Physiological cerebrovascular remodeling in response to chronic mild hypoxia: A role for activated protein C.

Activated protein C (APC) is a serine protease that promotes favorable changes in vascular barrier integrity and post-ischemic angiogenic remodeling in animal models of ischemic stroke, and its efficacy is currently being investigated in clinical ischemic stroke trials. Interestingly, application of sub-clinical chronic mild hypoxia (CMH) (8% O2) also promotes angiogenic remodeling and increased tight junction protein expression, suggestive of enhanced blood-brain barrier (BBB) integrity, though the role of APC in mediating the influence of CMH has not been investigated. To examine this potential link, we studied CMH-induced cerebrovascular remodeling after treating mice with two different reagents: (i) a function-blocking antibody that neutralizes APC activity, and (ii) exogenous recombinant murine APC. While CMH promoted endothelial proliferation, increased vascular density, and upregulated the angiogenic endothelial integrins α5ß1 and αvß3, these events were almost completely abolished by functional blockade of APC. Consistent with these findings, addition of exogenous recombinant APC enhanced CMH-induced endothelial proliferation, expansion of total vascular area and further enhanced the CMH-induced right-shift in vessel size distribution. Taken together, our findings support a key role for APC in mediating physiological remodeling of cerebral blood vessels in response to CMH.

ß4 integrin is not essential for localization of hemidesmosome proteins plectin and CD151 in cerebral vessels.

OBJECTIVE: In the central nervous system (CNS), ß4 integrin is predominantly expressed by endothelial cells lining arterioles. As ß4 integrin plays an essential role in epithelial tissues, organizing structural proteins into specialized adhesive structures called hemidesmosomes (HD), the aim of this study was to determine whether it plays a similar role in CNS endothelium. METHODS: Dual-immunofluorescence was used to examine the relationship between ß4 integrin expression and co-expression of the HD proteins plectin and CD151 in frozen sections of mouse brain, both under normoxic (control) conditions and following chronic mild hypoxia. The requirement of ß4 integrin for the localization of HD proteins was examined in transgenic mice lacking ß4 integrin expression specifically in endothelial cells (ß4-EC-KO mice). RESULTS: Immunofluorescence revealed that in the normal adult CNS, plectin and CD151 strongly co-localized with ß4 integrin in arterioles. However, in the chronic mild hypoxia model, in which extensive cerebrovascular remodeling is observed, plectin and CD151 were strongly upregulated on all cerebral vessels, but surprisingly, in capillaries, this occurred in a ß4 integrin-independent manner. Unexpectedly, absence of endothelial ß4 integrin (in ß4-EC-KO mice) had no impact on the expression level or distribution pattern of plectin and CD151 within stable or remodeling cerebral vessels. CONCLUSIONS: These results demonstrate that the HD proteins plectin and CD151 are closely associated with ß4 integrin on arterioles in normal brain, and are strongly upregulated on remodeling blood vessels. However, unlike its described role in the epidermis, ß4 integrin is not essential for localization or regulation of expression of plectin and CD151 in cerebral vessels.

Matrix metalloproteinase-9 mediates post-hypoxic vascular pruning of cerebral blood vessels by degrading laminin and claudin-5.

Vascular remodeling involves a highly coordinated break-down and build-up of the vascular basal lamina and inter-endothelial tight junction proteins. In light of the important role of matrix metalloproteinases (MMPs) in tissue remodeling, the goal of this study was to examine the role of MMP-9 in remodeling of cerebral blood vessels, both in hypoxia-induced angiogenesis and in the vascular pruning that accompanies the switch from hypoxia back to normoxia. In a chronic mild hypoxia model of cerebrovascular remodeling, gel zymography revealed that MMP-9 levels were increased, both during hypoxic-induced angiogenesis and in the post-hypoxic pruning response. Interestingly, compared to wild-type mice, MMP-9 KO mice showed no alteration in hypoxic-induced angiogenesis, but did show marked delay in post-hypoxic vascular pruning. In wild-type mice, vascular pruning was associated with fragmentation of vascular laminin and the tight junction protein claudin-5, while this process was markedly attenuated in MMP-9 KO mice. In vitro experiments showed that hypoxia stimulated MMP-9 expression in brain endothelial cells but not pericytes. These results show that while MMP-9 is not essential for hypoxic-induced cerebral angiogenesis, it plays an important role in post-hypoxic vascular pruning by degrading laminin and claudin-5.

Analysis of TNF-alpha-mediated cerebral pericyte remodeling.

As well as being a central regulator of inflammatory and immune-mediated events, TNF-α also influences vascular remodeling, resulting in alterations in the structure and function of blood vessels. In addition to endothelial cells, pericytes are another type of vascular cell that significantly contribute to the development, maturation, stabilization, and remodeling of blood vessels. To investigate the regulatory influence of different factors on pericyte behavior, we recently described a novel yet simple approach of isolating and culturing highly pure, high density cultures of mouse brain pericytes. In this chapter, we briefly describe this culture system, as well as methods for examining different aspects of pericyte behavior, including cell adhesion, cell migration, and cell proliferation. These assays can be used to examine the influence of TNF-α or any other factor on pericyte behavior.

Examining vascular remodeling in the hypoxic central nervous system.

The goal of this chapter is to highlight techniques used to determine the role of molecular mechanisms involved in remodeling of cerebral blood vessels. Enhanced vascularization in the central nervous system (CNS) is seen in many diseases including stroke, cancer, and multiple sclerosis (MS). However, despite the prevalence of this phenomenon in these different pathological conditions, the exact nature of how it occurs still remains unclear. To better understand the process of cerebrovascular remodeling, we use the chronic hypoxia model, in which a vigorous and robust angiogenic remodeling response takes place. In this model, mice are placed in a hypoxic chamber (8 % O2 for up to 14 days), which results in strong vascular remodeling and increased vessel density within the CNS. Using an immunofluorescent (IF)-based approach, different aspects of this vascular remodeling response can be examined. By employing this method, we have shown that chronic mild hypoxia triggers both angiogenic (capillary sprouting) and arteriogenic (widening of arterial vessels) responses. Furthermore, we have used this system to define both the expression pattern and potential role of candidate adhesion molecules in this vascular remodeling process. Thus, the techniques described in this chapter can be used to define the importance of different molecular mechanisms in vascular remodeling in the CNS.

Isolation and culture of primary mouse brain endothelial cells.

Blood vessels in the central nervous system (CNS) are unique in forming the blood-brain barrier (BBB), which confers high electrical resistance and low permeability properties, thus protecting neural cells from potentially harmful blood components. Endothelial cells, which form the inner cellular lining of all blood vessels, play a critical role in this process by forming tight adhesive interactions between each other. To study the properties of primary brain endothelial cells (BECs), a number of different methods have been described. In this chapter, we present a relatively simple method that produces high numbers of primary mouse BECs that are highly pure (greater than 99 % CD31-positive). In addition, we also describe an immunocytochemical approach to demonstrate the endothelial purity of these cultures.

Isolation and culture of primary pericytes from mouse brain.

Pericytes are perivascular cells that play an important role in the development, maturation, and remodeling of blood vessels. However, studies of this important cell type on vascular remodeling have been hindered due to the difficulty of culturing pericytes in adequate numbers to high purity. In this chapter, we present a novel yet simple method to isolate and culture large numbers of pure pericytes from the mouse central nervous system (CNS). In our approach, vascular cells obtained from adult mice brains are cultured initially under conditions optimized for endothelial cells. Following two passages, the medium is switched over to optimize pericyte growth. After growing the cells for 2-3 additional passages, this approach produces a largely homogeneous population of cells that express the pericyte markers NG2, PDGFß receptor, and CD146 but are negative for markers of endothelial cells (CD31), astrocytes (GFAP), and microglia (Mac-1), demonstrating a highly pure pericyte culture. Thus, our technique provides an effective method to culture CNS pericytes that is easy to establish and provides large numbers of highly pure pericytes for extended periods of time. This system provides a useful tool for those wishing to study pericyte behavior.

Extensive vascular remodeling in the spinal cord of pre-symptomatic experimental autoimmune encephalomyelitis mice; increased vessel expression of fibronectin and the α5ß1 integrin.

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 α5ß1 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 4-7days post-immunization, but after 14days, 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-α5ß1 integrin interaction in promoting vascular remodeling during demyelinating disease.

Microglia are the major source of TNF-α and TGF-ß1 in postnatal glial cultures; regulation by cytokines, lipopolysaccharide, and vitronectin.

Damage to the central nervous system (CNS) leads to increased production of TNF-α and TGF-ß1 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-ß1. In contrast, TGF-ß1 was produced by microglia and astrocytes, though at 10-fold higher levels by microglia. TGF-ß1 expression in microglia was increased by vitronectin and to a lesser extent by TNF-α and LPS, but astrocyte TGF-ß1 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-ß1 in postnatal glial cultures, and that microglial production of these antagonistic cytokines is tightly regulated by cytokines, LPS, and vitronectin.

TNF-α promotes cerebral pericyte remodeling in vitro, via a switch from α1 to α2 integrins.

BACKGROUND: There is increasing evidence to suggest that pericytes play a crucial role in regulating the remodeling state of blood vessels. As cerebral pericytes are embedded within the extracellular matrix (ECM) of the vascular basal lamina, it is important to understand how individual ECM components influence pericyte remodeling behavior, and how cytokines regulate these events. METHODS: The influence of different vascular ECM substrates on cerebral pericyte behavior was examined in assays of cell adhesion, migration, and proliferation. Pericyte expression of integrin receptors was examined by flow cytometry. The influence of cytokines on pericyte functions and integrin expression was also examined, and the role of specific integrins in mediating these effects was defined by function-blocking antibodies. Expression of pericyte integrins within remodeling cerebral blood vessels was analyzed using dual immunofluorescence (IF) of brain sections derived from the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). RESULTS: Fibronectin and collagen I promoted pericyte proliferation and migration, but heparan sulfate proteoglycan (HSPG) had an inhibitory influence on pericyte behavior. Flow cytometry showed that cerebral pericytes express high levels of α5 integrin, and lower levels of α1, α2, and α6 integrins. The pro-inflammatory cytokine tumor necrosis factor (TNF)-α strongly promoted pericyte proliferation and migration, and concomitantly induced a switch in pericyte integrins, from α1 to α2 integrin, the opposite to the switch seen when pericytes differentiated. Inhibition studies showed that α2 integrin mediates pericyte adhesion to collagens, and significantly, function blockade of α2 integrin abrogated the pro-modeling influence of TNF-α. Dual-IF on brain tissue with the pericyte marker NG2 showed that while α1 integrin was expressed by pericytes in both stable and remodeling vessels, pericyte expression of α2 integrin was strongly induced in remodeling vessels in EAE brain. CONCLUSIONS: Our results suggest a model in which ECM constituents exert an important influence on pericyte remodeling status. In this model, HSPG restricts pericyte remodeling in stable vessels, but during inflammation, TNF-α triggers a switch in pericyte integrins from α1 to α2, thereby stimulating pericyte proliferation and migration on collagen. These results thus define a fundamental molecular mechanism in which TNF-α stimulates pericyte remodeling in an α2 integrin-dependent manner.

Endothelial ß4 integrin is predominantly expressed in arterioles, where it promotes vascular remodeling in the hypoxic brain.

OBJECTIVE: Laminin is a major component of the vascular basal lamina, implying that laminin receptors, such as α6ß1 and α6ß4 integrins, may regulate vascular remodeling and homeostasis. Previous studies in the central nervous system have shown that ß4 integrin is expressed by only a fraction of cerebral vessels, but defining the vessel type and cellular source of ß4 integrin has proved controversial. The goal of this study was to define the class of vessel and cell type expressing ß4 integrin in cerebral vessels and to examine its potential role in vascular remodeling. APPROACH AND RESULTS: Dual-immunofluorescence showed that ß4 integrin is expressed predominantly in arterioles, both in the central nervous system and in peripheral organs. Cell-specific knockouts of ß4 integrin revealed that ß4 integrin expression in cerebral vessels is derived from endothelial cells, not astrocytes or smooth muscle cells. Lack of endothelial ß4 integrin had no effect on vascular development, integrity, or endothelial proliferation, but in the hypoxic central nervous system, its absence led to defective arteriolar remodeling and associated transforming growth factor-ß signaling. CONCLUSIONS: These results define high levels of ß4 integrin in arteriolar endothelial cells and demonstrate a novel link among ß4 integrin, transforming growth factor-ß signaling, and arteriolar remodeling in cerebral vessels.

Chronic cerebral hypoxia promotes arteriogenic remodeling events that can be identified by reduced endoglin (CD105) expression and a switch in ß1 integrins.

Chronic cerebral hypoxia leads to a strong vascular remodeling response, though little is known about which part of the vascular tree is modified, or whether this response includes formation of new arterial vessels. In this study, we examined this process in detail, analyzing how hypoxia (8% O(2) for 14 days) alters the size distribution of vessels, number of arteries/arterioles, and expression pattern of endoglin (CD105), a marker of angiogenic endothelial cells in tumors. We found that cerebral hypoxia promoted the biggest increase in the number of medium to large size vessels, and this correlated with increased numbers of alpha smooth muscle actin (α-SMA)-positive arterial vessels. Surprisingly, hypoxia induced a marked reduction in CD105 expression on brain endothelial cells (BECs) within remodeling arterial vessels, and these BECs also displayed an angiogenic switch in ß1 integrins (from α6 to α5), previously described for developmental angiogenesis. In vitro, transforming growth factor (TGF)-ß1 also promoted this switch of BEC ß1 integrins. Together, these results show that cerebral hypoxia promotes arteriogenesis, and identify reduced CD105 expression as a novel marker of arteriogenesis. Furthermore, our data suggest a mechanistic model whereby BECs in remodeling arterial vessels downregulate CD105 expression, which alters TGF-ß1 signaling, to promote a switch in ß1 integrins and arteriogenic remodeling.

A novel and simple method for culturing pericytes from mouse brain.

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, PDGFß-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 α-SMA expression, a marker of pericyte differentiation. Furthermore, switching the cells from pericyte medium into DMEM containing 10% FBS promoted α-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.

Upregulation of fibronectin and the α5ß1 and αvß3 integrins on blood vessels within the cerebral ischemic penumbra.

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 7 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, α5ß1 and αvß3 integrins. Taken together with our recent finding that the α5ß1 integrin plays an important role in promoting cerebral angiogenesis in response to hypoxia, these results suggest that stimulation of the fibronectin-α5ß1 integrin signaling pathway may provide a novel approach to amplifying the intrinsic angiogenic response to cerebral ischemia.

Microglia use multiple mechanisms to mediate interactions with vitronectin; non-essential roles for the highly-expressed αvß3 and αvß5 integrins.

BACKGROUND: As the primary resident immune cells, microglia play a central role in regulating inflammatory processes in the CNS. The extracellular matrix (ECM) protein vitronectin promotes microglial activation, switching microglia into an activated phenotype. We have shown previously that microglia express two vitronectin receptors, αvß3 and αvß5 integrins. As these integrins have well-defined roles in activation and phagocytic processes in other cell types, the purpose of the current study was to investigate the contribution of these two integrins in microglial activation. METHODS: Microglial cells were prepared from wild-type, ß3 integrin knockout (KO), ß5 integrin KO or ß3/ß5 integrin DKO mice, and their interactions and activation responses to vitronectin examined in a battery of assays, including adhesion, expression of activation markers, MMP-9 expression, and phagocytosis. Expression of other αv integrins was examined by flow cytometry and immunoprecipitation. RESULTS: Surprisingly, when cultured on vitronectin, microglia from the different knockout strains showed no obvious defects in adhesion, activation marker expression, MMP-9 induction, or phagocytosis of vitronectin-coated beads. To investigate the reason for this lack of effect, we examined the expression of other αv integrins. Flow cytometry showed that ß3/ß5 integrin DKO microglia expressed residual αv integrin at the cell surface, and immunoprecipitation confirmed this finding by revealing the presence of low levels of the αvß1 and αvß8 integrins. ß1 integrin blockade had no impact on adhesion of ß3/ß5 integrin DKO microglia to vitronectin, suggesting that in addition to αvß1, αvß3, and αvß5, αvß8 also serves as a functional vitronectin receptor on microglia. CONCLUSIONS: Taken together, this demonstrates that the αvß3 and αvß5 integrins are not essential for mediating microglial activation responses to vitronectin, but that microglia use multiple redundant receptors to mediate interactions with this ECM protein.

A dual role for microglia in promoting tissue inhibitor of metalloproteinase (TIMP) expression in glial cells in response to neuroinflammatory stimuli.

BACKGROUND: By neutralizing the effect of the matrix metalloproteinases (MMPs), the tissue inhibitors of matrix metalloproteinases (TIMPs) play a critical role in maintaining tissue proteolysis in balance. As the major reactive glial cell types in the central nervous system (CNS), microglia and astrocytes play fundamental roles in mediating tissue breakdown and repair. As such, it is important to define the TIMP expression profile in these cells, as well as the mechanisms of regulation by neuroinflammatory stimuli. METHODS: Primary mixed glial cultures (MGC), pure microglia, and pure astrocytes were used in this study. To study astrocytes, we employed a recently described pure astrocyte culture system, which has the major advantage of totally lacking microglia. The three different types of culture were treated with lipopolysaccharide (LPS) or individual cytokines, and cell culture supernatants assayed for TIMP-1 or TIMP-2 protein expression by western blot. RESULTS: LPS induced TIMP-1 expression in MGC, but not in pure astrocyte or microglial cultures. When pure astrocytes were treated with the cytokines IL-1ß, IFN-γ, TNF or TGF-ß1, only IL-1ß induced TIMP-1 expression. Significantly, astrocyte TIMP-1 expression was restored in LPS-treated astrocyte cultures after the addition of microglia, or conditioned medium taken from LPS-activated microglia (MG-CM). Furthermore, this effect was lost after depletion of IL-1ß from MG-CM. By contrast, TIMP-2 was constitutively expressed by astrocytes, whereas microglia expressed TIMP-2 only after exposure to serum. CONCLUSIONS: Taken together, these results demonstrate an important concept in glial interactions, by showing that microglia play a central role in regulating glial cell expression of TIMPs, and identify microglial IL-1ß as playing a key role in mediating microglial-astrocyte communication.