B r i e f r e p o r t4 IntroductionStroke is a leading cause of morbidity and mortality, whose prevalence increases dramatically with age. Despite its substantial heritable basis, only a small number of causative genes have so far been identified, generally for severe early-onset phenotypes (cerebral autosomal dominant or recessive arteriopathy with subcortical infarcts and leukoencephalopathy: CADASIL [NOTCH3], CARASIL [HTRA1], and porencephaly [COL4A1]) (1-3). Such cases have revealed important pathways that contribute to stroke, including the roles of Notch and TGF-β signaling.In the same way, the vascular basement membrane's contribution (COL4A1 and COL4A2) (3, 4) to juvenile stroke phenotypes further stimulated investigation of the cellular components (endothelial and mural cells) upon which brain vascular integrity depends. The demonstration that Notch signaling regulates pericyte numbers (5, 6) has in turn provided a mechanistic explanation for disorders such as CADASIL. These examples of juvenile stroke resulting from severe alterations in brain vascular development raise the intriguing possibility that milder changes contribute to late-onset disease and that a larger proportion of strokes have embryonic origins. It is therefore notable that the same genes regulate cerebral structural development and angiogenesis (7) and that the cell populations essential for cerebral vascular homeostasis (pericytes and vascular smooth muscle) are predominantly derived from the neural crest (8, 9). The increasing prevalence of stroke exerts disproportionately severe effects on the quality of life of affected individuals and their families. Consequently, phenotypes predictive of future stroke merit investigation, with the goal of developing treatments targeting causative pathways and preventing a frequently preterminal disease. One such phenotype is cerebral small-vessel disease (CSVD), which represents a major risk factor for both ischemic and hemorrhagic stroke (10-13). Characterized by perturbed perforating end-artery function, CSVD results in lesions apparent on MRI that encompass white matter hyperintensities (WMHs), dilated perivascular spaces, microbleeds, and lacunar infarcts. These markers of cerebrovascular pathology provide opportunities for gene discovery and for defining the mechanisms that contribute to subsequent stroke.Our study evaluated the hypothesis that the forkhead box transcription factor FOXC1, which patterns multiple organs including the CNS, contributes to CSVD. It was prompted by a higher incidence of self-reported stroke in some of our local pedigrees with FOXC1 mutations and supported experimentally by: (a) blood-stained hydrocephalus in murine Foxc1 -/-mutants, (b) related zebrafish foxc1 morphant phenotypes, and (c) the extenPatients with cerebral small-vessel disease (CSVD) exhibit perturbed end-artery function and have an increased risk for stroke and age-related cognitive decline. Here, we used targeted genome-wide association (GWA) analysis and defined a CSVD locus adjacent to the forkh...

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Background The transmembrane receptor Notch1 is a critical regulator of arterial differentiation and blood vessel sprouting. Recent evidence shows that functional blockade of Notch1 and its ligand, Dll-4, leads to postnatal lymphatic defects in mice. However, the precise role of the Notch signaling pathway in lymphatic vessel development has yet to be defined. Here we show the developmental role of Notch1 in lymphatic vascular morphogenesis by analyzing lymphatic endothelial cell (LEC)-specific conditional Notch1 knockout mice crossed with an inducible Prox1CreERT2 driver. Results LEC-specific Notch1 mutant embryos exhibited enlarged lymphatic vessels. The phenotype of lymphatic overgrowth accords with increased LEC sprouting from the lymph sacs and increased filopodia formation. Furthermore, cell death was significantly reduced in Notch1-mutant LECs, whereas proliferation was increased. RNA-seq analysis revealed that expression of cytokine/chemokine signaling molecules was upregulated in Notch1-mutant LECs isolated from E15.5 dorsal skin, whereas VEGFR3, VEGFR2, VEGFC, and Gja4 (Connexin 37) were downregulated. Conclusions The lymphatic phenotype of LEC-specific conditional Notch1 mouse mutants indicates that Notch activity in LECs controls lymphatic sprouting and growth during development. These results provide evidence that similar to postnatal and pathological lymphatic vessel formation, the Notch signaling pathway plays a role in inhibiting developmental lymphangiogenesis.

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In patients with biliary atresia (BA), the extent of intrahepatic biliary fibrosis negatively correlates with successful surgical bypass of the congenital cholangiopathy as well as subsequent transplant‐free survival. We recently linked the expansion of a population of prominin‐1 (Prom1)‐expressing hepatic progenitor cells to biliary fibrogenesis. Herein, we hypothesized that Prom1‐expressing progenitor cells play a role in BA‐associated fibrosis. Rhesus rotavirus (RRV)‐mediated experimental BA was induced in newborn mice homozygous for the transgene Prom1cre‐ert2‐nlacz, which was knocked in to the Prom1 gene locus, thus creating functional Prom1 knockout (KO) mice, and their wildtype (WT) littermates. Clinical data and tissue samples from BA infants from the Childhood Liver Disease Research Consortium were analyzed. Extrahepatic biliary obliteration was present in both WT and KO mice; there was no difference in serum total bilirubin (TBili) levels. The intrahepatic periportal expansion of the PROM1pos cell population, typically observed in RRV‐induced BA, was absent in KO mice. RRV‐treated KO mice demonstrated significantly fewer cytokeratin‐19 (CK19)‐positive ductular reactions (P = 0.0004) and significantly less periportal collagen deposition (P = 0.0001) compared with WT. RRV‐treated KO mice expressed significantly less integrin‐β6, which encodes a key biliary‐specific subunit of a transforming growth factor (TGF) β activator (P = 0.0004). Infants with successful biliary drainage (Tbili ≤1.5 mg/dL within 3 months postoperatively), which is highly predictive of increased transplant‐free survival, expressed significantly less hepatic PROM1, CK19, and COLLAGEN‐1α compared with those with TBili >1.5 (P < 0.05). Conclusion: Prom1 plays an important role in biliary fibrogenesis, in part through integrin‐mediated TGF pathway activation.

Forty-five horses were infected peripherally or intrathecally with enzootic or epizootic strains of Venezuelan equine encephalomyelitis (VEE) virus. Low titers of virus appeared in cerebrospinal fluid (CSF) after peripheral inoculation of enzootic or epizootic VEE virus strains. Intrathecal infection with either epizootic or enzootic VEE virus produced higher titers of virus in CSF than did peripheral infection. In contrast to peripheral infections with enzootic strains, intrathecal infections with these strains caused death. The animals that died had widespread histopathologic changes and large amounts of virus in brain tissue. The attenuated VEE virus vaccine strain, TC-83, also multiplied in the brain of horses inoculated intrathecally but caused no clinical disease and little histopathologic damage.