lammert E: Microvascular development: learning from pancreatic islets. Bioessays 26:1069, 2004 9. Boker A, Rothenberg L, Hernandez C, et al: Human islet transplantation: update. World J Surg 25:481, 2001 10. Shapiro AM: Eighty years after insulin: parallels with modern islet transplantation. JAMC 167:1398, 2002 11. Ryan EA, Lakey JR, Paty BW, et al: Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 51:2148, 2002 12. Vasin B, Reitz P, Xu G, et al: Effects of diabetes and hypoxia on gene markers of angiogenesis (HGF, cMet, uPA adn uPAR, TGF-a, TGF-␤, bFGF and vimentin) in cultured and transplanted rat islets. Diabetologia. 43:763, 2000 13. Vasir B, Jonas JC, Steil GM, et al: Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets. Transplantation 71:924, 2001 14. Narang AS, Cheng K, Henry J, et al: Vascular endothelial growth factor gene delivery for revascularization in transplanted human islets. Pharm Res 21:15, 2004 15. Sigris S, Mechine-Neuville A, Mandes K, et al: Influence of VEGF on the viability of encapsulated pancreatic rat islets after transplantation in diabetic mice. Cell Transplant 12:627, 2003 16. Meng XL, Riordan NH, Casciari JJ, et al: Effects of a high molecular mass Convolvulus arvensis extract on tumor growth and angiogenesis. P R Health Sci J 21:323, 2002 17. Riordan NH, Meng XL, Riordan HD: Antiangiogenic, antitumor and immunostimulatory effects of a non-toxic plant extract (PGM). Arlington, Va: Comprehensive Cancer Care; 2000 18. Robertson RP: Consequences on beta-cell function and reserve after long-term pancreas transplantation. Diabetes 53:633, 2004 19. Menger MD, Vajkoczy P, Beger C, et al: Orientation of microvascular blood flow in pancreatic islet isografts. J Clin Invest 93:2280, 1994 20. Mattsson G, Jansson L, Nordin A, et al: Impaired revascularization of transplanted mouse pancreatic islets is chronic and glucose-independent. Transplantation 75:736, 2003