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”Denna dagen, ett liv.” Thomas Thorild (1759-1808)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Olerud, J., Johansson, M., Christoffersson, G., Lawler, J.,

Welsh, N., Carlsson P-O. Thrombospondin-1: An islet endo-thelial cell signal of importance for beta-cell function. Submit-ted

II Johansson, Å., Olerud, J., Johansson, M., Carlsson, P-O. An-giostatic factors normally restrict islet endothelial cell prolifera-tion and migration: Implications for islet transplantation. Sub-mitted

III Olerud, J., Johansson, M., Lawler, J., Welsh, N., Carlsson, P-O. (2008) Improved vascular engraftment and graft function after inhibition of the angiostatic factor thrombospondin-1 in mouse pancreatic islets. Diabetes 57(7):1870-7

IV Johansson, M*., Olerud, J*., Jansson, L., Carlsson, P-O. (2008) Prolactin treatment improves engraftment and function of trans-planted pancreatic islets. Endocrinology. Dec 18. [Epub ahead of print]

*Shared contribution as first author

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11 Pancreatic islet morphology ..................................................................... 11 Angiogenesis ............................................................................................ 12 Endothelial cells ....................................................................................... 13 Islet microcirculation ................................................................................ 14 Islet transplantation .................................................................................. 14 Islet engraftment ....................................................................................... 15 Thrombospondin-1 ................................................................................... 16 Prolactin ................................................................................................... 18 RNA interference ..................................................................................... 19

Aims of thesis ............................................................................................... 21

Study Design and Methods ........................................................................... 22 Animals (Papers I-IV) .............................................................................. 22 Chemicals (Papers I-IV) ........................................................................... 22 Glucose and insulin tolerance tests (Paper I) ........................................... 22 Treatment with TGFbeta-1 activating recombinant protein (Paper I) ...... 23 Islet isolation and culture (Papers I-IV) ................................................... 23 Characterization of pancreatic islets and siRNA-transfected islet cells (Papers I and III) ...................................................................................... 24 Gene expression analysis (Papers I, III, IV) ............................................. 25 Protein expression analysis (Paper III) ..................................................... 25 Isolation and culture of islet endothelial cells (Papers I and II) ............... 25 Isolation and culture of liver endothelial cells (Paper II) ......................... 26 Islet conditioned culture medium (Paper II) ............................................. 26 Endothelial cell migration assay (Paper II) .............................................. 26 Endothelial cell proliferation assay (Paper II) .......................................... 27 Encapsulation (Paper I) ............................................................................ 27 Synthetic TSP-1 siRNA transfection of islet cells (Paper III) .................. 27 Islet transplantation (Papers I, III and IV) ................................................ 28 Measurements of blood flow and oxygen tension (Papers III and IV) ..... 28 Measurements of blood parameters (Papers I, III and IV) ....................... 29 Light microscopic evaluation (Papers I, III and IV) ................................. 29 Ki67 and TUNEL staining (Paper IV) ..................................................... 30 Function of transplanted encapsulated islets (Paper I) ............................. 31

Perfusion of islet grafts (Papers I and III) ................................................ 31 Minimal islet mass (Paper IV).................................................................. 31 Statistical analysis (Papers I-IV) .............................................................. 32

Results and Discussion ................................................................................. 33 Abundance of TSP-1 in different organs and its localization in pancreatic islets (Papers I and III) ............................................................................. 33 Role of TSP-1 in pancreatic islets (Paper I) ............................................. 33 Importance of the specific contribution of endothelium-derived TSP-1 in islets (Paper I) ........................................................................................... 34 Activation of TGFbeta-1 in pancreatic islets (Paper I) ............................ 35 In vitro evaluation of means to stimulate revascularization of transplanted islets (Paper II) ......................................................................................... 35 Protein and mRNA expression of angiogenic and angiostatic factors in islets treated with TSP-1 siRNA or prolactin (Papers III and IV) ............ 36 TSP-1 and PRL affect islet graft blood flow (Papers III and IV) ............. 36 TSP-1 and PRL affect oxygen tension (Papers III and IV) ...................... 37 TSP-1 and PRL affect islet vascular density (Papers III and IV) ............. 37 Influence of TSP-1 and PRL on beta-cell proliferation, apoptosis and islet graft volume (Papers III and IV) .............................................................. 38 Influence of TSP-1 and PRL on islet graft function (Papers III and IV) .. 39

Conclusions ................................................................................................... 40 Paper I ...................................................................................................... 40 Paper II ..................................................................................................... 40 Paper III .................................................................................................... 40 Paper IV ................................................................................................... 41

Swedish Summary ........................................................................................ 42

Acknowledgements ....................................................................................... 44

References ..................................................................................................... 46

Abbreviations

Alpha1-AT Alpha1-antitrypsin BS-1 Bandeiraea simplicifolia-1 BSA Bovine serum albumin CM Culture medium dsRNA double stranded DNA EC Endothelial cell ECM Extracellular matrix ELISA Enzyme linked immunosorbant assay FCS Fetal calf serum FGF-2 Fibroblast growth factor-2 GnRH Gonadotropin releasing hormon HBBS Hank´s balanced salt solution ICCM Islet conditioned culture medium IEQ Islet equivalents KRBH Krebs-Ringer bicarbonate buffer with

hepes LDH-A Lactate dehydrogenase-A miRNA micro-RNA MMP-9 Matrix metalloproteinase-9 NO Nitric oxide PBS Phosphate buffered saline PDGF Platelet derived growth factor PRL Prolactin RISC RNA induced silencing complex RNAi RNA interference siRNA small interfering RNA TBS Tris buffered saline TGFbeta Transforming growth factor beta TPU Tissue perfusion units TSP-1 Thrombospondin-1 TSR Thrombospondin type 1 repeat TUNEL Terminal dUTP nick and labelling UCP-2 Uncoupling protein-2 UE Ulex europaeus VEGF Vascular endothelial growth factor

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Introduction

Type 1 diabetes mellitus is an autoimmune multifactorial disease often lead-ing to severe complications. Today there is no cure for people suffering from type 1 diabetes, but most can be adequately treated with insulin injections. However, even with continuous insulin administration the patient has a high risk of developing complications, mainly angiopathy, retinopathy, nephropa-thy and neuropathy. To avoid this it is important to maintain the blood glu-cose concentration as close to normal as possible (1). One obvious method to achieve this is transplantation of insulin producing beta-cells. However, pa-tients with a transplant need immunosuppressive medicines to avoid rejec-tion and possibly also recurrence of disease. To avoid the difficult surgery and morbidity associated with whole-pancreas transplantation, implantation of isolated islets of Langerhans is an attractive option. However, there are several problems associated with this transplantation procedure. One major problem is that islets from at least two donors are generally needed to cure a patient when isolated islets are implanted, which means that the current shortage of donors is further emphasized. During and after islet transplanta-tion procedures it has been reported that up to 70 % of the beta-cell mass disappears (2-4). One of the most important factors for the reduction of beta-cell mass during early post-transplantation is probably hypoxia. For newly transplanted islets it can take up to 2 weeks before the revascularization have finished (5, 6), and even thereafter transplanted islets remain less vascula-rized and oxygenated than normal (7, 8). To sustain healthy islets it is impor-tant to establish a sufficient angiogenesis. If it would be possible to accele-rate or expand this process, this would most likely improve islet transplanta-tion results.

Pancreatic islet morphology The human pancreas contains approximately 1-2 million islets, which cor-respond to 1-2 % of the total volume (9). Each islet consist of 2-3000 endo-crine cells and varies between 100-400 �m in size (10). The islet endocrine cells are surrounded by a connective tissue capsule (9, 11). In rodents, insu-lin producing beta-cells are distributed mainly in the islet core, whereas glu-cagon producing alpha-cells, somatostatin producing delta-cells, ghrelin-cells, and the pancreatic polypeptide-cells are located more peripherally.

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This organisation may be important to regulate the insulin release from the beta-cells (12). In humans, the cell distribution is more heterogenous (13).

Besides endocrine cells, the islets also contain endothelial cells (ECs), dendritic cells, fibroblasts, macrophages and nerves. Blood vessels are es-sential during pancreatic development (14). In adult islets, they are crucial for glucose sensing, supply of nutrients, release of hormones and probably also to regulate beta-cell differentiation and growth (15). The endocrine pan-creas has a blood perfusion which is approximately 5-10 times higher than the exocrine pancreas suggesting that islets of Langerhans to fulfill its func-tions need a high blood supply (16). Approximately 10 % of the islet con-sists of blood vessels (17).

Angiogenesis The formation of new blood vessels occurs through vasculogenesis and an-giogenesis (18). In the embryo, and to some extent also in adults, blood ves-sels originate from angioblasts, a process referred to as vasculogenesis. However, during adulthood the majority of blood vessels forms through angiogenesis, which involves both sprouting, i.e. outgrowth of capillaries from pre-existing blood vessels, as well as in-growth of transcapillary tissue pillars into existing blood vessels, so called intussusception. The balance between anti-angiogenic and angiogenic factors regulates these processes in normal tissues (19).

The first step in angiogenesis involves vasodilation of blood vessels and EC activation adjacent to the angiogenic stimuli, resulting in increased vas-cular permeability and blood flow. This is partially due to an elevated nitric oxide (NO) production. Later, the ECs start to migrate and proliferate from the dilated blood vessels, processes which depend on stimulation by pro-angiogenic factors such as vascular endothelial growth factor (VEGF), angi-opoietin-2 and proteinases (20). The migration occurs toward areas low of oxygen tension where the hypoxic cells secrete pro-angiogenic factors. The newly formed blood vessels are sensitive to regression, and to avoid this, the ECs recruit supporting mesenchymal cells through its production of platelet-derived growth factor (PDGF) (21). ECs and microvessels then become sta-bilized, a mechanism involving transforming growth factor beta (TGFbeta) and angiopoietin-1. The mesenchymal cells differentiate into pericytes.

Under normal conditions, most ECs of the adult are in a quiescent state i.e. they replicate slowly. ECs are maintained quiescent by a close balance of positive and negative regulators of angiogenesis in tissues, resulting in no angiogenesis. Angiogenesis is extensively dependent on a so called “angi-ogenic switch” i.e. increase in angiogenic factors or decrease in angiostatic factors, or both. The most important angiogenic factors are VEGF, PDGF, matrix metalloproteinase-9 (MMP-9), and fibroblast growth factor-2 (FGF-

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2), whereas angiostatic factors include alpha1-antitrypsin (alpha1-AT), en-dostatin and thrombospondin-1 (TSP-1) (Figure 1).

Lack of adequate angiogenesis regulation can contribute to severe patho-logical conditions, which include hemangioblastoma, ischemic vascular dis-eases, ophthalmic and rheumatic diseases, psoriasis and tumor growth (22). However, also during certain physiological conditions, such as pregnancy and wound healing, angiogenesis takes place (23).

Figure 1: The balance between angiogenic and angiostatic factors regulates angi-ogenesis. An increased amount of angiogenic factors will lead to angiogenesis, whereas angiostatic factors inhibit this process. Under normal physiological condi-tions angiostatic factors are predominant and no angiogenesis occur.

Endothelial cells ECs are covering blood vessels, lymphatic vessels and anterior eye chamber. They are heterogenous depending on the location and size of the vessels (24, 25). Common to all blood vessels, irrespective of location and size, are sev-eral processes including blood flow regulation, hemostasis, capacity to serve as immunological barrier between tissues and blood, and angiogenesis (24).

Depending on morphology, capillaries can be divided into three different classes: continuous, fenestrated or discontinuous. Continuous capillaries do not have openings between the ECs. Fenestrated capillaries have permeable pores allowing passage of molecules and a quick transport of secreted hor-mones. Discontinuous capillaries have gaps between the ECs for cell trans-port between the interstitium and the blood stream. The vessel characteristics regulate to which class they belongs. Vessels that mediate the transport of released hormones into the blood are fenestrated. Such vessels are found in endocrine glands, which include endogenous pancreatic islets. Continuous ECs are found in skeletal muscle, heart, lung and brain, whereas disconti-nuous ECs are found in bone marrow, liver and spleen. Compared with fene-strated capillaries, discontinuous ECs lack basement membranes (26).

Some organs, e.g. the liver may have several forms of ECs depending on different functions. Whereas the portal vein transports oxygen-deprived blood rich in nutrients from the splanchnic region, tributaries of the hepatic arteries supplies the liver with oxygenized arterial blood. Interestingly, it has

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been shown that the liver ECs in the sinusoids are the major contributors to insulin uptake in the body and transport to the glycogen-rich hepatocytes (27).

The reciprocal interaction between islet ECs and beta-cells is likely of major importance regarding islet function. Studies have shown that beta-cells, which release VEGF-A, contribute to the formation of a dense network of fenestrated capillaries within islets (28, 29). Five percentage of the surface of islet capillaries consists of fenestrations (17). The richly fenestrated capil-laries are important for delivery of nutrients and oxygen to the islet cells, as well as for rapid delivery of hormones to the systemic circulation. In recent years it has become obvious that the islet ECs also may influence beta-cell function and growth through production of e.g. laminins as well as hepato-cyte growth factor (30, 31).

Islet microcirculation The number of blood vessels supplying islets may vary depending on the size of the islet. Larger islets are connected with up to three arterioles sepa-rate from those to the exocrine parenchyma, whereas smaller islets usually receive only one. Smaller islets are generally drained through small venules connected with exocrine capillaries, whereas the drainage of larger islets passes via postcapillary venules directly into the portal vein. It has been ob-served that compared with the exocrine capillary system the endocrine vas-cular system is wider and their ECs are 10 times more fenestrated (17). The increased fenestrations are believed to be important for high islet capillary permeability.

Despite that the endogenous islets comprise approximately 1 % of the pancreas their blood perfusion is 5-15 % of that to the whole pancreas. It is believed that the endocrine blood perfusion is autonomously regulated com-pared to that of the exocrine pancreas depending on the endocrine cell de-mand. There are several hypotheses in which order the cells in the endocrine pancreas are perfused (32). The most predominant hypothesis suggest that beta-cells are perfused before other endocrine cells (33-35).

The islet blood perfusion is controlled by nervous, endocrine and meta-bolic mechanisms (36).

Islet transplantation Today, patients suffering from type I diabetes need life-long insulin injec-tions. The only way to achieve insulin independency is transplantation of a sufficient number of beta-cells. However, even after islet transplantation, there is often a need for supplementary insulin therapy. The positive effect of transplantation is the improved fine adjustment of glucose homeostasis.

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The first pancreas-kidney transplantation was performed in 1966 (37). Transplantation of isolated islets was performed more than a decade later, in 1977 (38). Human islets have been implanted into several sites in the clinical settings. At present, most human islet transplantations are performed to the liver, using the “Edmonton” protocol (39). The major reasons for this are the minor surgical procedures involved and that the liver is the main target for insulin uptake. Because of the islet functional sensitivity to low oxygen ten-sion (40, 41), this site may however, not be optimal for the islets. Especially since newly transplanted islets are disconnected from vascular supply and the oxygen uptake is achieved through diffusion (42, 43). Furthermore, se-cretion of insulin to the liver could lead to glucolipotoxicity, resulting in beta-cell death due to elevated glucose and triaglycylglycerol levels (44). There are also immunological problems with the liver. The macrophages of the liver, Kupffer cells (45), located to the sinusoidal walls and the presence of instant blood mediated inflammatory reactions (46, 47) are likely to lead to an increased endocrine cell death post-transplantation. Immunosuppres-sive and anticoagulant treatments are necessary to avoid rejection and blood clot formation, respectively. It should be noted that at least 2 donors, i.e. more than 10,000 islet equivalents (IEQ)/kg body weight are needed for successful human islet transplantations (39), which underlines the fact that the liver is far from an optimal implantation site. After one year 80 % of the recipients are insulin-independent, a figure which declines to 10 % at five years post-transplantation (3).

Recently, Lund et al compared intramuscular and intraportal islet trans-plantations. To cure diabetic rats the double amount islets were required when transplanted to the intramuscular site, i.e. 4000 compared with 2000 IEQ at the intraportal site. This indicates that the intramuscular transplanta-tions are suboptimal, but the easy access of the transplantation site gives the opportunity for bioengineered matrices which could improve the transplanta-tion in the future (48).

In experimental studies in rodents, islets are most often transplanted be-neath the renal capsule, where they are easily retrieved. However, clinically the amount of islets needed to provide cure in humans is probably too large to be implanted at the renal subcapsular site.

Islet engraftment Engraftment is the adaptation of the transplanted tissue to the implantation organ, and encompasses revascularization, reinnervation and reorganization of stromal and endocrine cells. The engraftment process is crucial for islet survival and function. During isolation of pancreatic islets their blood ves-sels and nerve fibers become disconnected. In the immediate post-transplantation period, oxygen and nutrients must therefore enter the islets through diffusion. To achieve an optimal function and reduce endocrine cell

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death it is important to re-establish the vascular connections with the system-ic circulation. The revascularization is partially restored within 7-14 days post-transplantation (49-52). However, studies of rodent and human islets experimentally transplanted to the renal, splenic or hepatic subcapsular space have shown that the revascularization is suboptimal to achieve appropriate graft oxygenization (6, 42, 53).

During clinical transplantations islets are transplanted intraportally (39, 54), which make islet retrieval difficult and engraftment hard to evaluate. However, also intraportally implanted islets become poorly revascularized (55, 56) and oxygenated (57). Moreover, considering the fragmentation of the islets after embolization into the liver (58), the engraftment of intrapor-tally transplanted islets is likely to be disturbed.

Thrombospondin-1 Thrombospondin-1 (TSP-1) is one of many angiostatic factors, and was de-scribed as the first natural inhibitor of angiogenesis (59). It received its name due to its release when platelets were stimulated with thrombin (60). TSP-1 is one member of a group of five thrombospondins, which are matrix glyco-proteins.

The five thrombospondins can be divided into two subgroups with regard to their structure. All thrombospondins have conserved domains, which in-clude an epidermal growth factor-like (type-2 repeats), a Ca2+-binding (type-3 repeats) and a C-terminal domain. TSP-1, together with TSP-2, also has a N-terminal domain, a coiled-coil, a procollagen and a thrombospondin type 1 repeat (TSR) properdin domain (type-1 repeats) (Figure 2) (61). Figure 2: TSP-1 consists of four functional domains (modified from (62)).

Since TSP-1 was first discovered as a protein stored in alpha-granules of platelets, it has been showed to be involved in many different processes, including platelet aggregation, inflammatory response and regulation of an-giogenesis during wound repair and tumor growth (63). In activated plate-lets, TSP-1 release participates in the blood clot formation together with fibrin, plasminogen, urokinase and histidine-rich glycoprotein. It also takes part in the immune response by mediating contact between platelets, leuco-cytes and recognition of apoptotic neutrophils by macrophages (64), and indirectly by activation of TGFbeta-1 (65, 66). There are several processes

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involved in TSP-1 inhibitory functions on ECs such as interference with their proliferation, migration, proteolysis, capillary morphogenesis and growth factor mobilisation (67, 68).

Both TSP-1 and TSP-2 regulate extracellular matrix (ECM) by binding to collagens and fibronectin. TSP-1 also modulates ECM proteases like matrix MMP-9 and plasmin. Moreover, TSP-1 could up-regulate the formation of matrix proteins through TGFbeta (Figure 3) (62). Figure 3: TSP-1 regulates angiogenesis through the CD36 receptor on the endo-thelial cells, inhibition of MMP-9 and activation of TGFbeta (modified from (62)).

TSP-1 angiostatic activity is localized to its procollagen domain and TSR region. The TSR motif mediates interactions with several receptors including CD36 and beta1-integrins. Interaction with the CD36 receptor on ECs inhi-bits cell migration (69) and induces apoptosis in actively dividing cells (70). TSP-1 mediated apoptosis is regulated through both the intrinsic and extrin-sic pathway (71, 72). The intrinsic pathway involves up-regulation of Bax and decrease of Bcl-2 as a result of hypoxia, induction of reactive oxygen species, DNA damage or nutrient withdrawal. The extrinsic pathway in-volves formation of the TSP-1/CD36 complex which recruits and activates p59fyn leading to activation of caspase-3, which activates p38MAPK fol-lowed by up-regulation of Fas-ligands leading to apoptosis (73). TSP-1 is also believed to mediate its angiostatic properties through the cell surface proteoglycans, e.g. heparin sulfate, that act as a co-receptor for FGF-2 (74, 75). Mice treated with TSP-1 show an increased apoptosis in tumour ECs, whereas surrounding host capillaries are unaffected (70). Only the remodel-ling vasculature is sensitive to the inhibitors of angiogenesis. This is also shown in other studies where treatment with angiogenic inhibitors leads to normal vascular conditions without hemorrage (76). The beta1-integrins inhibit VEGF induced endothelial cell migration through the phosphoinositid 3 kinase signaling pathway (77).

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The serum response element in the promoter region of TSP-1 is believed to be responsible for regulation of synthesis of TSP-1 in many cell-types. TSP-1 is also classified as an immediate early response gene due its quick response in conditions of stress e.g., hypoxia and heat shock. Some of the factors responsible for up-regulation of TSP-1 are PDGF, TGFbeta, and FGF-2. However, it is down-regulated by the cytokines interleukin-1 beta and tumor necrosis factor alpha (reviewed in (63)). p53 has been reported to up-regulate TSP-1 in fibrosarcomas and breast carcinomas (78), whereas TSP-1 is down-regulated by Ha-ras, v-src, v-myc and c-jun (79-81).

TSP-1 has recently been shown to be present in islets (31, 82) and sub-jected to changes in gene expression during pregnancy and diabetes (31, 83). Hypoxia does not seem to influence TSP-1 gene expression in islets (82).

Prolactin Prolactin (PRL) is a 23 kDa peptide hormone, which is related to growth hormone and placental lactogen with regard to its sequence and domain structure (84). It is mainly secreted by lactotrophic cells in the adenohypo-physis (85), but also by various immune cells (86), mammary gland epithe-lium (87), the decidua (88) and the nonpregnant uterus (89). A total of more than 300 physiological activities have been reported for PRL, within such diverse areas as behavior, growth, immunity, metabolism, osmoregulation and reproduction (90-92).

PRL secretion can be regulated through several mechanisms. The main physiological stimuli increasing secretion of PRL from the adenohypophysis are nursing (93), stress (94) and high levels of estradiol (95). Besides estra-diol several other hormones stimulate PRL secretion, such as thyroid- and gonadotropin-releasing hormones (GnRH) (96, 97). Thyroid releasing hor-mone has been reported to stimulate PRL release both in vitro and in vivo (96, 98). Dopamine, on the other hand, is the major hypothalamic inhibitor of PRL synthesis and release (99). During late pregnancy elevated blood estradiol concentrations increase PRL, thereby preparing the mammary gland for lactation. Certain cancers, as well as the use of PRL- regulating substances, can lead to excessive PRL release, i.e. hyperprolactinemia, re-sulting in amenorrhea and galactorrhea in women. Anti-psychotic medicines could also lead to hyperprolactinemia by affecting dopamine release. Men suffering from hyperprolactinemia have an increased risk of developing hy-pogonadism and gynecomastia.

PRL has a diverse role in angiogenesis. The proteolytic 16 kDa fragment of native PRL inhibits angiogenesis (100, 101). Inhibition of EC prolifera-tion seems to involve inhibition of the Ras/Raf/MEK/MAPK signaling pathway, whereas stimulation of EC apoptosis is regulated through activa-tion of caspases and conversion of antiapoptotic Bcl-XL to proapoptotic Bcl-XS (102). Furthermore, PRL induces and increases expression of plasmino-

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gen activator inhibitor-1 (103), which inhibits ECM degradation through irreversibly binding to urokinase type plasminogen activator. On the other hand, the non-reduced 23 kDa form of PRL promotes angiogenesis (104). The dominating effect of PRL on angiogenesis depends on the model being used and the local vascular conditions (104), such as increased PRL receptor expression due to injury.

Exposure of pancreatic islets of rats, mice and humans to PRL increases DNA synthesis, insulin production and glucose- dependent insulin secretion in vitro (105, 106). In the long-term, PRL has been shown to stimulate glu-cose metabolism and associated enzymes both in vitro and in vivo (107), and to lead to increased beta-cell growth and insulin secretion (108). Even after one week of treatment with PRL, positive effects on beta-cell proliferation in rat islets persisted (105). This effect was also observed in primary cultures of human islets together with higher basal insulin release (109). Treatment with recombinant human PRL on primary cultures of human islets indicates that this response is mediated through janus kinase-2, and signal transducer and activator of transcription 1, 3 and 5 (109).

RNA interference RNA interference (RNAi) is a naturally occurring biologic process first dis-covered in 1998 when double-stranded RNA (dsDNA) was injected in Cae-norhabditis elegans, which resulted in sequence specific gene silencing (110). Today RNAi provides a large number of possibilities to manipulate gene expression both in vitro and in vivo. Depending on the RNAi construct and transfection vector being used, RNAi can be applied to both short and long-term gene silencing. The mechanism behind RNAi silencing involves two major steps (111). Firstly, dsRNA is recognized by the RNAi nuclease Dicer. Dicer consists of a helicase and a RNA III domain (112), which de-grades the dsRNA to 21-25 nucleotide long fragments called small interfer-ing RNA (siRNA) (113). In the second step siRNA is recognised and binds to a RNA-induced silencing complex (RISC). Within the RISC complex the siRNA will be unwinded thereby removing the sense strand. The unwound antisense strand binds to its complementary site on the target mRNA and the RNase enzyme Slicer degrades mRNA not bound to the siRNA complex (114). Exogenous synthetic siRNA or endogenously expressed siRNA could also be incorporated directly into the RISC complex, avoiding the Dicer cleaving step of long dsRNA.

Another way to achieve gene silencing is through the micro-RNA (miR-NA), closely related to siRNA (115), which regulates genes in both plants (116) and mammals (117). miRNA is a 22 nt long noncoding endogenous double-stranded RNA-molecule responsible for mRNA cleavage or transla-tional repression. Compared with siRNA, miRNA-induced silencing is more complex. miRNA is transcribed by polymerase II, and then cleaved by the

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RNase III endonuclease Drosha, giving rise to a stem loop intermediate (118). This is transported from the nucleus to the cytoplasm through the actions of Ran-GTP and the export receptor exportin-5 (119). Dicer cleaves the stem loop in the cytoplasm leaving a miRNA duplex that is incorporated into the RISC complex. After this, the silencing mechanism is the same as with siRNA. If the strand is 100% complementary, mRNA degradations will occur, whereas partial complementarity will lead to translational repression (111).

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Aims of thesis

Paper I In this study we analyzed where TSP-1 is mainly expressed within endogen-ous pancreatic islet cells. Furthermore, we evaluated the functional impor-tance of TSP-1 for pancreatic beta-cells postnatally. Paper II The aim of this study was to investigate which factors released from pan-creatic islets that normally impair the revascularization process after trans-plantation. Paper III The objective of this study was to investigate if islets chronically deficient of TSP-1, or with a transient reduction of TSP-1, have an improved revascula-rization and function post-transplantation. Paper IV In this paper we tested the hypothesis that prolactin could increase angioge-nesis and function of transplanted pancreatic islets.

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Study Design and Methods

Animals (Papers I-IV) Inbred, Wistar-Furth rats were purchased from B&K Universal, Sollentuna, Sweden, whereas inbred C57BL/6 and C57BL/6 (nu/nu) mice were pur-chased from M&B, Ry, Denmark. TSP-1 deficient (-/-) mice were kindly donated by Dr. Jack Lawler (Boston, MA, USA) and bred in a local animal facility (Biomedical Center, Uppsala University, Uppsala, Sweden). The TSP-1 deficient mice were obtained through homologous recombination in 129/Sv-derived ES cells implanted in C57BL/6 blastocysts. The offspring were back-crossed (N9) to a C57BL/6 genetic background. All animals had free access to food (R36, Lantmännen, Kinstad, Sweden) and tap water and were housed at a 12 hour dark/light cycle. Experiments were approved by the animal ethics committee for Uppsala University, Uppsala, Sweden.

Chemicals (Papers I-IV) All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless stated otherwise.

Glucose and insulin tolerance tests (Paper I) In glucose tolerance tests, wild-type C57BL/6 and TSP-1 deficient mice were injected with 2.5 g/kg body weight D-glucose (300 mg/ml; Fresenius Kabi, Uppsala, Sweden) into the tail vein. Just before glucose injection a blood glucose sample was taken which was labeled as time point 0. Blood glucose was then measured at 10, 30, 60 and 120 min after glucose adminis-tration. Blood samples for serum insulin analysis were collected 10 min after glucose injection and analyzed with an enzyme linked immunosorbant assay (ELISA; Mercodia, Uppsala, Sweden). In the insulin tolerance tests, similar sampling procedures were performed but human biosynthetic insulin (2 units insulin /kg body weight; Novorapid®; Novo Nordisk, Gentofte, Denmark) was injected instead of glucose, and intraperitoneally.

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Treatment with TGFbeta-1 activating recombinant protein (Paper I) The TGFbeta-1 activating recombinant protein TSR2+RFK, or the control peptide TSR2+QFK, was injected intraperitonally daily at a dose of 1 mg/kg in neonatal wild-type and TSP-1 deficient mice from day 2 after birth and for the following 3 weeks until glucose tolerance measurements were performed (120). The activating recombinant protein TSR2+RFK is the second type 1 repeat of TSP-1. Vehicle (10 mmol/l Tris-HCl (pH 7.6) in 500 mmol/l NaCl)-injected wild-type and TSP-1 deficient animals served as additional controls.

Islet isolation and culture (Papers I-IV) All animals were killed by cervical dislocation. Pancreas was then removed, cut into small pieces and placed into collagenase containing vials. The vials were shaken vigorously in a 37ºC water bath until fragmentation of the pan-creas had occurred. The collagenase digestion was terminated by addition of excess Hank’s balanced salt solution (HBSS; The National Bacteriological Laboratory, Stockholm, Sweden). The digest was washed three times with HBSS and islets were subsequently handpicked with braking pipettes under a stereo microscope. Handpicked islets were then cultured in groups of 100-150 in RPMI 1640 medium supplemented with 11 mmol/l D-glucose, 10 % (vol/vol) fetal calf serum (FCS), 2 mmol/L L-glutamine, 100 U/ml benzyl-penicillin (Roche Diagnostics Scandinavia, Bromma, Sweden) and 0.1 mg/ml streptomycin in 95%air/5%CO2 at 37ºC. Culture medium (CM) was changed every second day. Since it is very difficult to distinguish between exocrine and endocrine pancreas of neonatal mice, the collagenase digested pancreases were in these cases incubated in adhesive culture dishes over night in CM (air/CO2 95/5; 37°C) for the islets selectively to attach before islet isolation.

Human islets from heart-beating donors were kindly provided by Profes-sor Olle Korsgren (Human Islet Isolation Core Facility for the Nordic Coun-tries, Dept. Oncology, Radiology and Clinical Immunology, Uppsala Uni-versity, Uppsala, Sweden). Before delivery, functional perifusion studies were performed on batches of human islets from each donor, where insulin release was determined in response to 16.7 mmol/l glucose. The human islets were cultured in the same medium referred to above for rodent islets, with the exception that the glucose concentration was lower (5.6 mmol/l). The islets were cultured for 4-7 days before transplantation. All human islet ex-periments were approved by the human ethics committee for Uppsala Uni-versity, Uppsala, Sweden.

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Characterization of pancreatic islets and siRNA-transfected islet cells (Papers I and III) Pancreatic islets of adult male wild-type and TSP-1 deficient animals were investigated for metabolic function.

To investigate glucose-stimulated insulin release in islets, or siRNA-transfected islet cells reaggregated to islets, triplicates of 10 islets were transferred to glass vials containing 0.25 ml Krebs-Ringer bicarbonate buffer supplemented with 10 mmol/l HEPES and 2 mg/ml bovine serum albumin (BSA), hereafter designated as KRBH buffer. During the first hour the islets were incubated with 1.67 mmol/l glucose added to the KRBH buffer in 37°C (O2/CO2 95:5). Thereafter, the incubation medium was retrieved and re-placed with new KRBH buffer supplemented with 16.7 mmol/l glucose. After incubation for another hour, also this medium was retrieved. Obtained islet incubation media were stored at -20°C for later analysis of insulin con-centrations. Finally, the islet triplicates were retrieved, pooled in 200 �l dis-tilled water and homogenized through sonication. Fractions from the ho-mogenate were used for both DNA and insulin content analyses. DNA measurements were performed by fluorophotometry using a PicoGreen dsDNA Quantitation kit (Molecular probes, Eugene, OR, USA). In order to measure the insulin content a fraction from the homogenate was mixed with acid ethanol (0.18 mol/l HCl in 95% [vol/vol] ethanol), from which insulin was extracted overnight at 4°C. All insulin measurements were performed with an ELISA (Mercodia).

In order to measure glucose-stimulated (pro)insulin biosynthesis, dupli-cates of 10 islets were incubated for 2 hours at 37°C in 100 μl of KRBH buffer containing 50 μCi/ml of L-[4,5-3H]leucine (Amersham-Pharmacia, Buckinghamshire, UK) and 16.7 mmol/l glucose in an atmosphere of hu-midified air plus 5% CO2. After the incubation period the islets were washed in HBSS containing nonradioactive leucine (10 mmol/l) followed by sonica-tion in 200 μl of distilled water. The total protein biosynthesis was measured in trichloroacetic acid precipitates of the islet homogenate, whereas the amount labelled (pro)insulin was determined by an immunoabsorption tech-nique (121).

For the glucose oxidation rate analyses, triplicates of 10 islets were trans-ferred to glass vials containing 100 �l of KRBH buffer supplemented with D-[U-14C] glucose (Amersham-Pharmacia) and nonradioactive glucose at a final glucose concentration of 16.7 mmol/l (specific activity 0.5 mCi x mmol-1 x l-1). The islets were then incubated for 90 min at 37°C (O2/CO2, 95:5), and the islet glucose oxidation rates determined as described (122, 123).

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Gene expression analysis (Papers I, III, IV) Pancreatic islets, dispersed pancreatic islet cells, purified beta-cells (sorted by autofluorescence at 530 nm; FACS Calibur instrument, Becton Dicken-son, San Jose, CA, USA), insulinoma cell lines βTC-6 and INS-1, and puri-fied islet ECs (cf. below) were washed with phosphate buffered saline (PBS) followed by total RNA isolation using Ultraspec® (Biotecx Laboratories, Houston, TX, USA). First strand cDNA synthesis was performed with ran-dom nonamers (Sigma-Aldrich) and reverse transcriptase moloney murine leukemia virus H- (Finnzymes, Espoo, Finland). Semi-quantitative real-time PCR was obtained using a DyNAmoTM Capillary SYBR® Green qPCR kit (Finnzymes) and a Lightcycler system (Roche Diagnostics). PCR products were verified by agarose gel electrophoresis. Beta-actin and TATA-binding protein were used as house-keeping genes. Used primer sequences are listed in the respective papers (I, III and IV).

Protein expression analysis (Paper III) Whole islets, kidney and spleen homogenates, as well as dispersed islet cells transfected with scramble or TSP-1 siRNA and incubated 1-7 days, were washed in cold PBS following lyses in SDS sample buffer (2% SDS; 0.15 mol/l Tris pH 8.8; 10% glycerol; 5% �-mercaptoethanol; bromophenol blue; 2 mmol/l phenylmethylsulfonylfluoride), boiled for 2 min, and separated on a 6 % SDS-PAGE gel. Proteins were transferred to HypondTM-P membrane (GE Healthcare, Uppsala, Sweden). The membranes were blocked in 2.5% BSA at 4°C over night followed by incubation with TSP-1 (Ab-2, Ab-4 Lab-vision, Fremont, CA, USA; TX-17.10 Santa Cruz, Santa Cruz, CA, USA) and HSP60 antibodies (Stressgen, Ann Arbor, MI, USA) and probed with horseradish peroxidase antibody. The bound antibodies were visualized with Kodak image station 4000MM (Kodak, New Haven, CT, USA) using ECL+ (GE-Healthcare). The band intensities were calculated using Kodak Molecu-lar imaging software 4.5.1 SE.

Isolation and culture of islet endothelial cells (Papers I and II) Approximately 20 freshly isolated islets were added to a collagen matrix (1.8 mg collagen type 1/ml; Collagen GmbH, Nutacon, Leimuden, the Nether-lands) in a 24-well plate culture dish. Outgrowth of islet mesenchymal cells were stimulated in CM modified with 20% (vol/vol) FCS and 100 �g/ml EC growth supplement under normal culture conditions (124). ECs were then detached through trypsination and purified with Bandeiraea (Griffonia) sim-

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plicifolia (BS-1)-coated Dynabeads (Dynal Biotech, Oslo, Norway). Pre-vious findings have shown that the purity of the ECs are more than 90% as confirmed by positive uptake of 1,1´-dioctoadecyl-3,3,3´,3-tetramethyl-indocarbocyanine perochlorate acetylated low density lipoprotein and posi-tive staining for endothelial nitric oxide synthase, angiotensin converting enzyme, angiopoetin-2, BS-1 and CD31. Isolated cells were also verified to migrate towards VEGF (31, 124).

Isolation and culture of liver endothelial cells (Paper II) Wistar-Furth rats were anesthetized with 60 mg/kg pentobarbital followed by perfusion of the liver with 25 ml perfusion buffer (142 mmol/l sodium chlo-ride, 6.7 mmol/l potassium chloride, 10mmol/l HEPES (125)) and later with 25 mg collagenase from Clostridium Histolyticum (Roche Diagnostics, Mannheim, Germany) dissolved in 8 ml of HBSS. After the perfusion the liver was cut into pieces, transferred to a vial containing collagenase solution and shaken vigorously approximately 16 min. The homogenate was then purified with BS-1 coated dynabeads and transferred to a 24-well culture plate coated with collagen (cf. (124)).

Islet conditioned culture medium (Paper II) Wistar-Furth rat islets were cultured for six days with change of CM every second day. The collected islet conditioned culture medium (ICCM) from days 5-6 was centrifuged for 2 min at 800g to minimize cellular debris con-tamination, and thereafter kept at -70°C until use.

Endothelial cell migration assay (Paper II) To assess the migratory capacity of liver- and islet ECs a Boyden chamber with collagen coated polycarbonate membrane filter of 80 �m pore size (Whatman, Springfield Mill, UK) was used. To the lower chamber ICCM with or without 20 ng/ml VEGF, 1 �g/ml VEGF neutralizing antibody (Lab Vision, Fremont, CA, USA), 10 �g/ml endostatin (Chemicon, Themicon, CA, USA), 1 �g/ml TSP-1 (Lab Vision) and/or 10 �g/ml alpha1-AT was added. In the upper chamber the EC suspension was added. Liver- and islet ECs were fixed and stained with DAPI (Vector Laboratories, Burlingame, CA, USA) after 4 and 5 hours of incubation in normal culture condition, respectively. The number of migrating cells was counted under a fluores-cence microscope.

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Endothelial cell proliferation assay (Paper II) Equal volumes of EC suspension homogenates were plated to the wells in a collagen coated 24-well dish. Islet ECs were then counted in randomly cho-sen wells. In the remaining wells, culture medium or ICCM supplemented with VEGF, VEGF neutralizing antibodies, endostatin, TSP-1 and/or alpha1-AT (see above) was added to the ECs. The ECs were counted after 48 h. All counting was performed by the use of a Bürker chamber.

Encapsulation (Paper I) Neonatal mouse islets were encapsulated with alginate (guluronic acid 73% /mannuronic acid 27%) (Pronova Biopolymer, Drammen, Norway), in which divalent cations, such as Ca2+, crosslink guluronic acid blocks and thus form a gel (126, 127). The alginate solution contained 0.3 mol/l mannitol as an osmolyte, allowing the production of inhomogeneous capsules with a higher concentration of alginate at the surface of the bead than in the center. Algi-nate was pushed through a needle into a solution of 50 mmol/l CaCl2 and 1 mmol/l BaCl2 in the presence of a high voltage electrostatic field to enclose freshly isolated neonatal wild-type and TSP-1 deficient islets (cf. (127)). The diameter of the beads was approximately 700 μm.

Synthetic TSP-1 siRNA transfection of islet cells (Paper III) Freshly isolated islets were washed with PBS followed by less than 3 min trypsination. The trypsination was terminated with CM (see above), followed by 1 min DNAse-I treatment [(Amersham Life Science, Piscataway, NJ, USA) (33 mU/�l)]. The dispersed islet cells were carefully centrifuged, washed with RPMI 1640 and divided into two equal fractions. Each fraction was added to a non-adhesive dish that had been pre-coated with FCS for 30 min at room temperature and washed with RPMI 1640. A transfection rea-gent consisting of Lipofectamine® (Invitrogen, Lidingö, Sweden) and four TSP-1 sequences (Qiagen, Hilden, Germany) were added drop-wise to the FCS-treated dishes. The transfection medium was replaced with CM after three hours, followed by reaggregation with a vibrator over night in 37°C (air/CO2 95:5) before transplantation.

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Islet transplantation (Papers I, III and IV) Groups of 200-250 wild-type, TSP-1 deficient C57BL/6 or heterozygous TSP (+/-) islets, reaggregated TSP-1 siRNA or scramble siRNA-treated C57BL/6 islets, or 200 human islets were packed in a braking pipette and implanted beneath the left kidney capsule of syngeneic diabetic or non-diabetic C57BL/6 or non-diabetic C57BL/6 (nu/nu) mice. Recipients were anesthetized with 0.02 ml/g avertin (10g 97% [vol/vol] 2,2,2-tribromoethanol in 10 ml 2-methyl-2-butanol) intraperitoneally. Some of the groups of islets were pre-treated with 500 ng/ml ovine PRL, human PRL, or their respective vehicle for 24 h before transplantation. Some of the islet recipients were injected once daily with PRL (50 �g dissolved in 0.1 ml sa-line and 0.1 % BSA) or vehicle from one day before transplantation and for the following 7 days. Diabetic recipients were rendered hyperglycemic (> 16.7 mmol/l) by injection of 75 mg/kg alloxan i.v. 5 days before transplanta-tion.

Pancreatic islets encapsulated prior to transplantation were implanted intraperitoneally to non-diabetic C57BL/6 wild-type or TSP-1 deficient mice. These recipients were anesthetized with isoflurane (Abbott, Solna, Sweden) before a small incision of the skin and peritoneal membrane was performed. The encapsulated islets were injected to the peritoneal cavity in approximately 0.4 ml saline. One to two sutures were required to close the peritoneal membrane and 2-3 sutures closed the skin.

Measurements of blood flow and oxygen tension (Papers III and IV) One month post-transplantation some of the transplanted animals were anes-thetized with avertin and placed on an operating table maintained at body temperature (38ºC). Polyethylene catheters were inserted into the right caro-tid artery and left jugular vein. To compensate for loss of body fluid Ringer solution (5 ml x kg-1 x h-1) was continuously infused into the left jugular vein, whereas the right carotid artery was connected to a Statham P32dB pressure transducer (Statham Laboratories, Los Angeles, CA, USA) to measure mean arterial blood pressure. The graft-bearing left kidney was visualized through a subcostal flank incision, and was embedded in cotton wool soaked in Ringer solution, covered with mineral oil and placed in a plastic cup.

The blood perfusion of the islet graft and adjacent renal cortex was meas-ured by laser-Doppler flowmetry (PF 4001-2, Perimed, Stockholm, Sweden) with a needle probe (tip outer diameter, 0.45 mm; Perimed) (128). The laser-Doppler probe was positioned perpendicular to the immobilized tissue sur-face by the use of a micromanipulator, and care was taken to not cause any compression of the tissue. A mean value was calculated from at least three

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individual measurements and considered as one experiment. All blood flow measurements are given as arbitrary tissue perfusion units (TPU) instead of physical units because of difficulties in instrument calibration.

A modified Clark microelectrode (outer tip diameter, 2-6 �m; Unisense, Arhus, Denmark), inserted into the tissue by the use of a micromanipulator under a stereo microscope, was used to measure oxygen tension in islet grafts and adjacent renal parenchyma (128, 129). The mean value from at least 10 measurements was then calculated and considered as one experi-ment in the subsequent statistical analysis. In paper IV, multiple oxygen tension measurements were also performed in three or more superficial en-dogenous islets of each animal (128). The mean value from one animal was then considered to be one experiment.

During measurements, blood pressure, body temperature and tissue tem-perature were continuously monitored with MacLab Instrument (AD Instru-ments, Hastings, UK). A mean arterial blood pressure < 75 mm Hg was used as a pre-set exclusion criteria from the study.

Measurements of blood parameters (Papers I, III and IV) Blood samples were collected from a cut of the tail tip and blood glucose concentrations were analyzed with reagent strips (Medisense; Baxter Trave-nol, Deerfield, IL, USA). In paper I, blood glucose measurements were per-formed on adult C57BL/6 and TSP-1 deficient mice every second hour dur-ing 24h. All animals had free access to food (R36) and tap water during these measurements. After blood perfusion and oxygen tension measure-ments, blood samples were analysed for hematocrit and blood gases, where exclusion criteria were set to pH less than 7.30, pO2 less than 10 kPa, pCO2 more than 6.8 kPa and hematocrit less than 40.

Light microscopic evaluation (Papers I, III and IV) Pancreata or graft-bearing left kidneys were retrieved, fixed in 10 % (vol/vol) formalin and embedded in paraffin. Consecutive sections (5 �m thick) of pancreata or islet grafts were prepared and stained with insulin an-tibodies (Fitzgerald Industries International, Concord, MA, USA) the lectin BS-1 (Sigma-Aldrich) or with the lectin Ulex europaeus (UE; Sigma-Aldrich; only human islets) (130). All sections were counterstained with hematoxylin. In paper I, ten or more tissue sections from all parts of re-trieved TSP-1 deficient and wild-type pancreas were randomly chosen and evaluated. The fraction of the pancreas composed of endocrine tissue was measured by a direct point-counting method (131) and used for calculation

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of the endocrine mass compensating for differences in pancreatic weight between animals. The number of intersections overlapping islets and exo-crine tissue in the pancreas, respectively, was counted at a magnification of 100X using a light microscope. The beta-cell fraction of the islets in wild-type and TSP-1 deficient mice was estimated with a similar point-counting technique, where the number of intersections overlapping insulin-positive cells was determined. The area of the investigated islets was determined using a computerized system for morphometry (Scion Image, Scion, MD, USA). A total of at least 1089 intersections were counted in each pancreas. Also in grafts composed of wild-type islets, TSP-1 deficient islets or islet-cells transfected with siRNA the percentage of beta-cells was calculated with this point-counting technique.

In paper III, more than 12 islet graft tissue sections stained with BS-1 were randomly chosen from each animal and evaluated. In paper IV, more than 5 randomized sections stained with BS-1 or UE, respectively, was ana-lyzed from each animal. We have previously shown that UE only stains hu-man blood vessels, whereas BS-1 stains both human and rodent endothelium (6, 130). Due to the staining specificity the proportion of blood vessels that derive from the donor could be separated from those from the recipient. Sec-tions were evaluated by a point-counting method under a stereo microscope (magnification, 400x) (131). All sections were coded so that the observer was unaware of the origin of the sample. To evaluate total graft volume and fraction of endocrine cells in paper IV, every third section was van Gieson stained and analyzed with a morphometry computer program (Scion Image). In order to estimate the transplanted islet volume, the volume of 250 mouse islets after culture, but before transplantation, was also measured.

Ki67 and TUNEL staining (Paper IV) Sections were stained for the cell proliferation marker Ki67 (1:200; clone SP6; Lab vision, Fremont, CA, USA) according to the manufacturer´s in-structions. Thereafter, sections were double stained for insulin and counters-tained with hematoxylin (31). To calculate beta-cell proliferation, cells posi-tive for both Ki67 and insulin were divided with cells positive for only insu-lin. At least five sections (corresponding to 4156±437 cells) were evaluated in each graft.

To evaluate cell viability, terminal dUTP nick and labelling (TUNEL) staining was performed. Sections from two-day-old mouse islet grafts were prepared as described elsewhere (132). The ratio between TUNEL positive endocrine cells and total number of endocrine cells was used to calculate the fraction of apoptotic endocrine cells. At least five sections (corresponding to 4503±590 cells) were evaluated in each graft.

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Function of transplanted encapsulated islets (Paper I) One month post-transplantation, recipient animals were killed by cervical dislocation. The capsules were retrieved by filling the abdominal cavity with approximately 5-6 ml of cold saline supplemented with 2 mmol/l Ca2+ (Ba-lanced salt solution; SBL Vaccindistribution, Stockholm, Sweden) to wash out the capsules. Retrieved capsules were used for measurements of glucose-stimulated insulin release (cf. above), with each observation representing one animal.

Perfusion of islet grafts (Papers I and III) One month post-transplantation the graft bearing left kidney was removed together with a part of the aorta and inferior vena cava. The ureter and the renal vein were cut, while the aorta was cannulated and infused with a conti-nuously gassed (O2:CO2 = 95:5) Krebs-Ringer bicarbonate buffer supple-mented with 2.0% (wt/vol) each of BSA (fraction V; Miles Laboratories, Slough, UK) and dextran T70 (Pharmacia, Uppsala, Sweden). For different time periods during the perfusion, the medium contained either 2.8 or 16.7 mmol/l D-glucose. The medium was administered at a rate of 1 ml/min with-out recycling for 60 min with a perfusion pressure of ~40 mmHg. The perfu-sion experiments started with a 15-min period using medium containing 2.8 mmol/l glucose, which was followed by 30 min using 16.7 mmol/l glucose. The perfusions were concluded by a 15-min perfusion with medium contain-ing 2.8 mmol/l glucose. Fractions of 1 ml were collected at 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 35, 40, 45, 50, 55, and 60 min. Insulin concentrations of the effluent samples were measured by ELISA (Mercodia). The rate of insulin secretion was calculated by multiplying the insulin concentration in the sample by the flow rate (133). The area under curve (AUC) for insulin secretion was also determined from these values.

Minimal islet mass (Paper IV) In diabetic mice transplanted with 200 islets, body weight and blood glucose concentration were measured every fifth day for one month. The definition of recovery from disease was set to a non-fasting blood glucose < 11.1 mmol/l. The graft-bearing kidney was removed one month post-transplantation in animals cured from diabetes, in order to ascertain that hyperglycemia returned, thereby confirming that the cure of diabetes was not due to regained function in the endogenous pancreas.

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Statistical analysis (Papers I-IV) Values are given as means±SEM. Parametric data with only two groups were analysed using Student´s paired or unpaired two-tailed t-test (papers I-IV). When multiple comparisons were made between normally distributed data, a parametric analysis of variance was used (papers I, II and IV). In papers I, II and IV, unpaired and paired non-parametric data was analysed using Mann-Whitney´s Rank sum test. For comparisons of pair-wise nonpa-rametric data in paper III, Wilcoxon´s signed rank test was used, whereas Kruskal-Wallis test was used for multiple comparisons. The Kaplan-Meier log-rank test was used on data obtained from the minimal islet mass model in paper III. For all comparisons a p-value <0.05 was considered to be statis-tically significant. SigmaStat® (SPSS Science Software, Erfart, Germany) was used for the statistical analyses.

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Results and Discussion

Abundance of TSP-1 in different organs and its localization in pancreatic islets (Papers I and III) Freshly isolated islets had a low protein level of TSP-1 when compared to the kidney and the spleen.

Cultured islet ECs from both mice and rats had more than 15 times higher TSP-1 mRNA expression when compared with cultured islets and purified beta-cells. In rat and mouse beta-cell lines, �TC-6 and INS-1, TSP-1 could not be detected. This indicates that the main contributor of TSP-1 in islets is ECs.

Role of TSP-1 in pancreatic islets (Paper I) TSP-1 deficient mice had normal non-fasting blood glucose levels during a 24 h measurement period. However, when animals were administrated glu-cose intravenously, TSP-1 deficient mice showed impaired glucose tolerance as well as lower serum insulin concentration 10 min after glucose adminis-tration when compared to controls. No differences between TSP-1 deficient and wild-type mice were observed in an insulin tolerance test. Impaired beta-cell function in islets of TSP-1 deficient mice was confirmed in in vitro ex-periments, where islets in batch-type experiments had a poor glucose-stimulated insulin release despite having an increased insulin release during low glucose conditions (1.67 mmol/l glucose). The impaired beta-cell func-tion could be coupled to a mitochondrial dysfunction as islets of TSP-1 defi-cient mice had a lower capacity to oxidate glucose. Moreover, TSP-1 defi-cient islets had a higher mRNA expression of the enzymes uncoupling pro-tein-2 (UCP-2) and lactate dehydrogenase-A LDH-A than wild-type islets. An upregulation of UCP-2 could lead to an uncoupling of mitochondrial oxidative respiration by catalyzing a mitochondrial inner-membrane H+ leak that bypasses ATP synthase leading to decreased glucose induced ATP pro-duction and a decreased ability of glucose to inhibit KATP channels (134). Also the observed upregulation of LDH-A in TSP-1 deficient mice could lead to less ATP production and insulin secretion due to an increased cataly-zation of pyruvate into lactate (135). Increased levels of LDH-A could also explain the elevated insulin secretion in TSP-1 deficient mice when incu-

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bated in 1.67 mmol/l glucose (136). The mRNA expression of pyruvate car-boxylase, glucokinase, mithochondrial glycerol-phosphate dehydrogenase and insulin did not differ between TSP-1 deficient islets and wild-type islets, whereas pancreatic and duodenal homebox gene-1 tended to be decreased in the TSP-1 deficient islets (P=0.076).

Pro(insulin) biosynthesis was decreased in TSP-1 deficient islets, but there was no difference in insulin expression or insulin content of these islets when compared to wild-type islets.

Previous findings have shown that mice deficient of TSP-1 have an in-creased islet mass (65). This could be coupled to a lack of activation of TGFbeta-1 normally exerted by TSP-1 (65).

Importance of the specific contribution of endothelium-derived TSP-1 in islets (Paper I) In order to investigate whether it was endothelium-derived TSP-1, or TSP-1 of other origin, that was important for beta-cell function, we decided to use an islet transplantation model. By this procedure the ECs in the pre-cultured islets are mainly reconstituted from the islet transplant recipient (6) (paper IV). In perfusion experiments of graft-bearing kidneys one month post-transplantation, we observed that neonatal wild-type control islets trans-planted to TSP-1 deficient mice had a lower glucose-stimulated insulin re-lease when compared to if instead transplanted into wild-type mice. On the other hand TSP-1 deficient neonatal islets transplanted to wild-type mice, had a better glucose-stimulated insulin release when compared to if instead transplanted into TSP-1 deficient mice. Thus, TSP-1 deficient islets regained function when transplanted into wild-type mice, and thereby were revascula-rized by TSP-1 positive blood vessels, whereas wild-type islets lost function if transplanted to TSP-1 deficient mice and thereby were revascularized by TSP-1 negative blood vessels. All in all, these experiments indicate that it was indeed endothelial-derived TSP-1 that was important for beta-cell func-tion.

In order to exclude a possibility that a negative systemic milieu influ-enced beta-cell function in TSP-1 deficient mice, some neonatal islets were also microencapsulated before transplantation and were thereby isolated from ingrowth of blood vessels from the recipient. In microencapsulated islets some donor ECs remain or are even expanded (137). These islets were transplanted intraperitoneally, since encapsulated islets are more easily re-trieved for functional analysis from this site than from the renal subcapsular site. When glucose-stimulated insulin release measurements were performed on retrieved microencapsulated neonatal islets one month post-transplantation, wild-type islets secreted similar amounts of insulin in re-sponse to high glucose (16.7 mmol/l) independent of the origin of the host,

35

e.g. wild-type or TSP-1 deficient. Also encapsulated neonatal TSP-1 defi-cient islets showed similar glucose-stimulated insulin release independent of the host origin. However, compared to the transplanted encapsulated neonat-al wild-type islets, the microencapsulated neonatal TSP-1 deficient islets secreted less insulin. Therefore, from these combined experiments it seems to be that it is not a negative systemic milieu that impairs beta-cell function in TSP-1 deficient mice, but lack of TSP-1 positive islet blood vessels.

Activation of TGFbeta-1 in pancreatic islets (Paper I) In order to test the hypothesis that at least part of the dysfunction in TSP-1 deficient islets depend on insufficient TGFbeta-1 activation, we injected vehicle, control peptide (TSR2+QFK) or TGFbeta-1 activating peptide (TSR2+RFK; second type 1 repeat of TSP-1) intraperitonally to newborn wild-type and TSP-1 deficient mice for three weeks before glucose tolerance tests. There was no difference in glucose tolerance between the three differ-ent treatments in the wild-type group. However, the TSP-1 deficient mice treated with the TGFbeta-1 activating peptide, TSR2+RFK, responded better than corresponding vehicle treated mice. TSP-1 deficient mice treated with the TGFbeta-1 activating peptide had even a restored glucose tolerance when compared to wild-type mice. TSP-1 mediating TGFbeta-1 activation in islets therefore seems important to maintain beta-cell function.

In vitro evaluation of means to stimulate revascularization of transplanted islets (Paper II) When isolated rat liver ECs were exposed to ICCM the ECs started to mi-grate towards this stimulus. The effect was inhibited by addition of a VEGF-A neutralizing antibody. Migration of liver endothelial cells could be in-duced by adding VEGF-A in similar concentrations as present in ICCM to the CM. This indicates that beta-cells that produce VEGF could contribute to attraction of liver ECs following intraportal transplantation. When islet ECs were exposed to CM and ICCM the proliferation and migration was low, although the proliferation was slightly higher after exposure to ICCM. Addi-tion of a VEGF neutralizing antibody to the ICCM abolished this prolifera-tive effect. However, both the migratory and proliferatory capacity of islet ECs could be activated when neutralizing antibodies against the angiostatic factors alpha1-AT, TSP-1 or endostatin were added to the ICCM. Thus, a possibility to improve the intra-islet angiogenesis of transplanted islets may be by blocking angiostatic factors present in the islets.

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Protein and mRNA expression of angiogenic and angiostatic factors in islets treated with TSP-1 siRNA or prolactin (Papers III and IV) Dispersed islet cells that were transfected with TSP-1 siRNA had an ~40% reduction in TSP-1 expression 24 h post-transfection when compared to con-trol. The siRNA-treatment had a delayed effect on the protein level, with a 40 % decrease one week post-transfection. In islets treated with PRL for 24 h the TSP-1 mRNA expression was similarly decreased as with siRNA-treatment, whereas the expression of VEGF-A, alpha1-AT, endostatin, FGF-2, inhibitor of DNA binding-1, MMP-9, PDGF, and vasohibin did not differ in islets after PRL-treatment.

Although TSP-1 seems necessary for islet function (paper I), a transiently lowered expression of TSP-1 early post-transplantation could be beneficial for islet viability and long-term graft function due to improved revasculari-zation. One reason for the delayed reduction of TSP-1 protein levels follow-ing siRNA treatment could be the slow turnover rate of the ECM binding of the TSP-1 protein. Previous findings have shown that the effect of siRNA could sustain up to 3-4 weeks in non-dividing cells and less than 1 week in rapidly dividing cells both in vitro and in vivo (138). This suggests that in remnant donor ECs the reduction of TSP-1 using siRNA is probably short due to cell division as part of angiogenesis.

TSP-1 and PRL affect islet graft blood flow (Papers III and IV) In paper III, blood perfusion one month post-transplantation was similar in wild-type control islets and scramble siRNA-treated islet cells within the graft. The blood flow in the adjacent cortex was 50-60 % higher. Both grafts composed of TSP-1 deficient islets, TSP-1 (+/-) islets or TSP-1 siRNA-treated reaggregated islet cells had a markedly increased blood perfusion when compared to their respective controls. In paper IV, islets pre-treated with PRL or animals injected with PRL for the first 8 days after transplanta-tion had an increased islet graft blood flow by approximately 40 % one month post-transplantation when compared to control grafts. Increased blood flow was also observed in grafts composed of human islets cultured with addition of PRL for 24 h prior to transplantation when compared to control. The blood perfusion of native islets can not be determined with the used technique (laser-Doppler flowmetry) due to their small size. However, the renal cortex has a blood perfusion similar to that of native islets (53). Inte-restingly, since the majority of blood vessels in islet grafts are located in the vicinity of the transplanted islets, but not in the endocrine tissue (55), the

37

observed values for graft perfusion are likely to be an overestimation of the blood perfusion in the endocrine tissue.

TSP-1 and PRL affect oxygen tension (Papers III and IV) In paper III, oxygen tension was 7-8 mmHg one month post-transplantation in both grafts composed of whole wild-type islets or scrambled siRNA-treated reaggregated islet cells. There was an increased oxygen tension in both grafts composed of TSP-1 deficient islets, TSP-1 (+/-) islets or TSP-1 siRNA-transfected reaggregated islet cells.

In paper IV, the oxygen tension was 30 mmHg in the endogenous pan-creatic islets, whereas syngenically transplanted control islets had a partial pressure of oxygen of only ~5 mmHg. By contrast, islets treated with PRL before transplantation, or recipient animals treated with PRL for the first 8 days post-transplantation, had an islet transplant oxygen tension of ~15 mmHg at one month follow-up. Human islets pre-treated with PRL also had an increased oxygen tension when compared to controls one month post-transplantation.

In both papers the improved oxygen tension indicates a better environ-ment for islet beta-cells regarding cell viability and function. This is because transplanted islets, when compared to endogenous islets, normally work under more anaerobic conditions resulting in increased lactate formation and tissue acidosis predisposing for graft failure (139).

TSP-1 and PRL affect islet vascular density (Papers III and IV) The islet vascular density was similar (~7 %) in one month old grafts com-posed of wild-type islets and scramble siRNA-transfected islet cells. How-ever, in transplanted islets deficient of TSP-1, and in grafts of transplanted TSP-1 siRNA-transfected islets cells, the vascular density was increased with approximately 40 % one month post-transplantation. When TSP-1 (+/-) islets was transplanted the vascular density one month post-transplantation was in between the wild-type and TSP-1 deficient group. In paper IV, approximately 11 % of the endogenous mouse islet volume consisted of a vascular network, which should be compared to 5-6 % in one month-old syngeneic control mouse islet transplants. However, in grafts where islets had been pre-treated with PRL, or recipients had been given PRL intraperionetally for the first 8 days post-transplantation, there was a 1.5-fold increase in vascular volume. Human islets pre-treated with vehicle had the same vascular density as vehicle-treated mouse islets (6 %). An in-

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creased vascular density was also observed in grafts of human islets where the islets had been pre-treated with PRL before transplantation. The in-creased vascular network was a combination of both in-growth of recipient blood vessels and expansion of donor endothelial cells.

The improved blood flow and pO2 following interventions suggest that the newly formed blood vessels are functionally mature. Interestingly, one month post-transplantation genetically TSP-1 deficient islets had a pO2 of greater magnitude than expected by the increase in blood vessel number. This could be a result of better oxygen transport due to higher blood flow in individual blood vessels. One possible explanation could be that in absence of TSP-1, the effect of NO in vasodilation of blood vessels increases due to absence of the normal TSP-1 counteracting effect on NO (140). It should be noted that NO is a major mediator of blood flow in both endogenous and transplanted islets (141, 142).

The increased vascular density one month post-transplantation in papers III and IV indicates that both inhibition of TSP-1 and treatment with PRL are two possible strategies to improve islet engraftment and thereby function. Presently, there is however no commercially available pharmacological inhi-bitors of TSP-1. To reduce possible complications when PRL is admini-strated systemically, animals were treated post-transplantation with a PRL concentration that is in the same range as in a pregnant women (143) and mice (144). Even more appealing may be to use the incubation protocol with PRL for islets, since this produced almost similar results as the injection treatment of recipients. Mere incubation of islets for transplantation with PRL is likely to be a safe strategy with fewer side effects.

Influence of TSP-1 and PRL on beta-cell proliferation, apoptosis and islet graft volume (Papers III and IV) There was no difference in beta-cell percentage between islet grafts com-posed of wild-type islets, TSP-1 deficient islets or islet cells transfected with siRNA.

In both grafts where islets had been pre-treated with PRL, or recipients had been injected with PRL intraperitonally for the first 8 days post-transplantation, the endocrine volume one month post-transplantation was increased with 50 % and 100 %, respectively when compared to correspond-ing controls. In the recipient animals injected, with PRL, the graft endocrine volume was similar to the implanted islet mass one month earlier.

Endocrine cells in islets pre-treated with PRL had similar cell prolifera-tion as those in control islets when evaluated 2 days before transplantation. One explanation for the increased endocrine volume, besides improved vas-cular engraftment, may be direct effects of PRL on rates of proliferation and/or apoptosis in the transplanted pancreatic islets. We estimated prolifera-

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tion and apoptosis at day 2 after transplantation of mouse islets and found that beta-cell proliferation was not significantly affected, while endocrine cell apoptosis was suppressed in the grafts composed of PRL pre-treated islets. PRL has also previously been described to protect beta-cells against apoptosis (145) and up-regulate several anti-apoptotic genes (146).

Influence of TSP-1 and PRL on islet graft function (Papers III and IV) Perfusion studies of one month old grafts composed of TSP-1 siRNA-transfected islet cells showed an ~40% increase in total AUC for glucose stimulated insulin release when compared to control. Both the first and second peak of insulin release was improved in response to glucose stimula-tion.

The perfusion model enabled us to evaluate whether the improved vascu-lar engraftment in grafts composed of TSP-1 siRNA-treated islet cells ef-fected the insulin release kinetics. When the grafts were perfused with high glucose concentrations, there was a high first peak of stimulated insulin re-lease. The improved second phase of glucose-stimulated insulin release indi-cated that the insulin release during the first phase was not merely a “wash-out” effect of insulin retained in the islet graft due to low blood perfusion. The improved function of grafts composed of TSP-1 siRNA-treated islet cells suggest that compared to genetically TSP-1 deficient islets the islet cells do not lose their function when TSP-1 is only transiently suppressed. However, this could also be an effect of better endocrine cell viability due to better oxygenation and paracrine support of the beta-cells.

Alloxan-diabetic mice became more rapidly normoglycemic when they received PRL pre-treated islets than control islets, as analysed by a Kaplan-Meier log-rank test. All animals receiving 200 preincubated PRL-treated islets were cured from diabetes within 15 days post-transplantation, whereas only 43 % of the control animals became normoglycemic. One month post-transplantation less than 71 % of the control animals were normoglycemic. After kidney removal all mice reverted to hyperglycemia.

In vitro treatment of mouse islets with PRL had a positive effect in mak-ing mice normoglycemic early post-transplantation. This could be an effect of better function of implanted islets due to increased revascularization. However, also direct effects of PRL on beta-cells may contribute (145-147).

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Conclusions

To improve the results of pancreatic islet transplantation it is of major im-portance to improve islet engraftment. In the present thesis, the angiostatic factor TSP-1 is shown to be mainly expressed in ECs within pancreatic islets and to be important both for the functional development of pancreatic beta-cells and the re-establishment of a vascular network within the islets post-transplantation. TSP-1 mediates its effects on beta-cells at least partially through TGFbeta-1.

Besides by transient depletion of TSP-1, it is shown that exposure of is-lets to the hormone PRL could lead to improved vascular engraftment. These strategies could be valuable in clinical settings to reduce the amount of islets needed to restore normoglycemia at transplantation.

Paper I ECs are the main contributor of TSP-1 within pancreatic islets. TSP-1 is an important factor for beta-cell function and development. The effects of TSP-1 are mediated at least partially through TGFbeta-1.

Paper II Islet ECs normally have a very low capacity for proliferation and migration towards angiogenic stimuli. However, neutralizing the angiostatic factors alpha1-AT, TSP-1 or endostatin in islets increase islet EC proliferation and migration. Reduction of angiostatic factors in islets early post-transplantation could be a method to improve islet engraftment.

Paper III TSP-1 is shown to be an important factor to hinder the revascularization of experimentally transplanted islets, with effects on function and possibly sur-vival of the beta-cells. Substances that transiently inhibit TSP-1 may there-fore be important to evaluate for their capacity to improve results of islet transplantation.

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Paper IV PRL downregulates TSP-1 in islets and improves the engraftment of experi-mentally transplanted mouse and human islets. This suggests that PRL may be used to improve clinical islet transplantation results.

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Swedish Summary

Diabetes mellitus är en multifaktoriell sjukdom som grovt kan delas upp i typ 1 och typ 2 diabetes. Typ 1 diabetes är en autoimmun sjukdom där krop-pens eget immunförsvar förstör de insulinproducerande beta-cellerna i buk-spottkörteln. Behandlingsmetoden för patienter med typ 1 diabetes är livs-långa insulininjektioner. Trots insulinbehandling är det svårt att helt norma-lisera kroppens blodsockernivåer och det finns risk för att utveckla sekundä-ra komplikationer med bland annat blindhet och njursjukdom som följd. Enda sättet att normalisera glukoshomeostasen är genom att transplantera bukspottkörteln eller de Langerhanska öarna där beta-cellerna återfinns. Fördelen med att transplantera enbart de Langerhanska öarna är att det är ett betydligt mindre kirurgiskt ingrepp. Dock behövs fler donatorer, då upp till 70 % av de insulinproducerande cellerna försvinner i nära anslutning till transplantationen. En bidragande orsak till den ökade celldöden i de Langer-hanska öarna är förmodligen bristfällig syresättning, då det tar upp till 14 dagar innan blodkärlsystemet återbildats. Nybildningen av blodkärl blir inte helt komplett, vilket återspeglas i en lägre syresättning efter transplantation. Genom att stimulera blodkärlsnybildningen kan syresättningen och överlev-naden i de transplanterade Langerhanska öarna öka.

I denna studie visar vi att de Langerhanska öarna uttrycker det kärltill-växtinhiberande glykoproteinet thrombospondin-1 (TSP-1) främst i endotel-celler (blodkärlceller). Hos möss som saknar TSP-1 var insulinfrisättningen sämre. Behandling av dessa djur från födseln med den TGFbeta-1 aktiveran-de delen av TSP-1 kunde dock bevara insulinproduktionen, vilket tyder på att TSP-1 åtminstone delvis medierar sin effekt via aktivering av TGFbeta-1.

Genom att i odlingsskålar tillsätta neutraliserande antikroppar mot protei-nerna TSP-1, endostatin, samt alpha1-antitrypsin, som alla hämmar blod-kärlsnybildning, såg vi att endotelceller från Langerhanska öar kunde stimu-leras till celldelning och vandring mot insulinproducerande celler.

När vi övergående minskade bildningen av TSP-1 i Langerhanska öar ge-nom s.k. siRNA–behandling före transplantation, förbättrades den glukos-stimulerade insulinfrisättningen från transplantatet 1 månad efter transplanta-tion. Förutom detta så ökade även blodflödet och syresättningen i de trans-planterade öarna. Samma förbättringar sågs när Langerhanska öar, eller mot-tagarmöss av Langerhanska öar, behandlades med hormonet prolaktin innan transplantation.

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Dessa resultat betonar vikten av att vid transplantation av Langerhanska öar påskynda kärltillväxten och därigenom öka funktionen och överlevnaden hos de insulinproducerande cellerna. Dessa experimentellt utvecklade strate-gier kan förhoppningsvis framöver kunna bidra till att även förbättra resulta-ten inom klinisk ö-transplantation.

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Acknowledgements

I would like to thank my supervisors Nils Welsh and P-O Carlsson for sup-porting me through good and bad times. Leif Jansson for interesting conversations. He has always time for me. Stellan Sandler, Carina Carlsson, Micke Welsh, Nils Welsh and Josef for nice lunches. Arne Andersson for running the department in a professional way during hard times. I’m also happy that he has time for my questions regarding both laboratory work and things besides that. All scientists in Nils and P-O Carlssons group. Rikard Fred for helping me with theoretical questions, former PhD-student Robert Hägerkvist for both sport activities and good support at the lab, Andreea Barbu for being a help-ful “senior” in our group, Dariush Mokhtari for everything I have mentioned before and a really fun living at Studentvägen, Sara Bohman for learning me the technique to encapsulate islets, Åsa Johansson for learning me how to isolate endothelial cells, Joey Lau for helping me in histolab, Ulrika Petters-son for nice parties. My roommates during the past years. Magnus Johansson for showing me how thing works at the department, Joel Petersson for funny conversations and especially his attempt to make me clean my desk, Olof Schreiber for being happy all the time. Former PhD-students, especially Richard Olsson and Mattias Gäreskog. All lab technicians, especially Astrid Nordin for helping me with all differ-ent kinds of techniques, Eva Törnelius and Ing-Marie Mörsare for assistance with stainings. Birgitta Bodin for helping me with perfusion experiments. Björn Åkerblom for making funny after work events. All other PhD students at the department for making the workplace highly motivated.

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Senior scientists at the department for being good colleagues. Daniel Edgar, Hanna Wootz, Ole Forsberg, Jon Böhlmark, Martin Almlöf, Adam and Lina Strömstedt for happy moments. I will never forget you. My old friends from Gävle, especially Mandus Frykman, Göran Bergström, Martin Tiberg, Erik Westberg, Niklas Linde and Jakob Carlgren. Annika and Thorsten Persson for giving me new bikes all the time. My lovely family. Last and most important my wonderful girlfriend Ylva.

46

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