Blood-brain barrier genetic disruption leads to protective barrier formation at the Glia Limitans.

Inflammation of the central nervous system (CNS) induces endothelial blood-brain barrier (BBB) opening as well as the formation of a tight junction barrier between reactive astrocytes at the Glia Limitans. We hypothesized that the CNS parenchyma may acquire protection from the reactive astrocytic Glia Limitans not only during neuroinflammation but also when BBB integrity is compromised in the resting state. Previous studies found that astrocyte-derived Sonic hedgehog (SHH) stabilizes the BBB during CNS inflammatory disease, while endothelial-derived desert hedgehog (DHH) is expressed at the BBB under resting conditions. Here, we investigated the effects of endothelial Dhh on the integrity of the BBB and Glia Limitans. We first characterized DHH expression within endothelial cells at the BBB, then demonstrated that DHH is down-regulated during experimental autoimmune encephalomyelitis (EAE). Using a mouse model in which endothelial Dhh is inducibly deleted, we found that endothelial Dhh both opens the BBB via the modulation of forkhead box O1 (FoxO1) transcriptional activity and induces a tight junctional barrier at the Glia Limitans. We confirmed the relevance of this glial barrier system in human multiple sclerosis active lesions. These results provide evidence for the novel concept of "chronic neuroinflammatory tolerance" in which BBB opening in the resting state is sufficient to stimulate a protective barrier at the Glia Limitans that limits the severity of subsequent neuroinflammatory disease. In summary, genetic disruption of the BBB generates endothelial signals that drive the formation under resting conditions of a secondary barrier at the Glia Limitans with protective effects against subsequent CNS inflammation. The concept of a reciprocally regulated CNS double barrier system has implications for treatment strategies in both the acute and chronic phases of multiple sclerosis pathophysiology.

Increased Capillary Permeability in Heart Induces Diastolic Dysfunction Independently of Inflammation, Fibrosis, or Cardiomyocyte Dysfunction.

BACKGROUND: While endothelial dysfunction is suggested to contribute to heart failure with preserved ejection fraction pathophysiology, understanding the importance of the endothelium alone, in the pathogenesis of diastolic abnormalities has not yet been fully elucidated. Here, we investigated the consequences of specific endothelial dysfunction on cardiac function, independently of any comorbidity or risk factor (diabetes or obesity) and their potential effect on cardiomyocyte. METHODS: The ubiquitine ligase Pdzrn3, expressed in endothelial cells (ECs), was shown to destabilize tight junction. A genetic mouse model in which Pdzrn3 is overexpressed in EC (iEC-Pdzrn3) in adults was developed. RESULTS: EC-specific Pdzrn3 expression increased cardiac leakage of IgG and fibrinogen blood-born molecules. The induced edema demonstrated features of diastolic dysfunction, with increased end-diastolic pressure, alteration of dP/dt min, increased natriuretic peptides, in addition to limited exercise capacity, without major signs of cardiac fibrosis and inflammation. Electron microscopic images showed edema with disrupted EC-cardiomyocyte interactions. RNA sequencing analysis of gene expression in cardiac EC demonstrated a decrease in genes coding for endothelial extracellular matrix proteins, which could be related to the fragile blood vessel phenotype. Irregularly shaped capillaries with hemorrhages were found in heart sections of iEC-Pdzrn3 mice. We also found that a high-fat diet was not sufficient to provoke diastolic dysfunction; high-fat diet aggravated cardiac inflammation, associated with an altered cardiac metabolic signature in EC-Pdzrn3 mice, reminiscent of heart failure with preserved ejection fraction features. CONCLUSIONS: An increase of endothelial permeability is responsible for mediating diastolic dysfunction pathophysiology and for aggravating detrimental effects of a high-fat diet on cardiac inflammation and metabolism.

PHACTR-1 (Phosphatase and Actin Regulator 1) Deficiency in Either Endothelial or Smooth Muscle Cells Does Not Predispose Mice to Nonatherosclerotic Arteriopathies in 3 Transgenic Mice.

BACKGROUND: Genome-wide association studies have revealed robust associations of common genetic polymorphisms in an intron of the PHACTR-1 (phosphatase and actin regulator 1) gene (chr6p24), with cervical artery dissection, spontaneous coronary artery dissection, and fibromuscular dysplasia. The aim was to assess its role in the pathogenesis of cervical artery dissection or fibromuscular dysplasia. METHODS: Using various tissue-specific Cre-driver mouse lines, Phactr1 was deleted either in endothelial cells using 2 tissue-specific Cre-driver (PDGFB [platelet-derived growth factor B]-CreERT2 mice and Tie2 [tyrosine kinase with immunoglobulin and EGF homology domains]-Cre) and smooth muscle cells (smooth muscle actin-CreERT2) with a third tissue-specific Cre-driver. RESULTS: To test the efficacy of the Phactr1 deletion after cre-induction, we confirmed first, a decrease in Phactr1 transcription and Phactr1 expression in endothelial cell and smooth muscle cell isolated from Phactr1iPDGFB and Phactr1iSMA mice. Irrespective to the tissue or the duration of the deletion, mice did not spontaneously display pathological phenotype or vascular impairment: mouse survival, growth, blood pressure, large vessel morphology, or actin organization were not different in knockout mice than their comparatives littermates. Challenging vascular function and repair either by angiotensin II-induced hypertension or limb ischemia did not lead to vascular morphology or function impairment in Phactr1-deleted mice. Similarly, there were no more consequences of Phactr1 deletion during embryogenesis in endothelial cells. CONCLUSIONS: Loss of PHACTR-1 function in the cells involved in vascular physiology does not appear to induce a pathological vascular phenotype. The in vivo effect of the intronic variation described in genome-wide association studies is unlikely to involve downregulation in PHACTR-1 expression.

S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate.

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8+ T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic-epigenetic axis, to immune fate and function.

Therapies targeting Frizzled-7/ß-catenin pathway prevent the development of pathological angiogenesis in an ischemic retinopathy model.

Retinopathies remain major causes of visual impairment in diabetic patients and premature infants. Introduction of anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) has transformed therapy for these proliferative retinopathies. However, limitations associated with anti-VEGF medications require to unravel new pathways of vessel growth to identify potential drug targets. Here, we investigated the role of Wnt/Frizzled-7 (Fzd7) pathway in a mouse model of oxygen-induced retinopathy (OIR). Using transgenic mice, which enabled endothelium-specific and time-specific Fzd7 deletion, we demonstrated that Fzd7 controls both vaso-obliteration and neovascular phases (NV). Deletion of Fzd7 at P12, after the ischemic phase of OIR, prevented formation of aberrant neovessels into the vitreous by suppressing proliferation of endothelial cells (EC) in tufts. Next we validated in vitro two Frd7 blocking strategies: a monoclonal antibody (mAbFzd7) against Fzd7 and a soluble Fzd7 receptor (CRD). In vivo a single intravitreal microinjection of mAbFzd7 or CRD significantly attenuated retinal neovascularization (NV) in mice with OIR. Molecular analysis revealed that Fzd7 may act through the activation of Wnt/ß-catenin and Jagged1 expression to control EC proliferation in extra-retinal neovessels. We identified Fzd7/ß-catenin signaling as new regulator of pathological retinal NV. Fzd7 appears to be a potent pharmacological target to prevent or treat aberrant angiogenesis of ischemic retinopathies.

Microporous Hydroxyapatite-Based Ceramics Alter the Physiology of Endothelial Cells through Physical and Chemical Cues.

Incorporation of silicate ions in calcium phosphate ceramics (CPC) and modification of their multiscale architecture are two strategies for improving the vascularization of scaffolds for bone regenerative medicine. The response of endothelial cells, actors for vascularization, to the chemical and physical cues of biomaterial surfaces is little documented, although essential. We aimed to characterize in vitro the response of an endothelial cell line, C166, cultivated on the surface CPCs varying either in terms of their chemistry (pure versus silicon-doped HA) or their microstructure (dense versus microporous). Adhesion, metabolic activity, and proliferation were significantly altered on microporous ceramics, but the secretion of the pro-angiogenic VEGF-A increased from 262 to 386 pg/mL on porous compared to dense silicon-doped HA ceramics after 168 h. A tubulogenesis assay was set up directly on the ceramics. Two configurations were designed for discriminating the influence of the chemistry from that of the surface physical properties. The formation of tubule-like structures was qualitatively more frequent on dense ceramics. Microporous ceramics induced calcium depletion in the culture medium (from 2 down to 0.5 mmol/L), which is deleterious for C166. Importantly, this effect might be associated with the in vitro static cell culture. No influence of silicon doping of HA on C166 behavior was detected.

Targeting Pdzrn3 maintains adult blood-brain barrier and central nervous system homeostasis.

Blood brain barrier (BBB) disruption is a critical component of the pathophysiology of cognitive impairment of vascular etiology (VCI) and associated with Alzheimer's disease (AD). The Wnt pathway plays a crucial role in BBB maintenance, but there is limited data on its role in cognitive pathologies. The E3 ubiquitin ligase PDZRN3 is a regulator of the Wnt pathway. In a murine model of VCI, overexpressing Pdzrn3 in endothelial cell (EC) exacerbated BBB hyperpermeability and accelerated cognitive decline. We extended these observations, in both VCI and AD models, showing that EC-specific depletion of Pdzrn3, reinforced the BBB, with a decrease in vascular permeability and a subsequent spare in cognitive decline. We found that in cerebral vessels, Pdzrn3 depletion protects against AD-induced Wnt target gene alterations and enhances endothelial tight junctional proteins. Our results provide evidence that Wnt signaling could be a molecular link regulating BBB integrity and cognitive decline under VCI and AD pathologies.

Decrease of Pdzrn3 is required for heart maturation and protects against heart failure.

Heart failure is the final common stage of most cardiopathies. Cardiomyocytes (CM) connect with others via their extremities by intercalated disk protein complexes. This planar and directional organization of myocytes is crucial for mechanical coupling and anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure is alterations in the contact sites between CM. Yet no factor on its own is known to coordinate CM polarized organization. We have previously shown that PDZRN3, an ubiquitine ligase E3 expressed in various tissues including the heart, mediates a branch of the Planar cell polarity (PCP) signaling involved in tissue patterning, instructing cell polarity and cell polar organization within a tissue. PDZRN3 is expressed in the embryonic mouse heart then its expression dropped significantly postnatally corresponding with heart maturation and CM polarized elongation. A moderate CM overexpression of Pdzrn3 (Pdzrn3 OE) during the first week of life, induced a severe eccentric hypertrophic phenotype with heart failure. In models of pressure-overload stress heart failure, CM-specific Pdzrn3 knockout showed complete protection against degradation of heart function. We reported that Pdzrn3 signaling induced PKC ζ expression, c-Jun nuclear translocation and a reduced nuclear ß catenin level, consistent markers of the planar non-canonical Wnt signaling in CM. We then show that subcellular localization (intercalated disk) of junction proteins as Cx43, ZO1 and Desmoglein 2 was altered in Pdzrn3 OE mice, which provides a molecular explanation for impaired CM polarization in these mice. Our results reveal a novel signaling pathway that controls a genetic program essential for heart maturation and maintenance of overall geometry, as well as the contractile function of CM, and implicates PDZRN3 as a potential therapeutic target for the prevention of human heart failure.