Hedgehog signaling is required for maintaining the integrity of the blood-brain barrier.

The blood–brain barrier (BBB) restricts molecular and cellular trafficking between the blood and the CNS. Although astrocytes are known to control BBB permeability, the molecular determinants of this effect remain unknown. We show that angiotensinogen (AGT) produced and secreted by astrocytes is cleaved into angiotensin II (AngII) and acts on type 1 angiotensin receptors (AT₁) expressed by BBB endothelial cells (ECs). Activation of AT₁restricts the passage of molecular tracers across human BBB-derived ECs through threonine-phosphorylation of the tight junction protein occludin and its mobilization to lipid raft membrane microdomains. We also show that AGT knock-out animals have disorganized occludin strands at the level of the BBB and a diffuse accumulation of the endogenous serum protein plasminogen in the CNS, compared with wild-type animals. Finally, we demonstrate a reduction in the number of AGT-immunopositive perivascular astrocytes in multiple sclerosis (MS) lesions, which correlates with a reduced expression of occludin similarly seen in the CNS of AGT knock-out animals. Such a reduction in astrocyte-expressed AGT and AngII is dependent,in vitro, on the proinflammatory cytokines tumor necrosis factor-α and interferon-γ. Our study defines a novel physiological role for AngII in the CNS and suggests that inflammation-induced downregulation of AngII production by astrocytes is involved in BBB dysfunction in MS lesions.

Trafficking of antigen-presenting cells into the CNS is essential for lymphocyte reactivation within the CNS compartment. Although perivascular dendritic cells found in inflammatory lesions are reported to polarize naive CD4+ T lymphocytes into interleukin-17-secreting-cells, the origin of those antigen-presenting cells remains controversial. We demonstrate that a subset of CD14+ monocytes migrate across the inflamed human blood-brain barrier (BBB) and differentiate into CD83+CD209+ dendritic cells under the influence of BBB-secreted transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor. We also demonstrate that these dendritic cells secrete interleukin-12p70, transforming growth factor-beta and interleukin-6 and promote the proliferation and expansion of distinct populations of interferon-gamma-secreting Th1 and interleukin-17-secreting Th17 CD4+ T lymphocytes. We further confirmed the abundance of such dendritic cells in situ, closely associated with microvascular BBB-endothelial cells within acute multiple sclerosis lesions, as well as a significant number of CD4+ interleukin-17+ T lymphocytes in the perivascular infiltrate. Our data support the notion that functional perivascular myeloid CNS dendritic cells arise as a consequence of migration of CD14+ monocytes across the human BBB, through the concerted actions of BBB-secreted transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor.

Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescence-associated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fueling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations. Abbreviations: 3-MA: 3-methyladenine; ACTA2: actin, alpha 2, smooth muscle, aorta; AKT1: AKT serine/threonine kinase 1; p-AKT1: AKT1 Ser473 phosphorylation; t-AKT1: total AKT serine/threonine kinase 1; ATG4A: autophagy related 4A cysteine peptidase; ATG7: autophagy gene 7; C12FDG: 5-dodecanoylaminofluorescein Di-β-D-Galactopyranoside; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; Ctl: control; DAPI: 4ʹ,6-diamidino-2-phenylindole, dilactate; ECM: extracellular matrix; GSH: L-glutathione reduced; H₂O₂: hydrogen peroxide; HLF: adult human lung fibroblasts; Ho: Hoechst 33342 (2′‐[4‐ethoxyphenyl]‐5‐[4‐methyl‐1‐piperazinyl]‐2.5′‐bi‐1H‐benzimidazole); HSC: hepatic stellate cells; LY: LY294002; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTORC1/2: mechanistic target of rapamycin kinase complex 1/2; N: normal growth medium; NAC: N-acetyl-L-cysteine; PBS: phosphate-buffered saline; PDGFA: platelet derived growth factor subunit A; PRKCA/PKCα: protein kinase C alpha; PtdIns3K: class III phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homolog; R: rapamycin; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR: regulatory associated protein of MTOR complex 1; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SGK1: serum/glucocorticoid regulated kinase 1; shRNA: short hairpin RNA; siCtl: control siRNA; siRNA: small interfering RNA; SQSTM1: sequestosome 1; SS: serum-free (serum starvation) medium; TP53: tumor protein p53; TUBA: tubulin alpha; V: vehicle.