Damage to the central nervous system (CNS) leads to increased production of TNF-α and TGF-β1, cytokines that have pro- or anti-inflammatory actions, respectively. To define whether astrocytes or microglia express these cytokines, prior studies have used mixed glial cultures (MGC) to represent astrocytes, thought these results are inevitably complicated by the presence of contaminating microglia within MGC. To clarify the cellular source of these cytokines, here we employed a recently described method of preparing microglia-free astrocyte cultures, in which neural stem cells (NSC) are differentiated into astrocytes. Using ELISA to quantify cytokine production in three types of glial culture: MGC, pure microglia or pure astrocytes, this showed that microglia but not astrocytes, produce TNF-α, and that this expression is increased by LPS, IFN-γ, and to a lesser extent by vitronectin, but decreased by TGF-β1. In contrast, TGF-β1 was produced by microglia and astrocytes, though at 10-fold higher levels by microglia. TGF-β1 expression in microglia was increased by vitronectin and to a lesser extent by TNF-α and LPS, but astrocyte TGF-β1 expression was not regulated by any factor tested. In summary, our data reveal that microglia, not astrocytes are the major source of TNF-α and TGF-β1 in postnatal glial cultures, and that microglial production of these antagonistic cytokines is tightly regulated by cytokines, LPS, and vitronectin.

Pericytes play critical roles in the development, maturation and remodeling of blood vessels, and in the central nervous system (CNS), evidence suggests that pericytes also regulate blood flow and form an integral part of the blood-brain barrier. The study of this important cell type has been hampered by the lack of any pericyte-specific marker and by the difficulty of culturing pericytes in adequate numbers to high purity. Here we present a novel yet simple approach to isolate and culture large numbers of pericytes from the mouse CNS that nevertheless leads to very pure pericyte cultures. In our method, vascular cells obtained from adult mice brains are cultured initially under conditions optimized for endothelial cells, but after two passages switched to a medium optimized for pericyte growth. After growing the cells for 1-2 additional passages we obtained a largely homogeneous population of cells that expressed the pericyte markers NG2, PDGF -receptor, and CD146, but were negative for markers of endothelial cells (CD31), microglia (Mac-1) and astrocytes (GFAP). Under these conditions, pericytes could be grown to high passage number, and were maintained highly pure and largely undifferentiated, as determined by antigen expression profile and low levels of -SMA expression, a marker of pericyte differentiation. Furthermore, switching the cells from pericyte medium into DMEM containing 10% FBS promoted -SMA expression, demonstrating that high passage pericytes could still differentiate. Thus, we provide an alternative approach to the culture of CNS pericytes that is easy to establish and provides large numbers of highly pure pericytes for extended periods of time. This system should provide others working in the pericyte field with a useful additional tool to study the behavior of this fascinating cell type.

Fibronectin is a critical regulator of vascular modelling, both in development and in the adult. In the hypoxic adult central nervous system (CNS), fibronectin is induced on angiogenic vessels, and endothelial cells show strong induction of the two fibronectin receptors α5β1 and αvβ3 integrins. In a previous study, we found that the αvβ3 integrin is dispensable for hypoxic-induced cerebral angiogenesis, but a role for the endothelial α5β1 integrin was suggested. To directly investigate the role of endothelial α5 integrin in cerebral angiogenesis, wild-type mice and mice lacking α5 integrin expression in endothelial cells (α5-EC-KO) were subject to hypoxia (8% O2) for 0, 2, 4, 7 or 14 days. Quantification of cerebral vessel density and endothelial-specific proteins claudin-5 and Glut-1 revealed that α5-EC-KO mice displayed an attenuated angiogenic response, which correlated with delayed endothelial proliferation. α5-EC-KO mice showed no defect in the ability to organize a cerebrovascular fibronectin matrix, and no compensatory increase in vascular αvβ3 integrin expression. Consistent with these findings, primary α5KO brain endothelial cells (BEC) in culture exhibited delayed growth and proliferation. Taken together, these studies demonstrate an important angiogenic role for the α5β1 integrin in promoting BEC proliferation in response to cerebral hypoxia.

Following focal cerebral ischemia, blood vessels in the ischemic border, or penumbra, launch an angiogenic response. In light of the critical role for fibronectin in angiogenesis, and the observation that fibronectin and its integrin receptors are strongly upregulated on angiogenic vessels in the hypoxic CNS, the aim of this study was to establish whether angiogenic vessels in the ischemic CNS also show this response. Focal cerebral ischemia was established in C57/Bl6 mice by middle cerebral artery occlusion (MCA:O), and brain tissue analyzed seven days following re-perfusion, a time at which angiogenesis is ongoing. Within the ischemic core, immunofluorescent (IF) studies demonstrated vascular expression of MECA-32, a marker of leaky cerebral vessels, and vascular breakdown, defined by loss of staining for the endothelial marker, CD31, and the vascular adhesion molecules, laminin, dystroglycan and α6 integrin. Within the ischemic penumbra, dual-IF with CD31 and Ki67 revealed the presence of proliferating endothelial cells, indicating ongoing angiogenesis. Significantly, vessels in the ischemic penumbra showed strong upregulation of fibronectin and the fibronectin receptors, α5β1 and αvβ3 integrins. Taken together with our recent finding that the α5β1 integrin plays an important role in promoting cerebral angiogenesis in response to hypoxia, these results suggest that stimulation of the fibronectin-α5β1 integrin signalling pathway may provide a novel approach to amplifying the intrinsic angiogenic response to cerebral ischemia.

Alterations in vascular structure and function are a central component of demyelinating disease. In addition to blood-brain barrier (BBB) breakdown, which occurs early in the course of disease, recent studies have described angiogenic remodeling, both in multiple sclerosis tissue and in the mouse demyelinating model, experimental autoimmune encephalomyelitis (EAE). As the precise timing of vascular remodeling in demyelinating disease has yet to be fully defined, the purpose of the current study was to define the time-course of these events in the MOG35-55 EAE model. Quantification of endothelial cell proliferation and vessel density revealed that a large part of angiogenic remodeling in cervical spinal cord white matter occurs during the pre-symptomatic phase of EAE. At the height of vascular remodeling, blood vessels in the cervical spinal cord showed strong transient upregulation of fibronectin and the α5β1 integrin. In vitro experiments revealed that α5 integrin inhibition reduced brain endothelial cell proliferation under inflammatory conditions. Interestingly, loss of vascular integrity was evident in all vessels during the first 4–7 days post-immunization, but after 14 days, was localized predominantly to venules. Taken together, our data demonstrate that extensive vascular remodeling occurs during the pre-symptomatic phase of EAE and point to a potential role for the fibronectin-α5β1 integrin interaction in promoting vascular remodeling during demyelinating disease.