Reck enables cerebrovascular development by promoting canonical Wnt signaling.

The cerebral vasculature provides the massive blood supply that the brain needs to grow and survive. By acquiring distinctive cellular and molecular characteristics it becomes the blood-brain barrier (BBB), a selectively permeable and protective interface between the brain and the peripheral circulation that maintains the extracellular milieu permissive for neuronal activity. Accordingly, there is great interest in uncovering the mechanisms that modulate the formation and differentiation of the brain vasculature. By performing a forward genetic screen in zebrafish we isolated no food for thought (nft (y72)), a recessive late-lethal mutant that lacks most of the intracerebral central arteries (CtAs), but not other brain blood vessels. We found that the cerebral vascularization deficit of nft (y72) mutants is caused by an inactivating lesion in reversion-inducing cysteine-rich protein with Kazal motifs [reck; also known as suppressor of tumorigenicity 15 protein (ST15)], which encodes a membrane-anchored tumor suppressor glycoprotein. Our findings highlight Reck as a novel and pivotal modulator of the canonical Wnt signaling pathway that acts in endothelial cells to enable intracerebral vascularization and proper expression of molecular markers associated with BBB formation. Additional studies with cultured endothelial cells suggest that, in other contexts, Reck impacts vascular biology via the vascular endothelial growth factor (VEGF) cascade. Together, our findings have broad implications for both vascular and cancer biology.

CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis.

Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cγ1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis.

ApoB-containing lipoproteins regulate angiogenesis by modulating expression of VEGF receptor 1.

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.

Implanted microvessels progress through distinct neovascularization phenotypes.

We have previously demonstrated that implanted microvessels form a new microcirculation with minimal host-derived vessel investment. Our objective was to define the vascular phenotypes present during neovascularization in these implants and identify post-angiogenesis events. Morphological, functional and transcriptional assessments identified three distinct vascular phenotypes in the implants: sprouting angiogenesis, neovascular remodeling, and network maturation. A sprouting angiogenic phenotype appeared first, characterized by high proliferation and low mural cell coverage. This was followed by a neovascular remodeling phenotype characterized by a perfused, poorly organized neovascular network, reduced proliferation, and re-associated mural cells. The last phenotype included a vascular network organized into a stereotypical tree structure containing vessels with normal perivascular cell associations. In addition, proliferation was low and was restricted to the walls of larger microvessels. The transition from angiogenesis to neovascular remodeling coincided with the appearance of blood flow in the implant neovasculature. Analysis of vascular-specific and global gene expression indicates that the intermediate, neovascular remodeling phenotype is transcriptionally distinct from the other two phenotypes. Therefore, this vascular phenotype likely is not simply a transitional phenotype but a distinct vascular phenotype involving unique cellular and vascular processes. Furthermore, this neovascular remodeling phase may be a normal aspect of the general neovascularization process. Given that this phenotype is arguably dysfunctional, many of the microvasculatures present within compromised or diseased tissues may not represent a failure to progress appropriately through a normally occurring neovascularization phenotype.

fused-somites-like mutants exhibit defects in trunk vessel patterning.

We identified four mutants in two distinct loci exhibiting similar trunk vascular patterning defects in an F3 genetic screen for zebrafish vascular mutants. Initial vasculogenesis is not affected in these mutants, with proper specification and differentiation of endothelial cells. However, all four display severe defects in the growth and patterning of angiogenic vessels in the trunk, with ectopic branching and disoriented migration of intersegmental vessels. The four mutants are allelic to previously characterized mutants at the fused-somites (fss) and beamter (bea) loci, and they exhibit comparable defects in trunk somite boundary formation. The fss locus has been shown to correspond to tbx24; we show here that bea mutants are defective in the zebrafish dlC gene. Somitic expression of known vascular guidance factors efnb2a, sema3a1, and sema3a2 is aberrantly patterned in fss and bea mutants, suggesting that the vascular phenotype is due to loss of proper guidance cues provided by these factors.

Stimulated endothelial cell adhesion and angiogenesis with laminin-5 modification of expanded polytetrafluoroethylene.

Biomedical implants often exhibit poor clinical performance due to the formation of a periimplant avascular fibrous capsule. Surface modification of synthetic materials has been evaluated to accelerate the formation of functional microcirculation in association with implants. The current study used a flow-mediated protein deposition system to modify expanded polytetrafluoroethylene (ePTFE) with a laminin-5-rich conditioned growth medium and with medium from which laminin-5 had been selectively removed. An in vitro model of endothelial cell adherence determined that laminin-5 modification resulted in significantly increased adhesion of human microvessel endothelial cells to ePTFE. In vivo studies evaluating the periimplant vascular response to laminin-5-treated samples indicated that absorption of laminin-5-rich conditioned medium supported accelerated neovascularization of ePTFE implants. A flow system designed to treat porous implant materials facilitates laminin-5 modification of commercially available ePTFE, resulting in increased endothelial cell adhesion in vitro and increased vascularization in vivo.

Laminin-5-enriched extracellular matrix accelerates angiogenesis and neovascularization in association with ePTFE.

The performance of biomedical implant devices is often limited by inappropriate tissue responses associated with synthetic materials used in device construction. Adverse healing responses, in particular the lack of an extensive vascular supply in the peri-implant tissue, are believed to lead to the ultimate failure of many of these medical devices. Accelerated formation of new blood vessels in the peri-implant tissue and within porous polymeric implants is hypothesized to improve the performance of such biomedical implant devices. The current study evaluated the use of cell-mediated, extracellular matrix modification of expanded polytetrafluoroethylene (ePTFE) to increase vessel growth in peri-implant tissue and within the pores of the implants. Discs of ePTFE were modified through cell-mediated matrix deposition using epithelial and endothelial cell lines with variable deposition of collagen types, fibronectin, and laminin types. Cell matrix-modified discs, Matrigel-coated discs, and nonmodified discs were implanted in both the adipose and subcutaneous tissues of the rat. Following a 5-week implant period, samples were removed and evaluated histologically and morphometrically for the presence of blood vessels in the peri-implant tissue and within the pores of the polymer as well as for the presence of activated macrophages and monocytes. A significantly increased presence of activated macrophages and monocytes was associated only with the samples modified with the matrix from a human microvessel endothelial cell line. Increased vessel density was identified in association with those ePTFE samples modified with either the 804-G, HaCaT, or II-4 cell matrices, all of which have extracellular matrices enriched in the protein laminin-5.

Fishing for novel angiogenic therapies.

The zebrafish has recently emerged as an important model for the study of vascular embryogenesis. Its genetic accessibility, external development, and optically clear embryo are just a few of the features that set the zebrafish apart as a particularly well-suited model for studying vascular development. However, there is little precedent for its use as a tool for the experimental study of therapeutic angiogenesis. Here, we review the use of the zebrafish for studying vascular development and patterning, and discuss how the zebrafish might be used more directly as a model for developing and testing effective therapeutic angiogenesis approaches.

Accelerated endothelialization of interpositional 1-mm vascular grafts.

BACKGROUND: There is an increased need for alternative, synthetic, small-diameter vascular grafts due to a growing segment of the population who suffer from ischemic heart disease and lack suitable autologous vein grafts for use in coronary artery bypass grafting (CABG). We hypothesized that a cell-mediated extracellular matrix (ECM) modification of ePTFE would stimulate increased vascularization within the graft and thus promote lumenal endothelialization in a 1-mm rat abdomenal aortic implant model. METHODS AND RESULTS: Expanded polytetrafluoroethylene (ePTFE) vascular grafts (1 mm i.d.) were modified on the ablumenal surface with ECM deposited by the HaCaT or II-4 cell lines and implanted intrapositionally into the descending aorta of rats. Five weeks after implantation, all samples were patent and examination of the grafts demonstrated that the ECM modified samples exhibited extensive ablumenal vascularization and tissue incorporation compared to nonmodified samples. Also, ECM modified grafts had a cellular lining, while the nonmodified grafts were void of a cellular lining except for a limited pannus ingrowth. CONCLUSION: HaCaT and II-4 cell ECM modifications of ePTFE increase new blood vessel growth in association with the graft, and the II-4 cell modification results in formation of an endothelial monlayer on the lumenal surface of the graft.

A comparative evaluation of the tissue responses associated with polymeric implants in the rat and mouse.

End product application is an important consideration when evaluating a material in an in vivo setting (Didisheim, Cardiovasc Pathol 1993;2:1S-2S). Small animal models allow high through-put evaluation of biocompatability. Previous preclinical evaluations have often used a rat subcutaneous model for the characterization of material-tissue interaction. Recent advances in genetic manipulation have provided mouse models with selective expression of a wide range of critical proteins. The rat model does not have many of the resources (i.e., knockouts, SCID, nude) that are present in mouse strains. The availability of these mice provides a resource to delineate the mechanisms regulating the healing associated with implants. However, before the mouse models can be used, they must be validated with respect to their ability to accurately assess tissue responses to materials. In this study the tissue responses after the implantation of expanded polytetrafluoroethylene (ePTFE) were compared between rat and mouse. Discs of ePTFE (30-microm internodal distance) were implanted in subcutaneous and epididymal fat tissue of rats (Sprague-Dawley) and mice (129-SVJ). After 5 weeks the samples were removed and evaluated for vascular density, inflammation, and fibrous encapsulation. No difference in the vessel density was observed within the peri-implant subcutaneous and adipose tissue or within the porous material. However, a significant difference was found in the number of activated macrophages and giant cells between these two species. Implants in the rat exhibited greater numbers of activated inflammatory cells in the peri-implant tissue. The data indicate that the mouse and rat provide a comparable model for evaluating angiogenesis and neovascularization associated with synthetic porous implants.

Angiogenesis and neovascularization associated with extracellular matrix-modified porous implants.

Therapies directed toward stimulation of angiogenesis seek to accelerate the development of new blood vessels in tissues rendered dysfunctional because of an insufficient microvascular supply. The goal of the current study was the stimulation of an angiogenic response around and within porous biomedical implants, such as vascular grafts, constructed from a base polymer composed of expanded polytetrafluoroethylene (ePTFE). Similar to many biomaterials, ePTFE does not elicit a significant angiogenic response and the porous interstices of this material remain avascular after implantation. Studies were performed to evaluate the ability of a tumorigenic cell line, the 804-G rat kidney cell to secrete an angiogenic extracellular matrix on and within the porous structures of ePTFE. A rat model was used to evaluate and compare implant-associated healing responses between nonmodified materials and extracellular matrix-modified ePTFE. Results demonstrated that, in contrast to untreated ePTFE, the matrix-modified ePTFE stimulated both angiogenesis in implant-associated tissue and neovascularization of the pores within the ePTFE interstices. Deposition of an insoluble matrix stimulates an angiogenic response and has a potential application for the improvement of medical device function.