Daniel Nyqvist

Nrarp Coordinates Endothelial Notch and Wnt Signaling to Control Vessel Density in Angiogenesis

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.

The Wnt/β-Catenin Pathway Modulates Vascular Remodeling and Specification by Upregulating Dll4/Notch Signaling

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.

Noninvasive in vivo imaging of pancreatic islet cell biology

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.

Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes

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.

Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye

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.

Sox17 is indispensable for acquisition and maintenance of arterial identity

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.

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.

Donor Islet Endothelial Cells in Pancreatic Islet Revascularization

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.

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.

Deciphering the functional role of endothelial junctions by using in vivo models

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.