Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt/β-catenin signaling

ABSTRACTCentral nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/β-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/β-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/β-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of Fgfbp1 results in transient hypervascularization but delays BBB maturation in specific CNS regions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/β-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties in vitro. Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/β-catenin activity.

A Novel Laminin-Binding Protein Mediates Microbial-Endothelial Cell Interactions and Facilitates Dissemination of Lyme Disease Pathogens

Borrelia burgdorferi conserved gene products BB0406 and BB0405, members of a common B. burgdorferi paralogous gene family, share 59% similarity. Although both gene products can function as potential porins, only BB0405 is essential for infection. Here we show that, despite sequence homology and coexpression from the same operon, both proteins differ in their membrane localization attributes, antibody accessibility, and immunogenicity in mice. BB0406 is required for spirochete survival in mammalian hosts, particularly for the disseminated infection in distant organs. We identified that BB0406 interacts with laminin, one of the major constituents of the vascular basement membrane, and facilitates spirochete transmigration across host endothelial cell barriers. A better understanding of how B. burgdorferi transmigrates through dermal and tissue vascular barriers and establishes disseminated infections will contribute to the development of novel therapeutics to combat early infection.

Role and Molecular Mechanisms of Pericytes in Regulation of Leukocyte Diapedesis in Inflamed Tissues

Leukocyte recruitment is a hallmark of the inflammatory response. Migrating leukocytes breach the endothelium along with the vascular basement membrane and associated pericytes. While much is known about leukocyte-endothelial cell interactions, the mechanisms and role of pericytes in extravasation are poorly understood and the classical paradigm of leukocyte recruitment in the microvasculature seldom adequately discusses the involvement of pericytes. Emerging evidence shows that pericytes are essential players in the regulation of leukocyte extravasation in addition to their functions in blood vessel formation and blood-brain barrier maintenance. Junctions between venular endothelial cells are closely aligned with extracellular matrix protein low expression regions (LERs) in the basement membrane, which in turn are aligned with gaps between pericytes. This forms preferential paths for leukocyte extravasation. Breaching of the layer formed by pericytes and the basement membrane entails remodelling of LERs, leukocyte-pericyte adhesion, crawling of leukocytes on pericyte processes, and enlargement of gaps between pericytes to form channels for migrating leukocytes. Furthermore, inflamed arteriolar and capillary pericytes induce chemotactic migration of leukocytes that exit postcapillary venules, and through direct pericyte-leukocyte contact, they induce efficient interstitial migration to enhance the immunosurveillance capacity of leukocytes. Given their role as regulators of leukocyte extravasation, proper pericyte function is imperative in inflammatory disease contexts such as diabetic retinopathy and sepsis. This review summarizes research on the molecular mechanisms by which pericytes mediate leukocyte diapedesis in inflamed tissues.

Tumour-Derived Laminin α5 (LAMA5) Promotes Colorectal Liver Metastasis Growth, Branching Angiogenesis and Notch Pathway Inhibition

Hepatic metastatic growth is dependent upon stromal factors including the matrisomal proteins that make up the extracellular matrix (ECM). Laminins are ECM glycoproteins with several functions relevant to tumour progression including angiogenesis. We investigated whether metastatic colon cancer cells produce the laminins required for vascular basement membrane assembly as a mechanism for the promotion of angiogenesis and liver metastasis growth. qPCR was performed using human-specific primers to laminin chains on RNA from orthotopic human colorectal liver metastases. Laminin α5 (LAMA5) expression was inhibited in colon cancer cells using shRNA. Notch pathway gene expression was determined in endothelia from hepatic metastases. Orthotopic hepatic metastases expressed human laminin chains α5, β1 and γ1 (laminin 511), all of which are required for vascular basement membrane assembly. The expression of Laminin 511 was associated with reduced survival in several independent colorectal cancer cohorts and angiogenesis signatures or vessel density significantly correlated with LAMA5 expression. Colorectal cancer cells in culture made little LAMA5, but its levels were increased by culture in a medium conditioned by tumour-derived CD11b+ myeloid cells through TNFα/NFκB pathway signalling. Down-regulation of LAMA5 in cancer cells impaired liver metastatic growth and resulted in reduced intra-tumoural vessel branching and increased the expression of Notch pathway genes in metastasis-derived endothelia. This data demonstrates a mechanism whereby tumour inflammation induces LAMA5 expression in colorectal cancer cells. LAMA5 is required for the successful growth of hepatic metastases where it promotes branching angiogenesis and modulates Notch signalling.

Angiocentric astrocytoma in a cat

Gliomas are common primary central nervous system neoplasms of dogs and cats, but atypical glioma subtypes are rare. Herein we report an angiocentric astrocytoma in a 15-y-old spayed female domestic shorthaired cat that was euthanized after therapy-resistant seizures. Gross anatomic changes consisted of swelling of the rostral leptomeninges over the olfactory bulbs and rostral telencephalon. Histologically, polygonal-to-elongate atypical neoplastic cells were arranged along perivascular spaces within these areas. Neoplastic cells were positive for glial fibrillary acidic protein, S100 protein, and vimentin. Ultrastructurally, round-to-elongate neoplastic cells emitting long processes with aggregates of intermediary filaments expanded and occupied the spaces between the vascular basement membrane and the glia limitans; nuclei had marginal and central heterochromatin. Tight junctions connected the plasma membrane of neighboring cells. The cell morphology, immunohistochemistry, and ultrastructural findings were consistent with an astrocytoma; the exclusive perivascular arrangement of neoplastic cells with no parenchymal mass warranted the diagnosis of angiocentric astrocytoma.

Systemic Vascular Basement Membrane Markers Linked to Synovial Vascular Remodeling Are Bio-Markers of Hemarthrosis in Patients with Hemophilia

Introduction: Acute painful joint episodes in hemophilia are not always associated with hemarthrosis. Point-of-Care Musculoskeletal Ultrasound with Power Doppler (POC MSKUS/PD) is a fast and sensitive imaging modality to determine the presence of hemarthrosis and direct management. Systemic collagen cleavage products reflecting extracellular matrix, bone and soft tissue turnover may serve as an alternative tool for the detection of hemarthrosis. Based on previous observations of pronounced vascular remodeling within the synovial tissue during hemarthrosis in patients and hemophilic mouse models, we hypothesized that cleavage products of collagen IV, found in the basement membrane of blood vessels may differentiate between bleeding and non-bleeding states. Methods: Joint bleeding was induced in FVIII-/- mice (+/- FVIII-treatment before and shortly after injury). Synovial vascular remodeling was assessed 14 days later by MSKUS/PD and histology including αSmooth Muscle Actin (αSMA)-staining. Murine plasma samples were analyzed for C4M2 and P4NP-7S, which are type IV collagen turnover products. To determine human relevance, 31 adult patients with hemophilia (PWH), ≥ age 21, were studied prospectively for 3 years with MSKUS/PD during pain-free intervals and painful events for bleed status, vascular flow and 11 plasma markers of collagen turnover(C2M, ARGS, C3M, Pro-C3, C5M, Pro C5, C4M2, P4NP-7S, Col18N, DCN- CS, ColNeo10, NIC). Results: Type IV collagen turn-over markers were significantly elevated in plasma of FVIII-/- deficient mice 14 days after joint bleeding when compared to baseline (C4M2: 2.575 ng/mL vs. 1.94 ng/mL, p=0.029 and P4NP-7S: 80.3 ng/mL vs. 67.81 ng/mL, p= 0.0152 respectively ). This increase coincided with a significant increase in vessel number (2.3 fold, p=0.0008), vascular flow by PD (2.3 fold, p= 0.014) and αSMA staining (5.1 fold, p= 0.0007). C4M2 and P4NP7S plasma levels correlated negatively with αSMA staining, suggesting a role for αSMA in vessel stabilization (C4M2 p=0.02, Pearson r= -0.5707; P4NP-7S p= 0.0130, Pearson r= -0.6238). FVIII-treatment reduced plasma C4M2 and P4NP-7S levels to baseline (C4M2 1.98 ng/mL vs. 1.94 ng/mL, P4NP-7S 59.98 ng/mL vs. 67.81 ng/mL), but had no effect on the other vascular parameters. In patients, results from 91 visits were compiled. Twenty five were due to acute painful episodes with 16 confirmed hemarthroses. Of the 11 biomarkers evaluated, only C4M2 and P4NP-7S were transiently but significantly elevated in plasma during joint bleeding compared to pain free intervals or to non-bleeding painful episodes. Plasma C4M2 levels during acute painful bleeding episodes when compared to non-bleeding states were 41.65 vs. 29.69 ng/mL (p=0.0003), and P4NP-7S levels were 418.9 vs 278.1 ng/mL (p=0.0007), respectively. Baseline levels of C4M2 and P4NP-7S in PWH were above the lab reported normal range, indicating ongoing high vascular basement membrane turnover. Additionally, PWH who developed joint bleeding episodes had a higher level of collagen IV biomarkers at baseline when compared to PWH who did not experience joint bleeding (C4M2 33.91 vs 29.07 ng/mL, P4NP7s 320.6 vs 292.8 ng/mL), although this was not statistically significant. Hemarthrosis was accompanied by pronounced abnormal synovial microvascular flow evidenced by PD, while plasma levels of both biomarkers correlated positively with joint PD signal (C4M2, p= 0.0400, Pearson r=0.4409; P4NP7S-p=0.0141, Pearson r=0.5154). Conclusions: Our findings suggest that systemic vascular collagen turn-over products may be novel biomarker candidates to identify patients at high risk for joint bleeding and to diagnose acute hemarthrosis. Hemarthrosis in hemophilia is associated with pronounced synovial vascular remodeling. Cleavage products of type IV collagen, but not of any other collagen, were systemically elevated in PWH and distinguished bleeding from non-bleeding painful episodes. Type IV collagen is found exclusively in basement membranes and in the case of joint bleeding, the presumed source of these turnover markers is newly formed synovial blood vessels. Therefore, our findings support the concept that vascular instability during neovascularization is involved in the dynamics of hemophilic joint bleeding. These findings broaden the scope of potential diagnostic tools substantially, opening new avenues for personalized treatment of PWH. Disclosures von Drygalski: Bioverativ/Sanofi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria; HemaBiologics: Consultancy; Genentech: Consultancy; Biomarin: Consultancy, Honoraria; Uniqure: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Hematherix: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.