78 Endocrine Research
Authors
A. J. F. King1, C. E. Clarkin2, A. L. F. Austin1, L. Ajram1, J. K. Dhunna1, M. O. Jamil1, S. I. Ditta1, S. Ibrahim1, Z. Raza1, P. M. Jones1
Affiliations
1
Key words ▶ SB-431542 ● ▶ A-83-01 ● ▶ islet transplantation ● ▶ TGF-β ●
Abstract
received 07.08.2014 accepted 29.10.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1395567 Published online: November 27, 2014 Horm Metab Res 2015; 47: 78–83 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Dr. A. J. F. King Hodgkin Building King’s College London Guy’s campus London SE1 1UL UK Tel.: + 44/20/7848 6402 Fax: + 44/20/7848 6280 [email protected]
2
Diabetes Research Group, Division of Diabetes and Nutritional Sciences, King’s College, London, UK Centre for Biological Sciences, University of Southampton, Southampton General Hospital, Southampton, UK
▼
Islet transplantation is a potential treatment for Type 1 diabetes but long term graft function is suboptimal. The rich supply of intraislet endothelial cells diminishes rapidly after islet isolation and culture, which affects the revascularisation rate of islets after transplantation. The ALK5 pathway inhibits endothelial cell proliferation and thus inhibiting ALK5 is a potential target for improving endothelial cell survival. The aim of the study was to establish whether ALK5 inhibition prevents the loss of intraislet endothelial cells during islet culture and thus improves the functional survival of transplanted islets by enhancing their subsequent revascularisation after implantation. Islets were cultured for 48 h in the absence or presence of 2 different ALK inhibitors: SB-431542 or A-83-01. Their vascular density after culture was analysed using immu-
Introduction
▼
Although the success rate of clinical islet transplantation has markedly improved in the last few years, it is clear that the results are not optimal with many patients reverting to insulin dependence [1]. One factor that may affect the long term function and survival of transplanted islets is suboptimal revascularisation [2]. Endogenous islets are highly vascularised, allowing rapid glucose sensing and insulin secretion. During isolation from the pancreas, islets are separated from their vascular network and although endothelial cells are detectable within the islets immediately after isolation, their numbers diminish rapidly with culture [3–5]. In clinical islet transplantation, islets are routinely cultured to allow for microbial screening and recipient patient preparation [6–10]. It has been reported that intraislet endothelial cells
King AJ F et al. ALK5 Inhibition in Islet Transplantation … Horm Metab Res 2015; 47: 78–83
nohistochemistry. Islets pre-cultured with the ALK5 inhibitors were implanted into streptozotocin-diabetic mice for either 3 or 7 days and blood glucose concentrations were monitored and vascular densities of the grafts were analysed. Islets cultured with ALK5 inhibitors had higher vascular densities than control-cultured islets. Three days after implantation, endothelial cell numbers in islet grafts were minimal, irrespective of treatment during culture. Seven days after implantation, endothelial cells were evident within the islet grafts but there was no difference between control-cultured islets and islets pretreated with an ALK5 inhibitor. Blood glucose concentrations were no different between the treatment groups. In conclusion, inhibition of ALK5 improved intraislet endothelial cell numbers after islet culture, but this effect was lost in the early post-transplantation period.
contribute to the revascularisation process of islet grafts when freshly isolated islets are transplanted [4, 11, 12] whereas in the case of cultured islets, donor islet endothelial cells contribute minimally to the vasculature of islet grafts [12]. It has been suggested that freshly isolated islets have an increased rate of revascularisation [12, 13] and this has been associated with better graft outcome [13]. It is therefore potentially of interest to preserve endothelial cells within cultured islets to enhance graft revascularisation and hence islet survival and function. Transforming growth factor beta (TGFβ) is an important mediator in the survival of endothelial cells [14]. Its actions are mediated through an oligomeric receptor complex, which consists of 2 receptors: Type 1 (TβR1) and Type 2 (TβR2). These receptors have serine/threonine kinase activity and TβR2 activated by the bound TGFβ phosphorylates and activates TβR1 [also known
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
ALK5 Inhibition Maintains Islet Endothelial Cell Survival but does not Enhance Islet Graft Revascularisation or Function
Endocrine Research 79
Materials and Methods
▼
Islet transplantation
Islets were transplanted under the kidney capsule as described previously [21]. A lumber incision was made in isoflurane anaesthetised mice and the kidney exposed. One hundred and fifty control or ALK5-inhibitor treated islets were implanted under the kidney capsule using PE50 tubing (Becton Dickinson, Sparks, MD, USA) and a Hamilton syringe (Fisher, Pittsburg, PA, USA) in a suboptimal mass model. Blood glucose and weight were monitored before the animals were killed by cervical dislocation at either 3 or 7 days.
Histology
Animals
Male C57Bl/6 mice aged 8–12 weeks were used for islet isolation or as transplant recipients. Recipient mice were rendered diabetic by 180 mg/kg i.p. injection of streptozotocin (SigmaAldrich, Poole, UK) and those with a nonfasting blood glucose concentration of ≥ 20 mmol/l were used as recipients. Blood glucose concentrations were determined using a blood glucose meter and strips (Accu-Chek; Roche, Burgess Hill, UK) with blood obtained from a pin prick to the tail. All animal experiments were carried out after ethical approval from our institution and under licence in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986.
Islet isolation
Islets were isolated using collagenase digestion followed by gradient purification as previously described [18]. Briefly, mice were killed by cervical dislocation and the pancreatic duct was clamped at the ampulla of Vater. Two ml of collagenase (1 mg/ml, type XI; Sigma-Aldrich) was injected into the pancreatic duct. The pancreata were digested at 37 °C for 10 min before washing and purification of the digest using a density gradient (Histopaque-1077, Sigma-Aldrich). After washing, islets were handpicked and cultured as described below.
Islet culture and treatment
Islets were cultured in RPMI 1640 with 10 % foetal calf serum and 1 % penicillin/streptomycin. Islets were cultured for 48 h in the presence of either 10 μM SB-431542 {4-[4-(1,3-benzodioxol-5-yl)5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide} [19] or 10 μM A-83-01 [3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)1H-pyrazole-1-carbothioamide) [20]. The effective concentrations of the ALK5 inhibitors were based on our previous studies [16], and on those from other groups [20]. Control islets were cultured in media containing the equivalent volume of vehicle (0.1 % DMSO v/v). Islets were then used for transplantation studies, for measurement of insulin release or for quantification of endothelial cells as described in more detail below. Some islets were cultured for a further 3 or 7 days in parallel with the in vivo studies after which endothelial cells were quantified.
Insulin release
either 2 mM or 20 mM glucose. After 1 h, samples of the incubation medium were taken and stored at 20 °C until assayed for insulin content using in-house radioimmunoassay.
Islets were pre-incubated for 2 h in RPMI-1640 medium containing 2 mM glucose. Groups of 3 islets were incubated at 37 °C in a bicarbonate-buffered physiologic salt solution containing 2 mM calcium chloride and 0.5 mg/ml bovine serum albumin and
Cultured islets or kidneys containing islet grafts were fixed in 4 % formalin for 2 or 48 h respectively and paraffin-embedded. Endothelial cells were stained using CD34 antibodies as previously described in detail [21]. Briefly, antigen retrieval was carried out on the samples [2 min in 10 mM citric acid solution (pH 6.0) in a pressure cooker]. Sections were incubated for 1 h at room temperature with a monoclonal rat anti-CD34 antibody (1:500; AbD Serotec, Kidlington, UK) followed by a rabbit biotinylated anti-rat antibody (1:200; Vector Laboratories, Peterborough, UK). Sections were incubated with streptavidinhorseradish peroxidase (Dako, Ely, UK) and diaminobenzidine.
Intraislet endothelial cell quantification
Photographs were taken of the CD34-stained sections using an Olympus 2000 microscope and QCapturePro at either × 10 and × 20 magnifications. Image J software (http://rsbweb.nih. gov/ij/) was used to determine islet area and vascular density by an individual blinded to the origin of the sample.
Statistical analysis
Statistical analysis was carried out using SigmaPlot. When 2 groups were compared a t-test was used. ANOVAs were used to compare more than one group; a two-way ANOVA was used to analyse insulin release, a two-way repeated measures ANOVA was used to analyse blood glucose measurements and a one-way ANOVA on ranks was used to analyse vascular density after culture. p-Values 0.05 two way ANOVA, n = 8) and islet insulin content (Panel b, p > 0.05, one way ANOVA, n = 8) after 2 days culture in the presence or absence of 10 μM of the ALK5 inhibitors SB-431542 and A-83-01. Vascular density of islets after 2-day culture in the presence or absence of 10 μM SB-431542 or 10 μM A-83-01 (Panel c; * p 0.05 2 way repeated measures ANOVA, n = 3). Prior to implantation islets had been cultured for 2 days in the presence or absence of 10 μM SB-431542.
grafts to reverse the streptozotocin-induced hyperglycemia and maintain normoglycemia during the 7 day post-implantation ▶ Fig. 4b). period ( ●
Discussion
▼
The importance of endothelial cells in normal islet development and function has been well established [2], and there is some evidence that the rate and extent of islet revascularisation influences the outcome of islet transplantation to reverse diabetic hyperglycemia [13]. Endogenous islets are highly vascularised, but after isolation and culture the numbers of endothelial cells rapidly diminish [3–5]. After transplantation, the vascular density of the graft increases with time from initiation of revascularisation at 3–4 days until 28 days [12], when numbers of endothelial cells are partially replenished, but the vascular density is still considerably less than in endogenous islets [22]. This impaired vascular density of implanted islets has been associated with reduced functionality [13]. Strategies to improve vascular density in transplanted islets include implanting freshly isolated islets [3, 13], genetically manipulated islets [23, 24], or co-transplanting other cell types with the islets [21, 25]. Despite encouraging results in experimental studies each of these strategies has limitations when applied to the clinical setting. Freshly isolated islets are no longer used routinely for clinical islet transplantation because of the requirement of a period in tissue culture to allow microbial testing and preparation of the graft recipient. Genetic manipulation of islets to enhance revascularisation is still relatively inefficient and will require thorough safety testing before genetically
modified islets will be acceptable in a clinically setting. Similarly, co-transplanting other cells with islets to improve islet revascularisation requires stringent safety testing prior to becoming clinically applicable. The ability to improve vascular density of islet grafts by defined pharmacological treatment of the islets before transplantation avoids many of these problems, including potential off-target systemic effects of the drug. This strategy has had some experimental success in targeting beta cell function and survival, where several pharmacological interventions prior to transplantation have had a positive effect on transplantation outcome [26–28]. However, attempts to improve islet graft function by using proangiogenic factors such as VEGF and FGF during in vitro culture have failed to enhance the vascular density of the islets or their subsequent function in vivo [29]. We have previously shown that inhibition of the ALK5 activity prevents the impairment of endothelial cell viability in vitro [16], suggesting that ALK5 may be a useful target for maintaining endogenous endothelial cell viability in isolated islets during in vitro culture. In the present study, we have demonstrated that 2 structurally-dissimilar pharmacological inhibitors of ALK5 were partially effective in reducing the decline in intraislet endothelial cell number during a 2 day culture period. These observations confirm that the viability of endothelial cells within the islets is regulated by the TGFβ pathway, and offer a simple and defined pharmacological method for maintaining vascular density prior to transplantation. Pre-treatment with ALK5 inhibitors had no deleterious effects on islet β-cell function, as assessed by maintained insulin content and glucose-induced insulin secretion, which are perhaps the most important functional parameters for islet grafts. It has recently been reported that ALK5 inhibition enhanced glucose-induced insulin secretion from
King AJ F et al. ALK5 Inhibition in Islet Transplantation … Horm Metab Res 2015; 47: 78–83
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Fig. 3 Blood glucose concentrations of mice implanted with islets that had been pre-cultured in the presence or absence of the ALK5 inhibitor A-83-01 (Panel a, p > 0.05 two way repeated measures ANOVA, n = 4–5) or SB-431542 (Panel b, p > 0.05 two way repeated measures ANOVA, n = 4–5).
82 Endocrine Research
Conflict of Interest
▼
The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
References
1 Bruni A, Gala-Lopez B, Pepper AR, Abualhassan NS, Shapiro AJ. Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges. Diabetes Metab Syndr Obes 2014; 7: 211–223 2 Peiris H, Bonder CS, Coates PT, Keating DJ, Jessup CF. The beta-cell/EC axis: how do islet cells talk to each other? Diabetes 2014; 63: 3–11 3 Rackham CL, Dhadda PK, Chagastelles PC, Simpson SJ, Dattani AA, Bowe JE, Jones PM, King AJ. Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice. Cytotherapy 2013; 15: 449–459 4 Nyqvist D, Kohler M, Wahlstedt H, Berggren PO. Donor islet endothelial cells participate in formation of functional vessels within pancreatic islet grafts. Diabetes 2005; 54: 2287–2293 5 Parr EL, Bowen KM, Lafferty KJ. Cellular changes in cultured mouse thyroid glands and islets of Langerhans. Transplantation 1980; 30: 135–141 6 Hering BJ, Kandaswamy R, Harmon JV, Ansite JD, Clemmings SM, Sakai T, Paraskevas S, Eckman PM, Sageshima J, Nakano M, Sawada T, Matsumoto I, Zhang HJ, Sutherland DE, Bluestone JA. Transplantation of cultured islets from two-layer preserved pancreases in type 1 diabetes with anti-CD3 antibody. Am J Transplantat 2004; 4: 390–401 7 Shapiro AM, Ricordi C, Hering B. Edmonton’s islet success has indeed been replicated elsewhere. Lancet 2003; 362: 1242 8 Froud T, Ricordi C, Baidal DA, Hafiz MM, Ponte G, Cure P, Pileggi A, Poggioli R, Ichii H, Khan A, Ferreira JV, Pugliese A, Esquenazi VV, Kenyon NS, Alejandro R. Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosuppression: Miami experience. Am J Transplant 2005; 5: 2037–2046 9 Kin T, Senior P, O’Gorman D, Richer B, Salam A, Shapiro AM. Risk factors for islet loss during culture prior to transplantation. Transpl Int 2008; 21: 1029–1035 10 Goss JA, Schock AP, Brunicardi FC, Goodpastor SE, Garber AJ, Soltes G, Barth M, Froud T, Alejandro R, Ricordi C. Achievement of insulin independence in three consecutive type-1 diabetic patients via pancreatic islet transplantation using islets isolated at a remote islet isolation center. Transplantation 2002; 74: 1761–1766 11 Brissova M, Fowler M, Wiebe P, Shostak A, Shiota M, Radhika A, Lin PC, Gannon M, Powers AC. Intraislet endothelial cells contribute to revascularization of transplanted pancreatic islets. Diabetes 2004; 53: 1318–1325 12 Nyqvist D, Speier S, Rodriguez-Diaz R, Molano RD, Lipovsek S, Rupnik M, Dicker A, Ilegems E, Zahr-Akrawi E, Molina J, Lopez-Cabeza M, Villate S, Abdulreda MH, Ricordi C, Caicedo A, Pileggi A, Berggren PO. Donor islet endothelial cells in pancreatic islet revascularization. Diabetes 2011; 60: 2571–2577 13 Olsson R, Carlsson PO. Better vascular engraftment and function in pancreatic islets transplanted without prior culture. Diabetologia 2005; 48: 469–476 14 ten Dijke P, Arthur HM. Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol 2007; 8: 857–869 15 Rahimi RA, Leof EB. TGF-beta signaling: a tale of two responses. J Cell Biochem 2007; 102: 593–608 16 Clarkin CE, King AJ, Dhadda P, Chagastelles P, Nardi N, Wheeler-Jones CP, Jones PM. Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells. Stem Cells 2013; 31: 547–559 17 Liu Z, Kobayashi K, van Dinther M, van Heiningen SH, Valdimarsdottir G, van Laar T, Scharpfenecker M, Lowik CW, Goumans MJ, Ten Dijke P, Pardali E. VEGF and inhibitors of TGFbeta type-I receptor kinase synergistically promote blood-vessel formation by inducing alpha5integrin expression. J Cell Sci 2009; 122: 3294–3302 18 Kerby A, Bohman S, Westberg H, Jones P, King A. Immunoisolation of islets in high guluronic acid barium-alginate microcapsules does not improve graft outcome at the subcutaneous site. Artif Org 2012; 36: 564–570 19 Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 2002; 62: 65–74 20 Tojo M, Hamashima Y, Hanyu A, Kajimoto T, Saitoh M, Miyazono K, Node M, Imamura T. The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-beta. Cancer Sci 2005; 96: 791–800
King AJ F et al. ALK5 Inhibition in Islet Transplantation … Horm Metab Res 2015; 47: 78–83
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mouse islets when the inhibitor was present during glucose challenge [30]. However, our functional measurements were performed in the absence of the ALK5 inhibitors, after the pretreatment period with the inhibitors, to better reflect islet function in vitro immediately before implantation. Nonetheless, the available data suggest that using ALK5 inhibition as a strategy to maintain islet endothelial cells will not comprise islet secretory function and may even have acute beneficial effects on insulin release. It has been reported that endothelial cells in freshly isolated islets contribute more to graft revascularisation than those in cultured islets [4, 11, 12], resulting in higher vascular density and improved transplantation outcomes [3, 13], suggesting direct beneficial outcomes from implanting islets with higher numbers densities of endothelial cells. However, the enhanced endothelial cell density of cultured islets achieved by ALK5 inhibition was not maintained when the islets were transplanted into the in vivo environment. Thus, within 3 days of implantation endothelial cell number in ALK5-inhibitor pre-treated islets had declined to the similar low levels detected in untreated islets. Similarly, ALK5 inhibition had no effect on the revascularisation of transplanted islets within the first 7 days after implantation, presumably driven primarily via invasion of host endothelial cells, such that intraislet endothelial cell density was not different between ALK5-inhibitor pre-treated and control untreated islets. Consistent with the lack of effect on intraislet endothelial cells, ALK5 inhibition had no functional effects on the ability of the transplanted islets to regulate blood glucose levels at either 3 or 7 days after implantation, by which stage the grafts were maintaining normoglycemia. There are a number of reasons why the promising in vitro observations using ALK5 inhibitors did not translate into enhanced graft revascularisation and function in vivo. First, it is possible that ALK5 inhibition did not maintain a sufficient density of intraislet endothelial cells to have any significant functional effects in vivo, although this seems unlikely since pre-incubation with A-83-01 maintained endothelial cell density at approximately 75 % of that measured in freshly isolated islets. Second, the superior outcomes of studies using freshly isolated islets may reflect enhanced function of the intraislet endothelial cells rather than their higher density, in which case strategies aimed solely at maintaining endothelial cell number are unlikely to improve the functional outcomes of islet transplantation. Finally, it is possible that the intraislet endothelial cells play a relatively minor role in graft revascularisation, or that the extent of islet vascularisation is not critical for β-cell function, and the improved transplantation outcomes using freshly isolated islets reflect better β-cell function rather than any vascular parameter. This conclusion is consistent with a recent report that a prolonged 2-fold reduction in islet vascularisation in vivo had little effect on function [31], and may suggest that pharmacological strategies aimed at improving the outcomes of islet transplantation may be better targeted at the β-cell than at the endothelial cell.
Endocrine Research 83 27 Emamaullee JA, Shapiro AM. Interventional strategies to prevent betacell apoptosis in islet transplantation. Diabetes 2006; 55: 1907–1914 28 Sahraoui A, Jensen KK, Ueland T, Korsgren O, Foss A, Scholz H. Anakinra and tocilizumab enhance survival and function of human islets during culture: implications for clinical islet transplantation. Cell Transpl 2014; 23: 1199–1211 29 Olsson R, Maxhuni A, Carlsson PO. Revascularization of transplanted pancreatic islets following culture with stimulators of angiogenesis. Transplantation 2006; 82: 340–347 30 Wu H, Mezghenna K, Marmol P, Guo T, Moliner A, Yang SN, Berggren PO, Ibanez CF. Differential regulation of mouse pancreatic islet insulin secretion and Smad proteins by activin ligands. Diabetologia 2014; 57: 148–156 31 Reinert RB, Brissova M, Shostak A, Pan FC, Poffenberger G, Cai Q, Hundemer GL, Kantz J, Thompson CS, Dai C, McGuinness OP, Powers AC. Vascular endothelial growth factor-a and islet vascularization are necessary in developing, but not adult, pancreatic islets. Diabetes 2013; 62: 4154–4164
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21 Rackham CL, Chagastelles PC, Nardi NB, Hauge-Evans AC, Jones PM, King AJ. Co-transplantation of mesenchymal stem cells maintains islet organisation and morphology in mice. Diabetologia 2011; 54: 1127–1135 22 Mattsson G, Jansson L, Carlsson PO. Decreased vascular density in mouse pancreatic islets after transplantation. Diabetes 2002; 51: 1362–1366 23 Wang X, Meloche M, Verchere CB, Ou D, Mui A, Warnock GL. Improving islet engraftment by gene therapy. J Transpl 2011; 2011: 594851 24 Olerud J, Johansson M, Lawler J, Welsh N, Carlsson PO. Improved vascular engraftment and graft function after inhibition of the angiostatic factor thrombospondin-1 in mouse pancreatic islets. Diabetes 2008; 57: 1870–1877 25 Johansson U, Rasmusson I, Niclou SP, Forslund N, Gustavsson L, Nilsson B, Korsgren O, Magnusson PU. Formation of composite endothelial cell-mesenchymal stem cell islets: a novel approach to promote islet revascularization. Diabetes 2008; 57: 2393–2401 26 King A, Lock J, Xu G, Bonner-Weir S, Weir GC. Islet transplantation outcomes in mice are better with fresh islets and exendin-4 treatment. Diabetologia 2005; 48: 2074–2079
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Authors
A. J. F. King1, C. E. Clarkin2, A. L. F. Austin1, L. Ajram1, J. K. Dhunna1, M. O. Jamil1, S. I. Ditta1, S. Ibrahim1, Z. Raza1, P. M. Jones1
Affiliations
1
Key words ▶ SB-431542 ● ▶ A-83-01 ● ▶ islet transplantation ● ▶ TGF-β ●
Abstract
received 07.08.2014 accepted 29.10.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1395567 Published online: November 27, 2014 Horm Metab Res 2015; 47: 78–83 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Dr. A. J. F. King Hodgkin Building King’s College London Guy’s campus London SE1 1UL UK Tel.: + 44/20/7848 6402 Fax: + 44/20/7848 6280 [email protected]
2
Diabetes Research Group, Division of Diabetes and Nutritional Sciences, King’s College, London, UK Centre for Biological Sciences, University of Southampton, Southampton General Hospital, Southampton, UK
▼
Islet transplantation is a potential treatment for Type 1 diabetes but long term graft function is suboptimal. The rich supply of intraislet endothelial cells diminishes rapidly after islet isolation and culture, which affects the revascularisation rate of islets after transplantation. The ALK5 pathway inhibits endothelial cell proliferation and thus inhibiting ALK5 is a potential target for improving endothelial cell survival. The aim of the study was to establish whether ALK5 inhibition prevents the loss of intraislet endothelial cells during islet culture and thus improves the functional survival of transplanted islets by enhancing their subsequent revascularisation after implantation. Islets were cultured for 48 h in the absence or presence of 2 different ALK inhibitors: SB-431542 or A-83-01. Their vascular density after culture was analysed using immu-
Introduction
▼
Although the success rate of clinical islet transplantation has markedly improved in the last few years, it is clear that the results are not optimal with many patients reverting to insulin dependence [1]. One factor that may affect the long term function and survival of transplanted islets is suboptimal revascularisation [2]. Endogenous islets are highly vascularised, allowing rapid glucose sensing and insulin secretion. During isolation from the pancreas, islets are separated from their vascular network and although endothelial cells are detectable within the islets immediately after isolation, their numbers diminish rapidly with culture [3–5]. In clinical islet transplantation, islets are routinely cultured to allow for microbial screening and recipient patient preparation [6–10]. It has been reported that intraislet endothelial cells
King AJ F et al. ALK5 Inhibition in Islet Transplantation … Horm Metab Res 2015; 47: 78–83
nohistochemistry. Islets pre-cultured with the ALK5 inhibitors were implanted into streptozotocin-diabetic mice for either 3 or 7 days and blood glucose concentrations were monitored and vascular densities of the grafts were analysed. Islets cultured with ALK5 inhibitors had higher vascular densities than control-cultured islets. Three days after implantation, endothelial cell numbers in islet grafts were minimal, irrespective of treatment during culture. Seven days after implantation, endothelial cells were evident within the islet grafts but there was no difference between control-cultured islets and islets pretreated with an ALK5 inhibitor. Blood glucose concentrations were no different between the treatment groups. In conclusion, inhibition of ALK5 improved intraislet endothelial cell numbers after islet culture, but this effect was lost in the early post-transplantation period.
contribute to the revascularisation process of islet grafts when freshly isolated islets are transplanted [4, 11, 12] whereas in the case of cultured islets, donor islet endothelial cells contribute minimally to the vasculature of islet grafts [12]. It has been suggested that freshly isolated islets have an increased rate of revascularisation [12, 13] and this has been associated with better graft outcome [13]. It is therefore potentially of interest to preserve endothelial cells within cultured islets to enhance graft revascularisation and hence islet survival and function. Transforming growth factor beta (TGFβ) is an important mediator in the survival of endothelial cells [14]. Its actions are mediated through an oligomeric receptor complex, which consists of 2 receptors: Type 1 (TβR1) and Type 2 (TβR2). These receptors have serine/threonine kinase activity and TβR2 activated by the bound TGFβ phosphorylates and activates TβR1 [also known
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
ALK5 Inhibition Maintains Islet Endothelial Cell Survival but does not Enhance Islet Graft Revascularisation or Function
Endocrine Research 79
Materials and Methods
▼
Islet transplantation
Islets were transplanted under the kidney capsule as described previously [21]. A lumber incision was made in isoflurane anaesthetised mice and the kidney exposed. One hundred and fifty control or ALK5-inhibitor treated islets were implanted under the kidney capsule using PE50 tubing (Becton Dickinson, Sparks, MD, USA) and a Hamilton syringe (Fisher, Pittsburg, PA, USA) in a suboptimal mass model. Blood glucose and weight were monitored before the animals were killed by cervical dislocation at either 3 or 7 days.
Histology
Animals
Male C57Bl/6 mice aged 8–12 weeks were used for islet isolation or as transplant recipients. Recipient mice were rendered diabetic by 180 mg/kg i.p. injection of streptozotocin (SigmaAldrich, Poole, UK) and those with a nonfasting blood glucose concentration of ≥ 20 mmol/l were used as recipients. Blood glucose concentrations were determined using a blood glucose meter and strips (Accu-Chek; Roche, Burgess Hill, UK) with blood obtained from a pin prick to the tail. All animal experiments were carried out after ethical approval from our institution and under licence in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986.
Islet isolation
Islets were isolated using collagenase digestion followed by gradient purification as previously described [18]. Briefly, mice were killed by cervical dislocation and the pancreatic duct was clamped at the ampulla of Vater. Two ml of collagenase (1 mg/ml, type XI; Sigma-Aldrich) was injected into the pancreatic duct. The pancreata were digested at 37 °C for 10 min before washing and purification of the digest using a density gradient (Histopaque-1077, Sigma-Aldrich). After washing, islets were handpicked and cultured as described below.
Islet culture and treatment
Islets were cultured in RPMI 1640 with 10 % foetal calf serum and 1 % penicillin/streptomycin. Islets were cultured for 48 h in the presence of either 10 μM SB-431542 {4-[4-(1,3-benzodioxol-5-yl)5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide} [19] or 10 μM A-83-01 [3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)1H-pyrazole-1-carbothioamide) [20]. The effective concentrations of the ALK5 inhibitors were based on our previous studies [16], and on those from other groups [20]. Control islets were cultured in media containing the equivalent volume of vehicle (0.1 % DMSO v/v). Islets were then used for transplantation studies, for measurement of insulin release or for quantification of endothelial cells as described in more detail below. Some islets were cultured for a further 3 or 7 days in parallel with the in vivo studies after which endothelial cells were quantified.
Insulin release
either 2 mM or 20 mM glucose. After 1 h, samples of the incubation medium were taken and stored at 20 °C until assayed for insulin content using in-house radioimmunoassay.
Islets were pre-incubated for 2 h in RPMI-1640 medium containing 2 mM glucose. Groups of 3 islets were incubated at 37 °C in a bicarbonate-buffered physiologic salt solution containing 2 mM calcium chloride and 0.5 mg/ml bovine serum albumin and
Cultured islets or kidneys containing islet grafts were fixed in 4 % formalin for 2 or 48 h respectively and paraffin-embedded. Endothelial cells were stained using CD34 antibodies as previously described in detail [21]. Briefly, antigen retrieval was carried out on the samples [2 min in 10 mM citric acid solution (pH 6.0) in a pressure cooker]. Sections were incubated for 1 h at room temperature with a monoclonal rat anti-CD34 antibody (1:500; AbD Serotec, Kidlington, UK) followed by a rabbit biotinylated anti-rat antibody (1:200; Vector Laboratories, Peterborough, UK). Sections were incubated with streptavidinhorseradish peroxidase (Dako, Ely, UK) and diaminobenzidine.
Intraislet endothelial cell quantification
Photographs were taken of the CD34-stained sections using an Olympus 2000 microscope and QCapturePro at either × 10 and × 20 magnifications. Image J software (http://rsbweb.nih. gov/ij/) was used to determine islet area and vascular density by an individual blinded to the origin of the sample.
Statistical analysis
Statistical analysis was carried out using SigmaPlot. When 2 groups were compared a t-test was used. ANOVAs were used to compare more than one group; a two-way ANOVA was used to analyse insulin release, a two-way repeated measures ANOVA was used to analyse blood glucose measurements and a one-way ANOVA on ranks was used to analyse vascular density after culture. p-Values 0.05 two way ANOVA, n = 8) and islet insulin content (Panel b, p > 0.05, one way ANOVA, n = 8) after 2 days culture in the presence or absence of 10 μM of the ALK5 inhibitors SB-431542 and A-83-01. Vascular density of islets after 2-day culture in the presence or absence of 10 μM SB-431542 or 10 μM A-83-01 (Panel c; * p 0.05 2 way repeated measures ANOVA, n = 3). Prior to implantation islets had been cultured for 2 days in the presence or absence of 10 μM SB-431542.
grafts to reverse the streptozotocin-induced hyperglycemia and maintain normoglycemia during the 7 day post-implantation ▶ Fig. 4b). period ( ●
Discussion
▼
The importance of endothelial cells in normal islet development and function has been well established [2], and there is some evidence that the rate and extent of islet revascularisation influences the outcome of islet transplantation to reverse diabetic hyperglycemia [13]. Endogenous islets are highly vascularised, but after isolation and culture the numbers of endothelial cells rapidly diminish [3–5]. After transplantation, the vascular density of the graft increases with time from initiation of revascularisation at 3–4 days until 28 days [12], when numbers of endothelial cells are partially replenished, but the vascular density is still considerably less than in endogenous islets [22]. This impaired vascular density of implanted islets has been associated with reduced functionality [13]. Strategies to improve vascular density in transplanted islets include implanting freshly isolated islets [3, 13], genetically manipulated islets [23, 24], or co-transplanting other cell types with the islets [21, 25]. Despite encouraging results in experimental studies each of these strategies has limitations when applied to the clinical setting. Freshly isolated islets are no longer used routinely for clinical islet transplantation because of the requirement of a period in tissue culture to allow microbial testing and preparation of the graft recipient. Genetic manipulation of islets to enhance revascularisation is still relatively inefficient and will require thorough safety testing before genetically
modified islets will be acceptable in a clinically setting. Similarly, co-transplanting other cells with islets to improve islet revascularisation requires stringent safety testing prior to becoming clinically applicable. The ability to improve vascular density of islet grafts by defined pharmacological treatment of the islets before transplantation avoids many of these problems, including potential off-target systemic effects of the drug. This strategy has had some experimental success in targeting beta cell function and survival, where several pharmacological interventions prior to transplantation have had a positive effect on transplantation outcome [26–28]. However, attempts to improve islet graft function by using proangiogenic factors such as VEGF and FGF during in vitro culture have failed to enhance the vascular density of the islets or their subsequent function in vivo [29]. We have previously shown that inhibition of the ALK5 activity prevents the impairment of endothelial cell viability in vitro [16], suggesting that ALK5 may be a useful target for maintaining endogenous endothelial cell viability in isolated islets during in vitro culture. In the present study, we have demonstrated that 2 structurally-dissimilar pharmacological inhibitors of ALK5 were partially effective in reducing the decline in intraislet endothelial cell number during a 2 day culture period. These observations confirm that the viability of endothelial cells within the islets is regulated by the TGFβ pathway, and offer a simple and defined pharmacological method for maintaining vascular density prior to transplantation. Pre-treatment with ALK5 inhibitors had no deleterious effects on islet β-cell function, as assessed by maintained insulin content and glucose-induced insulin secretion, which are perhaps the most important functional parameters for islet grafts. It has recently been reported that ALK5 inhibition enhanced glucose-induced insulin secretion from
King AJ F et al. ALK5 Inhibition in Islet Transplantation … Horm Metab Res 2015; 47: 78–83
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Fig. 3 Blood glucose concentrations of mice implanted with islets that had been pre-cultured in the presence or absence of the ALK5 inhibitor A-83-01 (Panel a, p > 0.05 two way repeated measures ANOVA, n = 4–5) or SB-431542 (Panel b, p > 0.05 two way repeated measures ANOVA, n = 4–5).
82 Endocrine Research
Conflict of Interest
▼
The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
References
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mouse islets when the inhibitor was present during glucose challenge [30]. However, our functional measurements were performed in the absence of the ALK5 inhibitors, after the pretreatment period with the inhibitors, to better reflect islet function in vitro immediately before implantation. Nonetheless, the available data suggest that using ALK5 inhibition as a strategy to maintain islet endothelial cells will not comprise islet secretory function and may even have acute beneficial effects on insulin release. It has been reported that endothelial cells in freshly isolated islets contribute more to graft revascularisation than those in cultured islets [4, 11, 12], resulting in higher vascular density and improved transplantation outcomes [3, 13], suggesting direct beneficial outcomes from implanting islets with higher numbers densities of endothelial cells. However, the enhanced endothelial cell density of cultured islets achieved by ALK5 inhibition was not maintained when the islets were transplanted into the in vivo environment. Thus, within 3 days of implantation endothelial cell number in ALK5-inhibitor pre-treated islets had declined to the similar low levels detected in untreated islets. Similarly, ALK5 inhibition had no effect on the revascularisation of transplanted islets within the first 7 days after implantation, presumably driven primarily via invasion of host endothelial cells, such that intraislet endothelial cell density was not different between ALK5-inhibitor pre-treated and control untreated islets. Consistent with the lack of effect on intraislet endothelial cells, ALK5 inhibition had no functional effects on the ability of the transplanted islets to regulate blood glucose levels at either 3 or 7 days after implantation, by which stage the grafts were maintaining normoglycemia. There are a number of reasons why the promising in vitro observations using ALK5 inhibitors did not translate into enhanced graft revascularisation and function in vivo. First, it is possible that ALK5 inhibition did not maintain a sufficient density of intraislet endothelial cells to have any significant functional effects in vivo, although this seems unlikely since pre-incubation with A-83-01 maintained endothelial cell density at approximately 75 % of that measured in freshly isolated islets. Second, the superior outcomes of studies using freshly isolated islets may reflect enhanced function of the intraislet endothelial cells rather than their higher density, in which case strategies aimed solely at maintaining endothelial cell number are unlikely to improve the functional outcomes of islet transplantation. Finally, it is possible that the intraislet endothelial cells play a relatively minor role in graft revascularisation, or that the extent of islet vascularisation is not critical for β-cell function, and the improved transplantation outcomes using freshly isolated islets reflect better β-cell function rather than any vascular parameter. This conclusion is consistent with a recent report that a prolonged 2-fold reduction in islet vascularisation in vivo had little effect on function [31], and may suggest that pharmacological strategies aimed at improving the outcomes of islet transplantation may be better targeted at the β-cell than at the endothelial cell.
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