c-Myc directly induces both impaired insulin secretion and loss of ß-cell mass, independently of hyperglycemia in vivo.

c-Myc (Myc) is a mediator of glucotoxicity but could also independently compromise ß-cell survival and function. We have shown that after Myc activation in adult ß-cells in vivo, apoptosis is preceded by hyperglycemia, suggesting glucotoxicity might contribute to Myc-induced apoptosis. To address this question conditional Myc was activated in ß-cells of adult pIns-c-MycER(TAM) mice in vivo in the presence or absence of various glucose-lowering treatments, including exogenous insulin and prior to transplantation with wild-type islets. Changes in blood glucose levels were subsequently correlated with changes in ß-cell mass and markers of function/differentiation. Activation of c-Myc resulted in reduced insulin secretion, hyperglycemia and loss of ß-cell differentiation, followed by reduction in mass. Glucose-lowering interventions did not prevent loss of ß-cells. Therefore, Myc can cause diabetes by direct effects on ß-cell apoptosis even in the absence of potentially confounding secondary hyperglycemia. Moreover, as loss of ß-cell differentiation/function and hyperglycemia are not prevented by preventing ß-cell apoptosis, we conclude that Myc might contribute to the pathogenesis of diabetes by directly coupling cell cycle entry and ß-cell failure through two distinct pathways.

ImmunoFISH on isolated nuclei from paraffin-embedded biopsy material.

The detection of genetic abnormalities in paraffin sections by fluorescence in situ hybridization (FISH) is widely used in clinical practice to detect amplification of the ERB2 gene in breast carcinoma and various chromosomal translocations in lymphomas and soft tissue tumors. However, interpretation of FISH signals in tissue sections may be difficult due to overlapping nuclei and nuclear truncation artifacts. Some of these shortcomings may be avoided by the use of isolated nuclear preparations. However, identification of cell populations may be difficult in detached cells removed from their histological context. We have described an optimized immunoFISH technique on isolated nuclear suspension, which combines the benefits of studying isolated cells derived from paraffin embedded tissues by FISH analysis with the ability to detect cell lineage and other markers by immunofluorescence.

Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia?

Mutations in the C-terminal region of nucleophosmin in acute myeloid leukemia (AML) result in aberrant cytoplasmic nucleophosmin (cNPM) in leukemic blast cells which is detectable by immunocytochemistry in bone marrow trephine (BMT) biopsy sections. We tested whether cNPM is detectable by immunocytochemistry in air-dried smears of AML with nucleophosmin1 (NPM1) mutations. An immunoalkaline phosphatase method was developed using the OCI-AML3 cell line, known to have mutated NPM1, and assessed on blood and marrow smears of 60 AML cases. NPM was detectable in all blast cell nucleoli and cNPM in 21 of 31 of NPM1 mutated and 15 of 29 wild-type cases. Paired air-dried smears and BMT biopsies from the same case (mutated and wild-type) gave discrepancies in cNPM expression and there was no correlation in 10 of 22 cases. Due to the high false positive and negative rates for cNPM in cell smears, this method should not be used as a surrogate for NPM1 mutations in AML.

Beneficial role of pancreatic microenvironment for angiogenesis in transplanted pancreatic islets.

Pancreatic islets implanted heterotopically (i.e., into the kidney, spleen, or liver) become poorly revascularized following transplantation. We hypothesized that islets implanted into the pancreas would become better revascularized. Islets isolated from transgenic mice expressing enhanced yellow fluorescent protein (EYFP) in all somatic cells were cultured before they were implanted into the pancreas or beneath the renal capsule of athymic mice. Vascular density was evaluated in histological sections 1 month posttransplantation. EYFP was used as reporter for the transgene to identify the transplanted islets. Islet endothelial cells were visualized by staining with the lectin Bandeiraea simplicifolia (BS-1). Capillary numbers in intrapancreatically implanted islets were only slightly lower than those counted in endogenous islets, whereas islets implanted beneath the renal capsule had a markedly lower vascular density. In order to determine if this high graft vascular density at the intrapancreatic site reflected expansion of remnant donor endothelial cells or increased ingrowth of blood vessels from the host, also islets from Tie2-green fluorescent protein (GFP) mice (i.e., islets with fluorescent endothelial cells) were transplanted into the pancreas or beneath the renal capsule of athymic mice. These islet grafts revealed that the new vascular structures formed in the islet grafts contained very few GFP-positive cells, and thus mainly were of recipient origin. The reason(s) for the much better ingrowth of blood vessels at the intrapancreatic site merits further studies, because this may help us form strategies to overcome the barrier for ingrowth of host vessels also into islets in heterotopic implantation sites.

Growth hormone increases beta-cell proliferation in transplanted human and fetal rat islets.

OBJECTIVE: The aim of the study was to increase the number of human islet beta-cells after transplantation with injections of human growth hormone (hGH). INTERVENTIONS: Human islets and fetal rat islets were transplanted under the left kidney capsule and under the right kidney capsule, respectively in nude normoglycemic mice which were then given a daily injection of 200 microg hGH for 1-4 weeks. MAIN OUTCOME MEASURE: Beta-cell proliferation was determined using thymidine incorporation and the beta-cell area was assessed using light microscopy. RESULTS: Mice given hGH increased their body weight one week after transplantation and had a more efficient removal of glucose after 3 and 4 weeks. Treatment with hGH resulted in increased beta-cell proliferation in human and fetal rat beta-cells, and the beta-cell area tended to increase. However, serum insulin concentrations and pancreas insulin content remained unchanged. CONCLUSIONS: hGH increased the proliferation of transplanted human beta-cells as well as improving the glucose tolerance of the transplanted mice.

Labeling of multiple cell markers and mRNA using automated apparatus.

Double immunoenzymatic labeling of 2 different molecules in tissue sections is a widely used technique. However, it is time consuming since the 2 immunoenzymatic procedures are carried out in sequence, and they must also be optimally performed to avoid unwanted background labeling. In this paper, we report that double immunoenzymatic staining performed using automated immunostaining apparatus considerably reduces the requirements in terms of time and is also highly reproducible and free of background. Three tissue markers can also be visualized by performing (after immunoperoxidase labeling) 2 sequential immuno-alkaline phosphatase procedures using different substrates. Furthermore, single or double detection of mRNA by in situ hybridization can be combined with immunoenzymatic labeling. Finally, automated labeling could also be performed on peripheral blood and bone marrow smears, opening the possibility of using this procedure in the analysis of hematologic/cytology samples.

Distribution of intraportally implanted microspheres and fluorescent islets in mice.

The aim of the study was to evaluate the distribution of intraportally transplanted islets in mice. We initially administered 2000 polystyrene microspheres with a diameter of 50 microm intraportally into normoglycemic C57BL/6 mice. In separate experiments other mice were injected similarly with 300 microspheres each with a diameter of 100 or 200 microm. One week later the animals were killed, and the lungs and livers were removed and divided into lobes. The number of microspheres in each individual liver lobe and in the lungs was counted using a stereomicroscope. In other experiments, athymic C57BL/6 mice were similarly implanted with 250 islets isolated from transgenic mice expressing the enhanced yellow fluorescent protein in the islet cells. The distribution of microspheres and islets was independent of size, and fairly homogenous within the liver, with the exception of the caudate lobe, which contained fewer microspheres and islets, respectively. Approximately one third of all microspheres and islets were present as aggregates. Eighty-five to 90% of the implanted microspheres were identified in the liver sections, whereas 60-65% of the implanted islets were recovered. Aggregates or single fluorescent cells were observed in the liver of islet-implanted mice. We conclude that islets and microspheres implanted into the liver distribute fairly homogenously and quite a few of them exist as aggregates or, with respect to islets, as fragments.

Detection of genetic alterations by immunoFISH analysis of whole cells extracted from routine biopsy material.

The detection of genetic abnormalities (eg, translocations, amplifications) in paraffin-embedded samples by the fluorescence in situ hybridization (FISH) technique is usually performed on tissue sections. FISH analysis of nuclei extracted from paraffin-embedded samples is also possible, but the technique is not widely used, principally because of the extra labor involved and the loss of information on tissue architecture. In this article, we report that nuclei extracted from paraffin-embedded tissue often retain at least part of the surrounding cytoplasm. Consequently, immunocytochemical labeling for a range of cellular markers (eg, of lineage or proliferation) can be performed in combination with FISH labeling, allowing specific cell populations to be analyzed for genetic abnormalities. These cell preparations are largely free of the problems associated with tissue sections (eg, truncation artifact, signals in different focal planes) so that interpretation is easy and numerical chromosomal abnormalities are readily assessed. Cells isolated from paraffin sections can be stored in suspension so that arrays can be created as and when needed from a range of neoplasms for investigation by the immunoFISH technique (for example, for studying a new genetic abnormality). This procedure represents a novel methodology, which in some settings offers clear advantages over analysis of tissue sections.

Implantation site-dependent dysfunction of transplanted pancreatic islets.

OBJECTIVE: Clinical islet transplantations are performed through infusion of islets via the portal vein into the liver. This study aimed at characterizing the influence of the implantation microenvironment on islet graft metabolism and function. RESEARCH DESIGN AND METHODS: Islets were transplanted into their normal environment, i.e., the pancreas, or intraportally into the liver of mice. One month posttransplantation, the transplanted islets were retrieved and investigated for changes in function and gene expression. RESULTS: Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis, and glucose oxidation rate were markedly decreased in islets retrieved from the liver, both when compared with islets transplanted into the pancreas and endogenous islets. Islets transplanted into the pancreas showed normal insulin content, (pro)insulin biosynthesis, and glucose oxidation rate but increased basal insulin secretion and impaired glucose stimulation index. Gene expression data for retrieved islets showed downregulation of pancreatic and duodenal homeobox gene-1, GLUT-2, glucokinase, mitochondrial glycerol-phosphate dehydrogenase, and pyruvate carboxylase, preferentially in intraportally transplanted islets. CONCLUSIONS: Islets transplanted into their normal microenvironment, i.e., the pancreas, display gene expression changes when compared with endogenous islets but only moderate changes in metabolic functions. In contrast, site-specific properties of the liver markedly impaired the metabolic functions of intraportally transplanted islets.

Size-dependent revascularization of transplanted pancreatic islets.

For their survival and optimal function, pancreatic islets depend posttransplantation on a rapid and adequate revascularization. Native islets display a marked size-dependent heterogeneity in both angioarchitecture and degree of blood perfusion. This study evaluated whether there also are differences in the degree of revascularization of islets of different size when transplanted. Mouse pancreatic islets were isolated by collagenase digestion, and cultured in vitro for 4-7 days before transplantation. Groups of 200 islets with a diameter either exceeding or being below 100 microm were implanted beneath the left renal capsule of syngeneic C57 BL/6 mice. One month posttransplantation, graft-bearing kidneys were removed. Histological specimens were prepared and stained for endothelium with the lectin Bandeiraea simplicifolia. Pancreata from nontransplanted control animals were prepared similarly. The vascular density in transplanted islets was markedly lower than in native islets. However, islet transplants composed of small islets (<100 microm in diameter) had a vascular density in the endocrine tissue twice that in transplants of larger islets (>100 microm). The connective tissue stroma surrounding smaller islets was also more revascularized than in corresponding grafts with large islets. The vascular density in the connective tissue stroma surrounding the individual islets in the grafts was markedly higher than in the endocrine parts per se. These combined observations indicate that smaller islets have a higher capacity to stimulate regrowth of blood vessels following transplantation. Further studies on islet differences with regard to revascularization capacity may teach us strategies for treatment of transplanted islets to improve their revascularization.

Islet endothelial cells and pancreatic beta-cell proliferation: studies in vitro and during pregnancy in adult rats.

The growth of both tumors and nonneoplastic tissues may be influenced by signals from the vascular endothelium. In the present investigation we show that purified proliferating endothelial cells from pancreatic islets can stimulate beta-cell proliferation through secretion of hepatocyte growth factor (HGF). This secretion could be induced by soluble signals from the islets, such as vascular endothelial growth factor-A (VEGF-A) and insulin. During pregnancy, the pancreatic beta-cells display a highly reproducible physiological proliferation. We show that islet endothelial cell proliferation precedes beta-cell proliferation in pregnant animals. Vascular growth was closely associated with endocrine cell proliferation, and prominent expression of HGF was observed in islet endothelium on d 15 of pregnancy, i.e. coinciding with the peak of beta-cell proliferation. In summary, our results suggest the existence of an endothelial-endocrine axis within adult pancreatic islets, which is of importance for adult beta-cell proliferation.

Endothelial cells in endogenous and transplanted pancreatic islets: differences in the expression of angiogenic peptides and receptors.

BACKGROUND/AIMS: An important reason for the large amount of islets required for successful islet transplantation is likely to be inadequate engraftment of the transplanted islets. Thus, the revascularization is of major importance for graft survival. In order to study the expression of angiogenic peptides and receptors on islet endothelial cells (EC), we needed methods giving access to such endothelium. Therefore, we developed methods to isolate EC from islets transplanted intraportally or beneath the kidney capsule. METHODS: Pancreatic islets were isolated, cultured and syngeneically transplanted into the liver or beneath the kidney capsule of C57BL/6 mice. One month post-transplantation, the islets were retrieved and EC from these islets were explanted. EC were also collected from freshly isolated and cultured non-transplanted islets. The EC were purified with Dynabeads and identified with immunocytochemistry. Angiogenesis GEArray technology was used to study angiogenic gene expression. RESULTS: Several angiogenic genes were expressed in EC; e.g. endostatin, pigment-epithelial derived factor, vascular endothelial growth factor and angiopoietin-2, and their expression were affected by culture. CONCLUSION: The expression of angiogenesis-related genes in islet EC from non-transplanted islets is affected by culture. Moreover, we also describe a technique, which makes it possible to obtain EC from transplanted islets.

Pancreatic islet function in a transgenic mouse expressing fluorescent protein.

Pancreatic islet function and glucose homeostasis have been characterized in the transgenic YC-3.0 mouse, which expresses the yellow chameleon 3.0 (YC-3.0) protein under the control of the beta-actin and the cytomegalovirus promoters. Fluorescence from the enhanced yellow fluorescent protein (EYFP), one part of the yellow chameleon protein, was used as a reporter of transgene expression. EYFP was expressed in different quantities throughout most cell types, including islet endocrine and stromal cells. No adverse effects of the transgene on animal health, growth or fertility were observed. Likewise, in vivo glucose homeostasis, mean arterial blood pressure and regional blood flow values were normal. Furthermore, the transgenic YC-3.0 mouse had a normal beta-cell volume and mass as well as glucose-stimulated insulin release in vitro, compared with the C57BL/6 control mouse. Isolated islets from YC-3.0 animals continuously expressed the transgene and reversed hyperglycemia when transplanted under the renal capsule of alloxan-diabetic nude mice. We conclude that isolated pancreatic islets from YC-3.0 animals implanted into recipients without any EYFP expression, constitute a novel and versatile model for studies of islet engraftment.

The endothelial cells in islets of langerhans.

The blood vessels of the pancreatic islets are of crucial importance for oxygen and metabolite supply, and dispersal of secreted hormones. In addition to this, endothelial cells have an important role in the revascularization process after islet transplantation. Studies have reported signs of poor engraftment of transplanted islets, presumably due to impaired revascularization. The aims of this study were to investigate islet endothelial cells and the revascularization process of transplanted islets. The lectin Bandeiraea simplicifolia was found to consistently stain endothelium of both endogenous and transplanted pancreatic islets. By using this marker, we investigated the vascular density of both endogenous and transplanted islets of C57BL/6 mice. One month post-transplantation, a time point when the implants are assumed to be completely revascularized, the graft vascular density was decreased at all investigated implantation sites when compared to endogenous islets. Furthermore, most of the blood vessels were located in the graft connective tissue stroma. Similar results were obtained six months post-transplantation and in cured diabetic animals after one month. In order to evaluate the function of intraportally transplanted islets, we developed a method to retrieve such islets. Enzymatic and mechanic treatment of the liver enabled us to re-isolate the transplanted islets for further in vitro studies. These islets had decreased insulin release, insulin content and glucose oxidation rate when compared to non-transplanted control islets. To understand the role of islet endothelium in the revascularization of transplanted islets we performed angiogenesis microarray studies on islet endothelial cells, from non-cultured, cultured and transplanted islets. We found that the islet endothelium expressed mRNA for both inhibitors and inducers of angiogenesis, and that this expression differed with time. In conclusion, these results provide a useful platform for further studies on the islet endothelium.

Promoting islet cell function after transplantation.

Engraftment (i.e., the adaptation of transplanted pancreatic islets to their new surroundings with regard to revascularization, reinnervation, and reorganization of other stromal compartments) is of crucial importance for the survival and function of the endocrine cells. Previous studies suggest that transplantation induces both vascular and stromal dysfunctions in the implanted islets when compared with endogenous islets. Thus the vascular density and the blood perfusion of islet grafts is decreased and accompanied with a capillary hypertension. This leads to hypoxic conditions, with an associated shift toward anaerobic metabolism in grafted islets. An improved engraftment will prevent or compensate for the vascular/stromal dysfunction seen in transplanted islets and thereby augment survival of the islet implant. By such means the number of islets needed to cure the recipient will be lessened. This will increase the number of patients that can be transplanted with the limited material available.

Evidence of functional impairment of syngeneically transplanted mouse pancreatic islets retrieved from the liver.

A drawback in pancreatic islet transplantation is the large number of islets needed to obtain insulin independence in patients with diabetes. This most likely reflects extensive posttransplantation islet cell death and functional impairment of the remaining endocrine cells. We aimed to develop an experimental method to retrieve transplanted islets from the mouse liver, which would enable comparisons of transplanted and endogenous islets and provide valuable information on functional changes induced by intraportal transplantation. Transplanted islets were obtained by retrograde perfusion of the liver with collagenase. The identity of retrieved tissue as transplanted islets was confirmed by intravital staining, immunohistochemistry, and electron microscopy. The retrieved islets, irrespective of whether they had resided in diabetic or nondiabetic recipients, had a markedly lower insulin content and glucose-stimulated insulin release when compared with isolated endogenous islets. The glucose oxidation rate was also markedly lower in the retrieved islets, suggesting mitochondrial dysfunction. These disturbances in insulin content, insulin release, and glucose oxidation rate were not reversed by a few days of culture after retrieval. The results implicate changes in islet function after intraportal transplantation. Such dysfunction may contribute to the high number of islets needed for successful transplantation in diabetic individuals.

[Severe vascular dysfunction shown in transplanted islets]. / Kraftig vaskulär dysfunktion påvisas i transplanterade langerhanska öar.

Despite recent advances in clinical islet transplantation, a surprisingly large number of islets (approximately 1 million) are still required to obtain insulin independence in type 1 diabetes. The reasons for this are obscure and likely multifactorial. One explanation may be disturbances in engraftment of the transplanted islets, i.e. the adaptation of the islet transplant to its new surroundings with regard to e.g. revascularization and blood perfusion. Endogenous islets have a dense glomerular-like angioarchitecture. Transplantation of isolated islets causes a disruption of their vascular connections, making the islets dependent on the formation of new blood vessels for optimal function. Evidence from experimental islet transplantation indicates an insufficient revascularization of transplanted islets with subsequent chronically decreased blood perfusion and oxygen tension, which has metabolic consequences within the tissue.

Impaired revascularization of transplanted mouse pancreatic islets is chronic and glucose-independent.

BACKGROUND: Pancreatic islets are avascular immediately after transplantation and depend on revascularization. Recently, the authors found decreased vascular density in mouse islets 1 month after implantation into nondiabetic recipients. This study investigated possible differences in revascularization between islets implanted into nondiabetic and diabetic recipients, and also evaluated changes in vascular density up to 6 months posttransplantation. METHODS: Islets were syngenically transplanted beneath the renal capsule of normoglycemic or alloxan-diabetic C57BL/6 mice. One to 6 months later, the animals were killed and the grafts removed. Histologic slides were prepared and stained with Bandeiraea simplicifolia. RESULTS: The vascular density in all transplanted islets was decreased compared with native islets. There were no differences in the islet graft vascular density between nondiabetic and diabetic animals. No improvement over time occurred. CONCLUSIONS: The vascular density is decreased in islets implanted to cure diabetic recipients. No improvement occurs in transplanted islets after 1 month posttransplantation.

Low revascularization of experimentally transplanted human pancreatic islets.

Pancreatic islets are avascular immediately after transplantation. Although the islets are rapidly revascularized, it is uncertain whether the revascularization produces an adequate oxygenation of the transplanted islet tissue. We measured pO(2), blood flow and vascular density in mouse or human islets 1 month after transplantation to nude mice. Tissue pO(2) was measured with Clark microelectrodes. Blood perfusion was measured with laser-Doppler flow cytometry, whereas vascular density was determined in histological specimens stained for the lectin Bandeiraea simplicifolia (BS-1). Both the transplanted mouse and human islets had a pO(2) 15-20% of that in endogenous mouse islets. Moreover, the vascular density of the transplanted islets was decreased compared with that of endogenous mouse and human islets. Graft blood perfusion was approximately 50% of renal cortex blood flow. A negative correlation was found between donor age and blood perfusion of the human islet grafts. A similar correlation was seen between donor age and the total vascular density of these grafts. In conclusion, transplanted human islets had a markedly decreased vascular density and pO(2) compared with endogenous islets. This has potential implications for clinical islet transplantations, because poor vascular engraftment may significantly increase the number of islets needed to obtain insulin independence.

Histological markers for endothelial cells in endogenous and transplanted rodent pancreatic islets.

BACKGROUND/AIMS: To obtain a selective marker to identify endothelial cells is difficult, due to the heterogeneity of these cells. Most described markers perform well in some applications, but fail in others. The aim of this study was to identify a selective and specific marker for rodent microvascular endothelial cells, especially for use in studies on the vascular system of pancreatic islets. METHODS: A biotin-labelled form of the lectin Bandeiraea or Griffonia simplicifolia in combination with a streptAB-Complex with alkaline phosphatase was used to stain endothelium in paraffin-embedded tissue sections from C57BL/6 mice, Sprague-Dawley or Wistar-Furth rats. RESULTS: We were consistently able to selectively stain microvascular endothelial cells in lungs, small intestines, white and brown adipose tissue, pancreas and islets of Langerhans with the lectin Bandeiraea simplicifolia. Furthermore, we were able to visualise the vasculature in syngenically transplanted islets of Langerhans in Wistar-Furth rats and C57BL/6 mice. Attempts to stain rodent endothelial cells with antibodies against CD34, CD31, CD200, Ox43, von Willebrand factor and the lectin Ulex europaeus were not uniformly successful. CONCLUSION: The lectin Bandeiraea simplicifolia is a versatile marker for rodent endothelial cells, and may be used to study revascularisation after transplantation of pancreatic islet in rodents.