Mice completely lacking immunoproteasomes show major changes in antigen presentation.

The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes had not been available. Having now developed mice lacking the three immunoproteasome catalytic subunits, we found that the dendritic cells of these mice had defects in presenting several major histocompatibility complex (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes was markedly reduced in immunoproteasome-deficient animals compared with wild-type animals, whereas presentation of MHC class II peptides was unaffected. According to mass spectrometry, the repertoire of MHC class I-presented peptides was ∼50% different from that in wild-type mice, and these differences were sufficient to stimulate robust transplant rejection of wild-type cells in mutant mice. These results indicated that immunoproteasomes were more important in antigen presentation than previously thought.

Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations.

Despite antiretroviral therapy (ART), human immunodeficiency virus (HIV)-1 persists in a stable latent reservoir, primarily in resting memory CD4(+) T cells. This reservoir presents a major barrier to the cure of HIV-1 infection. To purge the reservoir, pharmacological reactivation of latent HIV-1 has been proposed and tested both in vitro and in vivo. A key remaining question is whether virus-specific immune mechanisms, including cytotoxic T lymphocytes (CTLs), can clear infected cells in ART-treated patients after latency is reversed. Here we show that there is a striking all or none pattern for CTL escape mutations in HIV-1 Gag epitopes. Unless ART is started early, the vast majority (>98%) of latent viruses carry CTL escape mutations that render infected cells insensitive to CTLs directed at common epitopes. To solve this problem, we identified CTLs that could recognize epitopes from latent HIV-1 that were unmutated in every chronically infected patient tested. Upon stimulation, these CTLs eliminated target cells infected with autologous virus derived from the latent reservoir, both in vitro and in patient-derived humanized mice. The predominance of CTL-resistant viruses in the latent reservoir poses a major challenge to viral eradication. Our results demonstrate that chronically infected patients retain a broad-spectrum viral-specific CTL response and that appropriate boosting of this response may be required for the elimination of the latent reservoir.

Neuronal surface P antigen (NSPA) modulates postsynaptic NMDAR stability through ubiquitination of tyrosine phosphatase PTPMEG.

BACKGROUND: Cognitive dysfunction (CD) is common among patients with the autoimmune disease systemic lupus erythematosus (SLE). Anti-ribosomal P autoantibodies associate with this dysfunction and have neuropathogenic effects that are mediated by cross-reacting with neuronal surface P antigen (NSPA) protein. Elucidating the function of NSPA can then reveal CD pathogenic mechanisms and treatment opportunities. In the brain, NSPA somehow contributes to glutamatergic NMDA receptor (NMDAR) activity in synaptic plasticity and memory. Here we analyze the consequences of NSPA absence in KO mice considering its structural features shared with E3 ubiquitin ligases and the crucial role of ubiquitination in synaptic plasticity. RESULTS: Electrophysiological studies revealed a decreased long-term potentiation in CA3-CA1 and medial perforant pathway-dentate gyrus (MPP-DG) hippocampal circuits, reflecting glutamatergic synaptic plasticity impairment in NSPA-KO mice. The hippocampal dentate gyrus of these mice showed a lower number of Arc-positive cells indicative of decreased synaptic activity and also showed proliferation defects of neural progenitors underlying less adult neurogenesis. All this translates into poor spatial and recognition memory when NSPA is absent. A cell-based assay demonstrated ubiquitination of NSPA as a property of RBR-type E3 ligases, while biochemical analysis of synaptic regions disclosed the tyrosine phosphatase PTPMEG as a potential substrate. Mice lacking NSPA have increased levels of PTPMEG due to its reduced ubiquitination and proteasomal degradation, which correlated with lower levels of GluN2A and GluN2B NMDAR subunits only at postsynaptic densities (PSDs), indicating selective trafficking of these proteins out of PSDs. As both GluN2A and GluN2B interact with PTPMEG, tyrosine (Tyr) dephosphorylation likely drives their endocytic removal from the PSD. Actually, immunoblot analysis showed reduced phosphorylation of the GluN2B endocytic signal Tyr1472 in NSPA-KO mice. CONCLUSIONS: NSPA contributes to hippocampal plasticity and memory processes ensuring appropriate levels of adult neurogenesis and PSD-located NMDAR. PTPMEG qualifies as NSPA ubiquitination substrate that regulates Tyr phosphorylation-dependent NMDAR stability at PSDs. The NSPA/PTPMEG pathway emerges as a new regulator of glutamatergic transmission and plasticity and may provide mechanistic clues and therapeutic opportunities for anti-P-mediated pathogenicity in SLE, a still unmet need.

Unique functions of the type II interleukin 4 receptor identified in mice lacking the interleukin 13 receptor alpha1 chain.

The interleukin 4 receptor (IL-4R) is a central mediator of T helper type 2 (T(H)2)-mediated disease and associates with either the common gamma-chain to form the type I IL-4R or with the IL-13R alpha1 chain (IL-13Ralpha1) to form the type II IL-4R. Here we used Il13ra1-/- mice to characterize the distinct functions of type I and type II IL-4 receptors in vivo. In contrast to Il4ra-/- mice, which have weak T(H)2 responses, Il13ra1-/- mice had exacerbated T(H)2 responses. Il13ra1-/- mice showed much less mortality after infection with Schistosoma mansoni and much more susceptibility to Nippostrongylus brasiliensis. IL-13Ralpha1 was essential for allergen-induced airway hyperreactivity and mucus hypersecretion but not for fibroblast or alternative macrophage activation. Thus, type I and II IL-4 receptors exert distinct effects on immune responses.

IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine.

Chronic mucosal inflammation and tissue damage predisposes patients to the development of colorectal cancer. This association could be explained by the hypothesis that the same factors and pathways important for wound healing also promote tumorigenesis. A sensor of tissue damage should induce these factors to promote tissue repair and regulate their action to prevent development of cancer. Interleukin 22 (IL-22), a cytokine of the IL-10 superfamily, has an important role in colonic epithelial cell repair, and its levels are increased in the blood and intestine of inflammatory bowel disease patients. This cytokine can be neutralized by the soluble IL-22 receptor, known as the IL-22 binding protein (IL-22BP, also known as IL22RA2); however, the significance of endogenous IL-22BP in vivo and the pathways that regulate this receptor are unknown. Here we describe that IL-22BP has a crucial role in controlling tumorigenesis and epithelial cell proliferation in the colon. IL-22BP is highly expressed by dendritic cells in the colon in steady-state conditions. Sensing of intestinal tissue damage via the NLRP3 or NLRP6 inflammasomes led to an IL-18-dependent downregulation of IL-22BP, thereby increasing the ratio of IL-22/IL-22BP. IL-22, which is induced during intestinal tissue damage, exerted protective properties during the peak of damage, but promoted tumour development if uncontrolled during the recovery phase. Thus, the IL-22-IL-22BP axis critically regulates intestinal tissue repair and tumorigenesis in the colon.

GPR17 gene disruption does not alter food intake or glucose homeostasis in mice.

G protein-coupled receptor 17 (GPR17) was recently reported to be a Foxo1 target in agouti-related peptide (AGRP) neurons. Intracerebroventricular injection of GPR17 agonists induced food intake, whereas administration of an antagonist to the receptor reduced feeding. These data lead to the conclusion that pharmacological modulation of GPR17 has therapeutic potential to treat obesity. Here we report that mice deficient in Gpr17 (Gpr17(-/-)) have similar food intake and body weight compared with their wild-type littermates. Gpr17(-/-) mice have normal hypothalamic Agrp mRNA expression, AGRP plasma levels, and metabolic rate. GPR17 deficiency in mice did not affect glucose homeostasis or prevent fat-induced insulin resistance. These data do not support a role for GPR17 in the control of food intake, body weight, or glycemic control.

Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease thought to be mediated by dysfunctional innate and/or adaptive immunity. This aberrant immune response leads to the secretion of harmful cytokines that destroy the epithelium of the gastrointestinal tract and thus cause further inflammation. Interleukin-22 (IL-22) is a T helper 17 (Th17) T cell-associated cytokine that is bifunctional in that it has both proinflammatory and protective effects on tissues depending on the inflammatory context. We show herein that IL-22 protected mice from IBD. Interestingly, not only was this protection mediated by CD4+ T cells, but IL-22-expressing natural killer (NK) cells also conferred protection. In addition, IL-22 expression was differentially regulated between NK cell subsets. Thus, both the innate and adaptive immune responses have developed protective mechanisms to counteract the damaging effects of inflammation on tissues.

ANGPTL4-αvß3 interaction counteracts hypoxia-induced vascular permeability by modulating Src signalling downstream of vascular endothelial growth factor receptor 2.

Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvß3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvß3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvß3 interaction enhances Src recruitment to integrin αvß3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvß3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvß3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvß3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes.

Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.

Mice with mutant Inf2 show impaired podocyte and slit diaphragm integrity in response to protamine-induced kidney injury.

Mutations in the INF2 (inverted formin 2) gene, encoding a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause human focal segmental glomerulosclerosis (FSGS). INF2 interacts directly with certain other mammalian diaphanous formin proteins (mDia) that function as RhoA effector molecules. FSGS-causing INF2 mutations impair these interactions and disrupt the ability of INF2 to regulate Rho/Dia-mediated actin dynamics in vitro. However, the precise mechanisms by which INF2 regulates and INF2 mutations impair glomerular structure and function remain unknown. Here, we characterize an Inf2 R218Q point-mutant (knockin) mouse to help answer these questions. Knockin mice have no significant renal pathology or proteinuria at baseline despite diminished INF2 protein levels. INF2 mutant podocytes do show impaired reversal of protamine sulfate-induced foot process effacement by heparin sulfate perfusion. This is associated with persistent podocyte cytoplasmic aggregation, nephrin phosphorylation, and nephrin and podocin mislocalization, as well as impaired recovery of mDia membrane localization. These changes were partially mimicked in podocyte outgrowth cultures, in which podocytes from knockin mice show altered cellular protrusions compared to those from wild-type mice. Thus, in mice, normal INF2 function is not required for glomerular development but normal INF2 is required for regulation of the actin-based behaviors necessary for response to and/or recovery from injury.

Mice lacking ANGPTL8 (Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis.

Angiopoietin-like protein (ANGPTL)8 (alternatively called TD26, RIFL, Lipasin, and Betatrophin) is a newly recognized ANGPTL family member that has been implicated in both triglyceride (TG) and glucose metabolism. Hepatic overexpression of ANGPTL8 causes hypertriglyceridemia and increased insulin secretion. Here we examined the effects of inactivating Angptl8 on TG and glucose metabolism in mice. Angptl8 knockout (Angptl8(-/-)) mice gained weight more slowly than wild-type littermates due to a selective reduction in adipose tissue accretion. Plasma levels of TGs of the Angptl8(-/-) mice were similar to wild-type animals in the fasted state but paradoxically decreased after refeeding. The lower TG levels were associated with both a reduction in very low density lipoprotein secretion and an increase in lipoprotein lipase (LPL) activity. Despite the increase in LPL activity, the uptake of very low density lipoprotein-TG is markedly reduced in adipose tissue but preserved in hearts of fed Angptl8(-/-) mice. Taken together, these data indicate that ANGPTL8 plays a key role in the metabolic transition between fasting and refeeding; it is required to direct fatty acids to adipose tissue for storage in the fed state. Finally, glucose and insulin tolerance testing revealed no alterations in glucose homeostasis in mice fed either a chow or high fat diet. Thus, although absence of ANGPTL8 profoundly disrupts TG metabolism, we found no evidence that it is required for maintenance of glucose homeostasis.

Conditionals by inversion provide a universal method for the generation of conditional alleles.

Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.

ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys.

Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia.

Generation of fertile and fecund F0 XY female mice from XY ES cells.

Known examples of male to female sex reversal in mice are caused by either strain incompatibilities or mutations in genes required for male sex determination. The resultant XY females are often sterile or exhibit very poor fertility. We describe here embryonic stem (ES) cell growth conditions that promote the production of healthy, anatomically normal fertile and fecund female F0 generation mice completely derived from gene-targeted XY male ES cells. The sex reversal is a transient trait that is not transmitted to the F1 progeny. Growth media with low osmolality and reduced sodium bicarbonate, maintained throughout the gene targeting process, enhance the yield of XY females. As a practical application of the induced sex reversal, we demonstrate the generation of homozygous mutant mice ready for phenotypic studies by the breeding of F0 XY females with their isogenic XY male clonal siblings, thereby eliminating one generation of breeding and the associated costs.

Atypical angiopoietin-like protein that regulates ANGPTL3.

Angiopoietin-like proteins (ANGPTLs) play major roles in the trafficking and metabolism of lipids. Inactivation of ANGPTL3, a gene located in an intron of DOCK7, results in very low levels of LDL-cholesterol (C), HDL-C and triglyceride (TAG). We identified another ANGPTL family member, ANGPTL8, which is located in the corresponding intron of DOCK6. A variant in this family member (rs2278426, R59W) was associated with lower plasma LDL-C and HDL-C levels in three populations. ANGPTL8 is expressed in liver and adipose tissue, and circulates in plasma of humans. Expression of ANGPTL8 was reduced by fasting and increased by refeeding in both mice and humans. To examine the functional relationship between the two ANGPTL family members, we expressed ANGPTL3 at physiological levels alone or together with ANGPTL8 in livers of mice. Plasma TAG level did not change in mice expressing ANGPTL3 alone, whereas coexpression with ANGPTL8 resulted in hypertriglyceridemia, despite a reduction in circulating ANGPTL3. ANGPTL8 coimmunoprecipitated with the N-terminal domain of ANGPTL3 in plasma of these mice. In cultured hepatocytes, ANGPTL8 expression increased the appearance of N-terminal ANGPTL3 in the medium, suggesting ANGPTL8 may activate ANGPTL3. Consistent with this scenario, expression of ANGPTL8 in Angptl3(-/-) mice failed to promote hypertriglyceridemia. Thus, ANGPTL8, a paralog of ANGPTL3 that arose through duplication of an ancestral DOCK gene, regulates postprandial TAG and fatty acid metabolism by controlling activation of its progenitor, and perhaps other ANGPTLs. Inhibition of ANGPTL8 provides a new therapeutic strategy for reducing plasma lipoprotein levels.

Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung.

Mice with a functional human immune system have the potential to allow in vivo studies of human infectious diseases and to enable vaccine testing. To this end, mice need to fully support the development of human immune cells, allow infection with human pathogens, and be capable of mounting effective human immune responses. A major limitation of humanized mice is the poor development and function of human myeloid cells and the absence of human immune responses at mucosal surfaces, such as the lung. To overcome this, we generated human IL-3/GM-CSF knock-in (hIL-3/GM-CSF KI) mice. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis because of elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34(+) hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the pulmonary alveolar proteinosis syndrome. Moreover, human alveolar macrophages mounted correlates of a human innate immune response against influenza virus. The hIL-3/GM-CSF KI mice represent a unique mouse model that permits the study of human mucosal immune responses to lung pathogens.

Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo.

Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are broadly used to study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells are suboptimal in these hosts. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. We generated RAG2(-/-)γ(c)(-/-) mice in which we replaced the gene encoding mouse TPO by its human homolog. Homozygous humanization of TPO led to increased levels of human engraftment in the bone marrow of the hosts, and multilineage differentiation of hematopoietic cells was improved, with an increased ratio of myelomonocytic verus lymphoid lineages. Moreover, maintenance of human stem and progenitor cells was improved, as demonstrated by serial transplantation. Therefore, RAG2(-/-)γ(c)(-/-) TPO-humanized mice represent a useful model to study human hematopoiesis in vivo.

Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor.

The vasculature of the CNS is structurally and functionally distinct from that of other organ systems and is particularly prone to developmental abnormalities and hemorrhage. Although other embryonic tissues undergo primary vascularization, the developing nervous system is unique in that it is secondarily vascularized by sprouting angiogenesis from a surrounding perineural plexus. This sprouting angiogenesis requires the TGF-ß and Wnt pathways because ablation of these pathways results in aberrant sprouting and hemorrhage. We have genetically deleted Gpr124, a member of the large family of long N-terminal group B G protein-coupled receptors, few members of which have identified ligands or well-defined biologic functions in mammals. We show that, in the developing CNS, Gpr124 is specifically expressed in the vasculature and is absolutely required for proper angiogenic sprouting into the developing neural tube. Embryos lacking Gpr124 exhibit vascular defects characterized by delayed vascular penetration, formation of pathological glomeruloid tufts within the CNS, and hemorrhage. In addition, they display defects in palate and lung development, two processes in which TGF-ß and/or Wnt pathways also play important roles. We also show that TGF-ß stimulates Gpr124 expression, and ablation of Gpr124 results in perturbed TGF-ß pathway activation, suggesting roles for Gpr124 in modulating TGF-ß signaling. These results represent a unique function attributed to a long N-terminal group B-type G protein-coupled receptor in a mammalian system.

Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke.

AIMS: Given the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke. METHODS AND RESULTS: Using a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema. Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2-VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area. CONCLUSION: These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke.