Search

Introduction of non-native species is an important cause of biodiversity decline in rivers. Separated by mountains and salt water, the freshwater fish fauna in Italy has experienced a natural isolation from fish in continental Europe. As a consequence, several Italian fish species have diverged from their European sister species, likely with unique adaptations to the local environment. Relatedly, the region is also susceptible to the invasion of non-native fish, and today almost half the fish species present are of non-native origin. Several of these non-native species have Italian congeners susceptible to competition and hybridizations, and, in the long run, displacements and extinctions. One such example is the Italian gudgeon (Gobio benacensis) and its European congener European gudgeon (Gobio gobio). During the last few decades the European gudgeon was introduced in Italian waters and has since spread rapidly, causing progressive declines in the Italian species. As for several other similar species pairs, little is known about potential differences in ecology and behavior. Here we study differences between Italian and European gudgeons in a controlled laboratory environment, using a combined open field and provoked escape response test, as well as tracking their sympatric survival over time in the hatchery. The smaller Italian gudgeon displayed a lower maximum swimming speed compared to the larger European gudgeon. The Italian gudgeon also experienced substantially higher sympatric mortality, resulting in only European gudgeon surviving to the end of the experiment. Contrary to expectations, no difference was seen in boldness and the Italian gudgeon displayed a higher movement activity, moving a larger distance in an open field test, compared to its European sister species. The reported differences could play a role in the replacement process, and may also have impacts on the surrounding ecosystem, and the prey and predators that have coevolved with the Italian species. [ABSTRACT FROM AUTHOR]

The behavior of early (June-July) and late (August-September) migrating, adult Atlantic salmon, in The River Klarälven, Sweden, was analyzed using radio telemetry. River Klarälven is a regulated river without functioning fishways, instead upstream migrating salmon are trapped and trucked past eight hydropower plants before released back to the river. We distinguished two parts of the spawning migration, that is, one part being the migration from the place where the fish was released to the spawning grounds. The other part was a holding phase on the spawning grounds with little or no movements before spawning. The late salmon spent less of their total time on holding, 36.2%, and more on migration, 63.8%, compared with early migrating salmon, which distributed their time rather evenly between migration, 47.5%, and holding, 52.5%. In total, early salmon used 30% more time migrating and 156% more time holding than late salmon. Some Atlantic salmon (Salmo salar) fell back over the hydropower plant after release and got excluded from spawning. The fallback rates of transported, tagged spawners were higher in the early than in the late group in both years. The fallback rate in 2012 was 42.8% of the early group and 15.1% in the late. In 2013, there were 51.7 % fallbacks in the early group and 3.4% in the late. The salmon fell back on average 9 days after being released in 2012 and 16 days in 2013. A high mean daily discharge on the day of release increased the probability of becoming a fallback. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Repeat salmonid spawners may make large contributions to total recruitment and long term population stability. Despite their potential importance, relatively little is known about this phase of the life history for anadromous populations, and nothing has been reported for landlocked populations. Here, we studied post-spawning behaviour and survival of landlocked Atlantic salmon in relation to downstream dam passage in the River Klarälven, Sweden. Eight hydropower stations separate the feeding grounds in Lake Vänern from the spawning grounds in the River Klarälven, and no measures to facilitate downstream migration are present in the river. Forty-nine percent of the salmon survived spawning and initiated downstream migration. Females and small fish had higher post-spawning survival than males and large fish. The post-spawners migrated downstream in autumn and spring and remained relatively inactive in the river during winter. Downstream migration speed in the free flowing part of the river was highly variable with a median of 9.30 km/day. Most fish passed the first hydropower station via upward-opening spill gates after a median residence time in the forebay of 25 min. However, no tagged fish survived passage of all eight hydropower stations to reach Lake Vänern. This result underscores the need for remedial measures to increase the survival of downstream migrating kelts. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Fatigue curves quantify fish swimming performance, providing information about the time ([Formula: see text]) fish can swim against a steady flow velocity ( U f ) before fatiguing. Such curves represent a key tool for many applications in ecological engineering, especially for fish pass design and management. Despite years of research, though, our current ability to model fatigue curves still lacks theoretical foundations and relies primarily on fitting empirical data, as obtained from time-consuming and costly experiments. In the present article, we address this shortcoming by proposing a theoretical analysis that builds upon concepts of fish hydrodynamics to derive scaling laws linking statistical properties of [Formula: see text] to velocities U f , pertaining to the so-called burst range. Theoretical arguments, in the present study, suggest that the proposed scaling laws may hold true for all fish species and sizes. A new experimental database obtained from over 800 trials and five small-sized Cypriniformes support theoretical predictions satisfactorily and calls for further experiments on more fish species and sizes to confirm their general validity.

Social facilitation is a well-known phenomenon where the presence of organisms belonging to the same species enhances an individual organism's performance in a specific task. As far as fishes are concerned, most studies on social facilitation have been conducted in standing-water conditions. However, for riverine species, fish are most commonly located in moving waters, and the effects of hydrodynamics on social facilitation remain largely unknown. To bridge this knowledge gap, we designed and performed flume experiments where the behaviour of wild juvenile Italian riffle dace (Telestes muticellus) in varying group sizes and at different mean flow velocities, was studied. An artificial intelligence (AI) deep learning algorithm was developed and employed to track fish positions in time and subsequently assess their exploration, swimming activity, and space use. Results indicate that energy-saving strategies dictated space use in flowing waters regardless of group size. Instead, exploration and swimming activity increased by increasing group size, but the magnitude of this enhancement (which quantifies social facilitation) was modulated by flow velocity. These results have implications for how future research efforts should be designed to understand the social dynamics of riverine fish populations, which can no longer ignore the contribution of hydrodynamics., (© 2024. The Author(s).)

The Italian spined loach (Cobitis bilineata) is an elongated, small-sized (<12 cm) spined loach native to northern Italy, Slovenia and Croatia. As for loaches in general, little is known about the individual movements of this loach in nature. Passive integrated transponders (PIT-tags) are small (typically 7-32 mm), relatively cheap and allow tracking of individual fish movements and behaviour. A fundamental assumption in animal telemetry is that the performance of a tagged animal does not deviate substantially from its natural performance. Although PIT-tagged fish often display high survival and tag retention, the effect varies between species and contexts, and few studies have looked at behavioural effects of PIT-tagging. Here we demonstrate a PIT-tagging methodology for spined loaches, and compare survival, activity and provoked escape response (maximum swimming speed) between tagged and control fish. We also track tag retention in the tagged fish. Italian spined loaches tagged with 12 mm PIT-tags displayed high tag retention and no extra mortality, and no effects of tagging on activity or maximum swimming speed were observed. The tag-to-fish weight and length ratios in our study ranged from 2% to 5% and from 10% to 16%, respectively, and we conclude that PIT-tagging, within these ratios, appears suitable for Italian spined loach., (© 2022 Fisheries Society of the British Isles.)

Submarine power cables carry electricity over long distances. Their geographic distribution, number, and areal coverage are increasing rapidly with the development of, for example, offshore wind facilities. The flow of current passing through these cables creates a magnetic field (MF) that can potentially affect marine organisms, particularly those that are magnetosensitive. The lumpfish ( Cyclopterus lumpus ) is a migratory species that is widely distributed in the North Atlantic Ocean and Barents Sea. It migrates between coastal spawning grounds and pelagic offshore feeding areas. We tested whether lumpfish respond to MFs of the same intensity as those emitted by high voltage direct current (HVDC) submarine power cables. Laboratory experiments were conducted by placing juvenile lumpfish in an artificial MF gradient generated by a Helmholtz coil system. The intensity of the artificial MF used (230 µT) corresponded to the field at 1 m from a high-power submarine cable. The fish were filmed for 30 min with the coil either on or off. Swimming speeds, and presence in the different parts of a raceway, were extracted from the videos and analyzed. Juvenile lumpfish activity, defined as the time that the fish spent swimming relative to stationary pauses (attached to the substrate), and the distance travelled, were unaffected by exposure to the artificial MF. The swimming speed of juvenile lumpfish was reduced (by 16%) when the coil was on indicating that the fish could either sense the MF or the induced electric field created by the movement of the fish through the magnetic field. However, it seems unlikely that a 16% decrease in swimming speed occurring within 1 m of HVDC cables would significantly affect Atlantic lumpfish migration or homing., Competing Interests: Steven Shema is employed by Grótti ehf, Reykjavik., (© 2023 Durif et al.)

Electromagnetic surveys generate electromagnetic fields to map petroleum deposits under the seabed with unknown consequences for marine animals. The electric and magnetic fields induced by electromagnetic surveys can be detected by many marine animals, and the generated fields may potentially affect the behavior of perceptive animals. Animals using magnetic cues for migration or local orientation, especially during a restricted time-window, risk being affected by electromagnetic surveys. In electrosensitive animals, anthropogenic electric fields could disrupt a range of behaviors. The lack of studies on effects of the electromagnetic fields induced by electromagnetic surveys on the behavior of magneto- and electrosensitive animals is a reason for concern. Here, we review the use of electric and magnetic fields among marine animals, present data on survey generated and natural electromagnetic fields, and discuss potential effects of electromagnetic surveys on the behavior of marine animals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Type 2 diabetes mellitus (T2DM) affects millions of people and is linked with obesity and lipid accumulation in peripheral tissues. Increased lipid handling and lipotoxicity in insulin producing β-cells may contribute to β-cell dysfunction in T2DM. The vascular endothelial growth factor (VEGF)-B regulates uptake and transcytosis of long-chain fatty acids over the endothelium to tissues such as heart and skeletal muscle. Systemic inhibition of VEGF-B signaling prevents tissue lipid accumulation, improves insulin sensitivity and glucose tolerance, as well as reduces pancreatic islet triglyceride content, under T2DM conditions. To date, the role of local VEGF-B signaling in pancreatic islet physiology and in the regulation of fatty acid trans-endothelial transport in pancreatic islet is unknown. To address these questions, we have generated a mouse strain where VEGF-B is selectively depleted in β-cells, and assessed glucose homeostasis, β-cell function and islet lipid content under both normal and high-fat diet feeding conditions. We found that Vegfb was ubiquitously expressed throughout the pancreas, and that β-cell Vegfb deletion resulted in increased insulin gene expression. However, glucose homeostasis and islet lipid uptake remained unaffected by β-cell VEGF-B deficiency.

Objective: Activation of endothelial β-catenin signaling by neural cell-derived Norrin or Wnt ligands is vital for the vascularization of the retina and brain. Mutations in members of the Norrin/β-catenin pathway contribute to inherited blinding disorders because of defective vascular development and dysfunctional blood-retina barrier. Despite a vital role for endothelial β-catenin signaling in central nervous system health and disease, its contribution to central nervous system angiogenesis and its interactions with downstream signaling cascades remains incompletely understood. Approach and Results: Here, using genetically modified mouse models, we show that impaired endothelial β-catenin signaling caused hypovascularization of the postnatal retina and brain because of deficient endothelial cell proliferation and sprouting. Mosaic genetic analysis demonstrated that endothelial β-catenin promotes but is not required for tip cell formation. In addition, pharmacological treatment revealed that angiogenesis under conditions of inhibited Notch signaling depends upon endothelial β-catenin. Importantly, impaired endothelial β-catenin signaling abrogated the expression of the VEGFR (vascular endothelial growth factor receptor)-2 and VEGFR3 in brain microvessels but not in the lung endothelium., Conclusions: Our study identifies molecular crosstalk between the Wnt/β-catenin and the Notch and VEGF-A signaling pathways and strongly suggest that endothelial β-catenin signaling supports central nervous system angiogenesis by promoting endothelial cell sprouting, tip cell formation, and VEGF-A/VEGFR2 signaling.

Preclinical trials of cancer drugs in animal models are important for drug development. The Rip1Tag2 (RT2) transgenic mouse, a model of pancreatic neuroendocrine tumours (PNET), has provided immense knowledge about PNET biology, although tumour progression occurs in a location inaccessible for real-time monitoring. To overcome this hurdle we have developed a novel platform for intravital 3D imaging of RT2 tumours to facilitate real-time studies of cancer progression. Pre-oncogenic islets retrieved from RT2 mice were implanted into the anterior chamber of the eye (ACE) of host mice, where they engrafted on the iris, recruited blood vessels and showed continuous growth. Noninvasive confocal and two-photon laser-scanning microscopy through the transparent cornea facilitated high-resolution imaging of tumour growth and angiogenesis. RT2 tumours in the ACE expanded up to 8-fold in size and shared hallmarks with tumours developing in situ in the pancreas. Genetically encoded fluorescent reporters enabled high-resolution imaging of stromal cells and tumour cell migration. Sunitinib treatment impaired RT2 tumour angiogenesis and growth, while overexpression of the vascular endothelial growth factor (VEGF)-B increased tumour angiogenesis though tumour growth was impaired. In conclusion, we present a novel platform for intravital high-resolution and 3D imaging of PNET biology and cancer drug assessment.

Objective- The Wnt/β-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/β-catenin signaling by induced overexpression of Axin1, an inhibitor of β-catenin signaling, specifically in endothelial cells ( Axin1 iEC - OE ). AOE (Axin1 overexpression) in Axin1 iEC - OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/β-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC - OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, β-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific β-catenin-responsive ECM signature was also repressed in Axin1 iEC - OE and endothelial cell-specific β-catenin-knockout mice ( Ctnnb1-KO iEC ) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/β-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-β-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies., Competing Interests: EB, MG, MC, FF, PP, ML, CB, DP, AD, CG, GJ, CL, GB, SF, MM, MI, CG, RG, DN, DG No competing interests declared, JM, JS Affiliated with Gene Tools, LLC. Funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. ED Reviewing editor, eLife, CG Consultant for Gene-Tools, LLC. Funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript., (© 2019, Angiolini et al.)

Background: Glioblastoma (GBM) is an aggressive form of brain cancer with poor prognosis. Although murine animal models have given valuable insights into the GBM disease biology, they cannot be used in high-throughput screens to identify and profile novel therapies. The only vertebrate model suitable for large-scale screens, the zebrafish, has proven to faithfully recapitulate biology and pathology of human malignancies, and clinically relevant orthotopic zebrafish models have been developed. However, currently available GBM orthotopic zebrafish models do not support high-throughput drug discovery screens., Methods: We transplanted both GBM cell lines as well as patient-derived material into zebrafish blastulas. We followed the behavior of the transplants with time-lapse microscopy and real-time in vivo light-sheet microscopy., Results: We found that GBM material transplanted into zebrafish blastomeres robustly migrated into the developing nervous system, establishing an orthotopic intracranial tumor already 24 hours after transplantation. Detailed analysis revealed that our model faithfully recapitulates the human disease., Conclusion: We have developed a robust, fast, and automatable transplantation assay to establish orthotopic GBM tumors in zebrafish. In contrast to currently available orthotopic zebrafish models, our approach does not require technically challenging intracranial transplantation of single embryos. Our improved zebrafish model enables transplantation of thousands of embryos per hour, thus providing an orthotopic vertebrate GBM model for direct application in drug discovery screens.

Vascular lumen formation is a fundamental step during angiogenesis; yet, the molecular mechanisms underlying this process are poorly understood. Recent studies have shown that neural and vascular systems share common anatomical, functional and molecular similarities. Here we show that the organization of endothelial lumen is controlled at the post-transcriptional level by the alternative splicing (AS) regulator Nova2, which was previously considered to be neural cell-specific. Nova2 is expressed during angiogenesis and its depletion disrupts vascular lumen formation in vivo. Similarly, Nova2 depletion in cultured endothelial cells (ECs) impairs the apical distribution and the downstream signalling of the Par polarity complex, resulting in altered EC polarity, a process required for vascular lumen formation. These defects are linked to AS changes of Nova2 target exons affecting the Par complex and its regulators. Collectively, our results reveal that Nova2 functions as an AS regulator in angiogenesis and is a novel member of the 'angioneurins' family.

The functional diversity of the arterial and venous endothelia is regulated through a complex system of signalling pathways and downstream transcription factors. Here we report that the transcription factor Sox17, which is known as a regulator of endoderm and hemopoietic differentiation, is selectively expressed in arteries, and not in veins, in the mouse embryo and in mouse postnatal retina and adult. Endothelial cell-specific inactivation of Sox17 in the mouse embryo is accompanied by a lack of arterial differentiation and vascular remodelling that results in embryo death in utero. In mouse postnatal retina, abrogation of Sox17 expression in endothelial cells leads to strong vascular hypersprouting, loss of arterial identity and large arteriovenous malformations. Mechanistically, Sox17 acts upstream of the Notch system and downstream of the canonical Wnt system. These data introduce Sox17 as a component of the complex signalling network that orchestrates arterial/venous specification.

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved β-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

Objective: Freshly isolated pancreatic islets contain, in contrast to cultured islets, intraislet endothelial cells (ECs), which can contribute to the formation of functional blood vessels after transplantation. We have characterized how donor islet endothelial cells (DIECs) may contribute to the revascularization rate, vascular density, and endocrine graft function after transplantation of freshly isolated and cultured islets., Research Design and Methods: Freshly isolated and cultured islets were transplanted under the kidney capsule and into the anterior chamber of the eye. Intravital laser scanning microscopy was used to monitor the revascularization process and DIECs in intact grafts. The grafts' metabolic function was examined by reversal of diabetes, and the ultrastructural morphology by transmission electron microscopy., Results: DIECs significantly contributed to the vasculature of fresh islet grafts, assessed up to 5 months after transplantation, but were hardly detected in cultured islet grafts. Early participation of DIECs in the revascularization process correlated with a higher revascularization rate of freshly isolated islets compared with cultured islets. However, after complete revascularization, the vascular density was similar in the two groups, and host ECs gained morphological features resembling the endogenous islet vasculature. Surprisingly, grafts originating from cultured islets reversed diabetes more rapidly than those originating from fresh islets., Conclusions: In summary, DIECs contributed to the revascularization of fresh, but not cultured, islets by participating in early processes of vessel formation and persisting in the vasculature over long periods of time. However, the DIECs did not increase the vascular density or improve the endocrine function of the grafts.

During development, microvessels acquire specialized functions to meet the requirements of different tissues and organs. The vasculature of the brain constitutes one of the best examples of an organ-specific and highly specialized microvasculature, in which the endothelial cells that line blood vessels form an active permeability barrier and transport system called the blood-brain barrier (BBB); little is known, however, about the molecular mechanisms that instruct endothelial cells toward a BBB phenotype. Now Kuhnert et al. reveal that the orphan heterotrimeric GTP-binding protein-coupled receptor GPR124/TEM5 acts as an organ-specific regulator of brain angiogenesis, required for normal endothelial cell sprouting, migration, and expression of the BBB marker Glut-1 in the forebrain and neural tube. These findings add to our knowledge of brain vascularization and may open up possibilities for new therapeutic regimes to treat several diseases, including stroke, brain tumors, and vascular malformations.

The Wnt/beta-catenin pathway is evolutionary conserved signaling system that regulates cell differentiation and organogenesis. We show that endothelial specific stabilization of Wnt/beta-catenin signaling alters early vascular development in the embryo. The phenotype resembles that induced by upregulation of Notch signaling, including lack of vascular remodeling, altered elongation of the intersomitic vessels, defects in branching, and loss of venous identity. Both in vivo and in vitro data show that beta-catenin upregulates Dll4 transcription and strongly increases Notch signaling in the endothelium, leading to functional and morphological alterations. The functional consequences of beta-catenin signaling depend on the stage of vascular development and are lost when a gain-of-function mutation is induced at a late stage of development or postnatally. Our findings establish a link between Wnt and Notch signaling in vascular development. We propose that early and sustained beta-catenin signaling prevents correct endothelial cell differentiation, altering vascular remodeling and arteriovenous specification., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections.

Endothelial cell-to-cell junctions are vital for the formation and integrity of blood vessels. The main adhesive junctional complexes in endothelial cells, adherens junctions and tight junctions, are formed by transmembrane adhesive proteins that are linked to intracellular signalling partners and cytoskeletal-binding proteins. Gene inactivation and blocking antibodies in mouse models have revealed some of the functions of the individual junctional components in vivo, and are increasing our understanding of the functional role of endothelial cell junctions in angiogenesis and vascular homeostasis. Adherens-junction organization is required for correct vascular morphogenesis during embryo development. By contrast, the data available suggest that tight-junction proteins are not essential for vascular development but are necessary for endothelial barrier function.

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.

There is clearly a demand for an experimental platform that enables cell biology to be studied in intact vascularized and innervated tissue in vivo. This platform should allow observations of cells noninvasively and longitudinally at single-cell resolution. For this purpose, we use the anterior chamber of the mouse eye in combination with laser scanning microscopy (LSM). Tissue transplanted to the anterior chamber of the eye is rapidly vascularized, innervated and regains function. After transplantation, LSM through the cornea allows repetitive and noninvasive in vivo imaging at cellular resolution. Morphology, vascularization, cell function and cell survival are monitored longitudinally using fluorescent proteins and dyes. We have used this system to study pancreatic islets, but the platform can easily be adapted for studying a variety of tissues and additional biological parameters. Transplantation to the anterior chamber of the eye takes 25 min, and in vivo imaging 1-5 h, depending on the features monitored.

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.

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.

Pancreatic islet transplantation has emerged as a therapy for type 1 diabetes and is today performed using both freshly isolated and cultured islets. Islet blood vessels are disrupted during islet isolation; therefore, proper revascularization of the transplanted islets is of great importance for islet graft function and survival. We have studied intraislet endothelial cells after islet isolation, during islet culture, and following islet transplantation. By isolating islets from the transgenic Tie2-GFP (green fluorescent protein) mouse, characterized by an endothelial cell-specific expression of GFP, living endothelial cells could be studied in intact islets utilizing two-photon laser-scanning microscopy (TPLSM). Intraislet endothelial cells were found to survive islet transplantation but to rapidly disappear during islet culture. By transplanting freshly isolated Tie2-GFP islets and applying a novel ex vivo model for simultaneous perfusion and TPLSM imaging of the graft-bearing kidneys, GFP fluorescent endothelial cells were found to extensively contribute to vessels within the islet graft vasculature. Real-time imaging of the flow through the islet graft vasculature confirmed that the donor-derived vessels were functionally integrated. Hence, intraislet endothelial cells have the capability of participating in revascularization of pancreatic islets subsequent to transplantation. Therefore, preservation of intraislet endothelial cell mass may improve long-term graft function.

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.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-gamma (PLC-gamma). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-alpha and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-gamma activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1-mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-gamma activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1-mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1-mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.