Cerebral arterioles express the laminin subunits α4 and α5 in conjunction with α6ß4 integrin, but strongly downregulate laminin α4 during hypoxia-induced arteriogenic remodeling.

Previous studies have shown that expression of the endothelial laminin receptor α6ß4 integrin in the brain is uniquely restricted to arterioles. As exposure to chronic mild hypoxia (CMH, 8 % O2) stimulates robust angiogenic and arteriogenic remodeling responses in the brain, the goal of this study was to determine how CMH influences cerebrovascular expression of the ß4 integrin as well as its potential ligands, laminin 411 and 511, containing the α4 and α5 laminin subunits respectively, and then define how aging impacts this expression. We observed the following: (i) CMH launched a robust arteriogenic remodeling response both in the young (10 weeks) and aged (20 months) brain, correlating with an increased number of ß4 integrin+ vessels, (ii) while the laminin α4 subunit is expressed evenly across all cerebral blood vessels, laminin α5 was highly expressed preferentially on ß4 integrin+ arterioles, (iii) CMH-induced arteriolar remodeling was associated with strong downregulation of the laminin α4 subunit but no change in the laminin α5 subunit, (iv) in addition to its expression on arterioles, ß4 integrin was also expressed at lower levels on capillaries specifically in white matter (WM) tracts but not in the grey matter (GM), and (v), these observations were consistent in both the brain and spinal cord, and age had no obvious impact. Taken together, our findings suggest that laminin 511 may be a specific ligand for α6ß4 integrin and that dynamic switching of the laminin subunits α4 and α5 might play an instructive role in arteriogenic remodeling. Furthermore, ß4 integrin expression differentiates WM from GM capillaries, highlighting a novel and important difference.

Hypoxia-induced vascular remodeling responses in the brain are much more robust than other organs.

Exposure to chronic mild hypoxia (CMH; 8-10% O2) promotes a robust vascular remodeling response in the brain resulting in 50% increased vessel density over a period of two weeks. It is currently unknown whether blood vessels in other organs show similar responses. To address this question, mice were exposed to CMH for 4 days and various markers of vascular remodeling were examined in the brain along with heart, skeletal muscle, kidney, and liver. In contrast to brain, where CMH strongly promoted endothelial proliferation, none of the peripheral organs showed this response and in heart and liver, CMH notably reduced endothelial proliferation. While the MECA-32 endothelial activation marker was strongly induced by CMH in brain, in peripheral organs it was constitutively expressed either on a sub-population of vessels (heart and skeletal muscle) or on all vessels (kidney and liver), and notably, CMH did not affect expression. Endothelial expression of the tight junction proteins claudin-5 and ZO-1 were markedly increased on cerebral vessels, but in the peripheral organs examined, CMH either had no effect or reduced ZO-1 expression (liver). Finally, while CMH had no impact on the number of Mac-1 positive macrophages in the brain, heart, or skeletal muscle, this number was markedly decreased in the kidney but increased in the liver. Our findings show that the vascular remodeling responses to CMH are organ-specific, with the brain showing a strong angiogenic response and enhanced tight junction protein expression, but heart, skeletal muscle, kidney, and liver failing to show these responses.

Reducing herbicide input and optimizing spray method can minimize nontarget impacts on native grassland plant species.

Invasive plants threaten biodiversity worldwide and effective management must control the target invader while conserving biodiversity. Herbicide is often used to control invasive plants, but potential negative impacts on biodiversity have led to spot spraying being recommended over boom spraying to minimize the exposure of nontarget species to chemicals. We examined the influence of herbicide application methods on off-target plant communities in threatened temperate grasslands of southeastern Australia, where spraying with the broadleaf herbicide fluroxypr is commonly used to control St. John's wort, Hypericum perforatum L. It is well established that fluroxypr effectively controls H. perforatum but few studies have examined its impact on native forbs. A spray drift experiment using water-sensitive cards indicated that ground surface coverage was higher for spot spraying (91%-99%) than for boom spraying (5%-31%). We established a replicated, 3-year, before-after-control-impact experiment across 48 1-m2 quadrats to determine how three herbicide application methods (spot spray, fine boom and coarse boom) affected nontarget native forbs, the group most likely to be affected by broadleaf herbicides. This experiment was conducted in grasslands where H. perforatum was almost absent, so responses would reflect the direct impacts of the chemical, rather than structural changes resulting from removal of the target invader. Spot spraying decreased the probability of occurrence of native leguminous forbs, while increasing the occurrence of exotic leguminous forbs and the richness of all exotic species and exotic annual forbs. Spot spraying reduced the occurrence of the native Desmodium varians and the abundance of the native Chrysocephalum apiculatum. During this 3-year study, native species appeared to be impacted either directly by fluroxypr or indirectly by increased competition with exotic species. Where herbicide application is deemed crucial in these grasslands, we recommend boom spraying when H. perforatum density is moderate to high. Spot spraying should only be used when the density of H. perforatum is very low. Given the regional variation in H. perforatum density, the spatial scale of invasion, soil depth, and conservation values, we present a decision tree to assist managers in evaluating the costs and benefits of chemical control, indicating situations where alternative or modified methods could be used.

Chronic mild hypoxia accelerates recovery from preexisting EAE by enhancing vascular integrity and apoptosis of infiltrated monocytes.

While several studies have shown that hypoxic preconditioning suppresses development of the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), no one has yet examined the important clinically relevant question of whether mild hypoxia can impact the progression of preexisting disease. Using a relapsing-remitting model of EAE, here we demonstrate that when applied to preexisting disease, chronic mild hypoxia (CMH, 10% O2) markedly accelerates clinical recovery, leading to long-term stable reductions in clinical score. At the histological level, CMH led to significant reductions in vascular disruption, leukocyte accumulation, and demyelination. Spinal cord blood vessels of CMH-treated mice showed reduced expression of the endothelial activation molecule VCAM-1 but increased expression of the endothelial tight junction proteins ZO-1 and occludin, key mechanisms underlying vascular integrity. Interestingly, while equal numbers of inflammatory leukocytes were present in the spinal cord at peak disease (day 14 postimmunization; i.e., 3 d after CMH started), apoptotic removal of infiltrated leukocytes during the remission phase was markedly accelerated in CMH-treated mice, as determined by increased numbers of monocytes positive for TUNEL and cleaved caspase-3. The enhanced monocyte apoptosis in CMH-treated mice was paralleled by increased numbers of HIF-1α+ monocytes, suggesting that CMH enhances monocyte removal by amplifying the hypoxic stress manifest within monocytes in acute inflammatory lesions. These data demonstrate that mild hypoxia promotes recovery from preexisting inflammatory demyelinating disease and suggest that this protection is primarily the result of enhanced vascular integrity and accelerated apoptosis of infiltrated monocytes.

Spinal Cord Blood Vessels in Aged Mice Show Greater Levels of Hypoxia-Induced Vascular Disruption and Microglial Activation.

In response to chronic mild hypoxia (CMH, 8% O2), spinal cord blood vessels launch a robust angiogenic response that is associated with transient disruption of the blood-spinal cord barrier (BSCB) which, in turn, triggers a microglial vasculo-protective response. Because hypoxia occurs in many age-related conditions, the goal of this study was to define how aging influences these responses by comparing events in young (8-10 weeks) and aged (20 months) mice. This revealed that aged mice had much greater (3-4-fold) levels of hypoxic-induced BSCB disruption than young mice and that, while the early stage of the angiogenic response in aged mice was no different to young mice, the maturation of newly formed vessels was significantly delayed. Interestingly, microglia in the spinal cords of aged mice were much more activated than young mice, even under normoxic conditions, and this was further enhanced by CMH, though, surprisingly, this resulted in reduced microglial clustering around leaky blood vessels and diminished vasculo-protection. Vascular disruption was associated with loss of myelin in spinal cord white matter (WM) in both young and aged mice. Furthermore, it was notable that the spinal cord of aged mice contained a lower density of Olig2+ oligodendroglial cells even under normoxic conditions and that CMH significantly reduced the density of Olig2+ cells in spinal cord WM of the aged, but not the young, mice. These results demonstrate that spinal cord blood vessels of aged mice are much more vulnerable to the damaging effects of hypoxia than young mice, in part due to the reduced vasculo-protection conferred by chronically activated microglial cells. These observations may have implications for the pathogenesis and/or treatment of spinal cord diseases such as amyotrophic lateral sclerosis (ALS) and suggest that an improvement in microglial function could offer therapeutic potential for treating these age-related conditions.

The GFAP Monoclonal Antibody GA-5 Identifies Astrocyte Remodeling and Glio-Vascular Uncoupling During the Evolution of EAE.

To examine how astrocyte activation is regulated at different phases of relapsing-remitting EAE, we performed an immunofluorescent analysis of the spinal cord using the anti-glial fibrillary acidic protein (GFAP) monoclonal antibody GA-5. In keeping with previous studies, gray matter astrocytes showed strongly increased GFAP expression during the peak phase of disease (14 days post-immunization), which remained elevated during the remission phase (21-28 days post-immunization). In sharp contrast, during the peak phase of disease, the GA-5 signal in sub-meningeal white matter transiently disappeared in areas containing high levels of infiltrating leukocytes, but during the remission phase, the GFAP signal was fully restored. Parallel staining of the same sections with a polyclonal GFAP antibody confirmed elevated GFAP expression in the gray matter but no loss of signal in white matter. Interestingly, loss of GA-5 signal in sub-meningeal white matter was strongly associated with vascular disruption as defined by extravascular fibrinogen leak and by glio-vascular uncoupling, as defined by dissociation of AQP4-positive astrocyte endfeet and CD31-positive blood vessels. GA-5-negative areas were also associated with demyelination. These findings demonstrate a novel staining pattern of a GFAP antibody during EAE progression and suggest that the GFAP epitope recognized by the GA-5 monoclonal antibody transiently disappears as white matter astrocytes undergo remodeling during the peak phase of EAE. They also suggest that the GA-5 antibody provides a novel tool to identify astrocyte remodeling in other neurological conditions.

Hypoxia in multiple sclerosis; is it the chicken or the egg?

Over the past 50 years, intense research effort has taught us a great deal about multiple sclerosis. We know that it is the most common neurological disease affecting the young-middle aged, that it affects two to three times more females than males, and that it is characterized as an autoimmune disease, in which autoreactive T lymphocytes cross the blood-brain barrier, resulting in demyelinating lesions. But despite all the knowledge gained, a key question still remains; what is the initial event that triggers the inflammatory demyelinating process? While most research effort to date has focused on the immune system, more recently, another potential candidate has emerged: hypoxia. Specifically, a growing number of studies have described the presence of hypoxia (both 'virtual' and real) at an early stage of demyelinating lesions, and several groups, including our own, have begun to investigate how manipulation of inspired oxygen levels impacts disease progression. In this review we summarize the findings of these hypoxia studies, and in particular, address three main questions: (i) is the hypoxia found in demyelinating lesions 'virtual' or real; (ii) what causes this hypoxia; and (iii) how does manipulation of inspired oxygen impact disease progression?

A critical role for microglia in maintaining vascular integrity in the hypoxic spinal cord.

Hypoxic preconditioning reduces disease severity in a mouse model of multiple sclerosis (MS), in part by enhancing the barrier properties of spinal cord blood vessels. Because other studies have shown that similar levels of hypoxia transiently increase permeability of central nervous system (CNS) blood vessels, the goal of this study was to define the impact of chronic mild hypoxia (CMH, 8% O2) on the integrity of spinal cord blood vessels and the responses of neighboring glial cells. Using extravascular fibrinogen as a marker of vascular disruption, we found that CMH triggered transient vascular leak in spinal cord blood vessels, particularly in white matter, which was associated with clustering and activation of Mac-1-positive microglia around disrupted vessels. Microglial depletion with the colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX5622, while having no effect under normoxic conditions, profoundly increased vascular leak in both white and gray matter during CMH, and this was associated with disruption of astrocyte-vascular coupling and enhanced loss of tight junction proteins. Microglial repair of leaky blood vessels was blocked by a peptide that inhibits the interaction between fibrinogen and its Mac-1 integrin receptor. These findings highlight an important role for microglia in maintaining vascular integrity in the hypoxic spinal cord and suggest that a fibrinogen-Mac-1 interaction underpins this response. As relative hypoxia is experienced in many situations including high altitude, lung disease, obstructive sleep apnea, and age-related CNS ischemia/hypoxia, our findings have important implications regarding the critical role of microglia in maintaining vascular integrity in the CNS.

Chronic mild hypoxia promotes profound vascular remodeling in spinal cord blood vessels, preferentially in white matter, via an α5ß1 integrin-mediated mechanism.

Spinal cord injury (SCI) leads to rapid destruction of neuronal tissue, resulting in devastating motor and sensory deficits. This is exacerbated by damage to spinal cord blood vessels and loss of vascular integrity. Thus, approaches that protect existing blood vessels or stimulate the growth of new blood vessels might present a novel approach to minimize loss or promote regeneration of spinal cord tissue following SCI. In light of the remarkable power of chronic mild hypoxia (CMH) to stimulate vascular remodeling in the brain, the goal of this study was to examine how CMH (8% O2 for up to 7 days) affects blood vessel remodeling in the spinal cord. We found that CMH promoted the following: (1) endothelial proliferation and increased vascularity as a result of angiogenesis and arteriogenesis, (2) increased vascular expression of the angiogenic extracellular matrix protein fibronectin as well as concomitant increases in endothelial expression of the fibronectin receptor α5ß1 integrin, (3) strongly upregulated endothelial expression of the tight junction proteins claudin-5, ZO-1 and occludin and (4) astrocyte activation. Of note, the vascular remodeling changes induced by CMH were more extensive in white matter. Interestingly, hypoxic-induced vascular remodeling in spinal cord blood vessels was markedly attenuated in mice lacking endothelial α5 integrin expression (α5-EC-KO mice). Taken together, these studies demonstrate the considerable remodeling potential of spinal cord blood vessels and highlight an important angiogenic role for the α5ß1 integrin in promoting endothelial proliferation. They also imply that stimulation of the α5ß1 integrin or controlled use of mild hypoxia might provide new approaches for promoting angiogenesis and improving vascular integrity in spinal cord blood vessels.

Endothelial α6ß4 integrin protects during experimental autoimmune encephalomyelitis-induced neuroinflammation by maintaining vascular integrity and tight junction protein expression.

BACKGROUND: Extracellular matrix (ECM) proteins play critical functions regulating vascular formation and function. Laminin is a major component of the vascular basal lamina, and transgenic mice deficient in astrocyte or pericyte laminin show defective blood-brain barrier (BBB) integrity, indicating an important instructive role for laminin in cerebral blood vessels. As previous work shows that in the normal brain, vascular expression of the laminin receptor α6ß4 integrin is predominantly restricted to arterioles, but induced on all vessels during neuroinflammation, it is important to define the role of this integrin in the maintenance of BBB integrity. METHODS: α6ß4 integrin expression was analyzed using dual immunofluorescence (dual-IF) of brain sections taken from the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To investigate the role of endothelial α6ß4 integrin, transgenic mice lacking ß4 integrin in endothelial cells (ß4-EC-KO) and wild-type (WT) littermates were subject to EAE, and clinical score and various neuropathological parameters were examined by immunofluorescence. In addition, ß4 integrin null brain endothelial cells (BECs) were examined in culture for expression of tight junction proteins using immunocytochemistry and flow cytometry. RESULTS: Cerebrovascular expression of ß4 integrin was markedly upregulated during EAE progression, such that by the acute stage of EAE (day 21), the vast majority of blood vessels expressed ß4 integrin. In the EAE model, while the ß4-EC-KO mice showed the same time of disease onset as the WT littermates, they developed significantly worse clinical disease over time, resulting in increased clinical score at the peak of disease and maintained elevated thereafter. Consistent with this, the ß4-EC-KO mice showed enhanced levels of leukocyte infiltration and BBB breakdown and also displayed increased loss of the endothelial tight junction proteins claudin-5 and ZO-1. Under pro-inflammatory conditions, primary cultures of ß4KO BECs also showed increased loss of claudin-5 and ZO-1 expression. CONCLUSIONS: Taken together, our data suggest that α6ß4 integrin upregulation is an inducible protective mechanism that stabilizes the BBB during neuroinflammatory conditions.

Cerebral ischemia-induced angiogenesis is dependent on tumor necrosis factor receptor 1-mediated upregulation of α5ß1 and αVß3 integrins.

BACKGROUND: The pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), is expressed in ischemic tissue and is known to modulate angiogenesis; however, the role of the two distinct TNF-α receptors, TNFR1 and TNFR2, in mediating angiogenic signaling after cerebral ischemic stroke is relatively unknown. METHODS: C57BL6 mice were subject to 90 min of ischemia by temporary occlusion of the middle cerebral artery (MCAO) and given daily intra-cerebroventricular injections of antibodies against TNFR1, TNFR2 or control IgG (doses of 10, 50, and 100 ng/day) for 4 days following 90 min MCAO. Vascular remodeling and α5ß1 and αVß3 integrin expression were then examined in the brains of these mice after 4, 7, and 14 days post-ischemia. In parallel in vitro studies, flow cytometry was used to determine the influence of TNF-α on proliferation and integrin expression of human brain microvascular endothelial cells (HBMECs). RESULTS: The post-ischemic cerebral angiogenic response was inhibited by antibodies against TNFR1 but not TNFR2, and this correlated with reduced endothelial proliferation and decreased α5ß1 and αVß3 integrin expression after 4 and 7 days post-ischemia. Consistent with these findings, in vitro studies showed that TNF-α induced endothelial proliferation and upregulation of α5ß1 and αVß3 integrins was abrogated by anti-TNFR1 but not anti-TNFR2 antibodies in cultured HBMECs. In addition, blocking antibodies to α5ß1 and αVß3 integrins significantly inhibited TNF-α-induced HBMEC proliferation. CONCLUSIONS: Our results suggest that TNFR1-mediated signaling plays a critical role in triggering angiogenic integrins and subsequent angiogenic responses following cerebral ischemia. These novel findings could form a platform for future therapeutic strategies aimed at stimulating angiogenesis following cerebral ischemia.

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.

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.

ß1 integrins play a critical role maintaining vascular integrity in the hypoxic spinal cord, particularly in white matter.

Interactions between extracellular matrix (ECM) proteins and ß1 integrins play an essential role maintaining vascular integrity in the brain, particularly under vascular remodeling conditions. As blood vessels in the spinal cord are reported to have distinct properties from those in the brain, here we examined the impact of ß1 integrin inhibition on spinal cord vascular integrity, both under normoxic conditions, when blood vessels are stable, and during exposure to chronic mild hypoxia (CMH), when extensive vascular remodeling occurs. We found that a function-blocking ß1 integrin antibody triggered a small degree of vascular disruption in the spinal cord under normoxic conditions, but under hypoxic conditions, it greatly enhanced (20-fold) vascular disruption, preferentially in spinal cord white matter (WM). This resulted in elevated microglial activation as well as marked loss of myelin integrity and reduced density of oligodendroglial cells. To understand why vascular breakdown is localized to WM, we compared expression levels of major BBB components of WM and grey matter (GM) blood vessels, but this revealed no obvious differences. Interestingly however, hypoxyprobe staining demonstrated that the most severe levels of spinal cord hypoxia induced by CMH occurred in the WM. Analysis of brain tissue revealed a similar preferential vulnerability of WM tracts to show vascular disruption under these conditions. Taken together, these findings demonstrate an essential role for ß1 integrins in maintaining vascular integrity in the spinal cord, and unexpectedly, reveal a novel and fundamental difference between WM and GM blood vessels in their dependence on ß1 integrin function during hypoxic exposure. Our data support the concept that the preferential WM vulnerability described may be less a result of intrinsic differences in vascular barrier properties between WM and GM, and more a consequence of differences in vascular density and architecture.

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.

Periodontal health of palatally displaced canines treated with open or closed surgical technique: a multicenter, randomized controlled trial.

INTRODUCTION: The aim of this study was to investigate differences in the periodontal outcomes of palatally displaced canines (PDC) exposed with either an open or a closed surgical technique. METHODS: A multicenter, randomized controlled trial was undertaken in 3 hospitals in the United Kingdom, involving 2 parallel groups. Patients with unilateral PDC were randomly allocated to receive either an open or a closed surgical exposure. Periodontal health was assessed 3 months after removal of fixed appliances. Parameters measured included clinical attachment levels, recession, alveolar bone levels, and clinical crown height. RESULTS: Data from 62 participants (closed, 29; open, 33) were analyzed. There was no difference between PDC exposed with an open vs a closed surgical technique (mean difference, 0.1 mm; 95% confidence interval [CI], -0.2-0.5). There was, however, a statistical difference in mean attachment loss between the operated and unoperated (contralateral) canines (mean difference, 0.5 mm; 96% CI, 0.4-0.7; P <0.001). Twenty of the 62 subjects had some recession on the palatal aspect of the operated canine, whereas only 4 subjects had some visible root surface on the palatal aspect on the unoperated side (P = 0.001). CONCLUSIONS: There is a periodontal impact when a unilateral PDC is exposed and aligned. This impact is small and unlikely to have clinical relevance in the short term; however, the long-term significance is unknown. When the open and closed techniques were compared, no difference in periodontal health was found.

The importance of laminin at the blood-brain barrier.

The blood-brain barrier is a unique property of central nervous system blood vessels that protects sensitive central nervous system cells from potentially harmful blood components. The mechanistic basis of this barrier is found at multiple levels, including the adherens and tight junction proteins that tightly bind adjacent endothelial cells and the influence of neighboring pericytes, microglia, and astrocyte endfeet. In addition, extracellular matrix components of the vascular basement membrane play a critical role in establishing and maintaining blood-brain barrier integrity, not only by providing an adhesive substrate for blood-brain barrier cells to adhere to, but also by providing guidance cues that strongly influence vascular cell behavior. The extracellular matrix protein laminin is one of the most abundant components of the basement membrane, and several lines of evidence suggest that it plays a key role in directing blood-brain barrier behavior. In this review, we describe the basic structure of laminin and its receptors, the expression patterns of these molecules in central nervous system blood vessels and how they are altered in disease states, and most importantly, how genetic deletion of different laminin isoforms or their receptors reveals the contribution of these molecules to blood-brain barrier function and integrity. Finally, we discuss some of the important unanswered questions in the field and provide a "to-do" list of some of the critical outstanding experiments.

ß1 integrin is essential for blood-brain barrier integrity under stable and vascular remodelling conditions; effects differ with age.

BACKGROUND: Maintaining a tight blood-brain barrier (BBB) is an important prerequisite for the preservation of neurological health, though current evidence suggests it declines with age. While extracellular matrix-integrin interactions play critical roles in regulating the balance between vascular stability and remodeling, it remains to be established whether manipulation of integrin function weakens or strengthens vascular integrity. Indeed, recent reports have generated conflicting outcomes in this regard. METHODS: Here, in young (8-10 weeks) and aged (20 months) mice, we examined the impact of intraperitoneal injection of a function-blocking ß1 integrin antibody, both under normoxic conditions, when the BBB is stable, and during chronic mild hypoxic (CMH; 8% O2) conditions, when a vigorous vascular remodeling response is ongoing. Brain tissue was examined by immunofluorescence (IF) for markers of vascular remodeling and BBB disruption, and microglial activation and proliferation. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post-hoc test. RESULTS: In both young and aged mice, ß1 integrin block greatly amplified hypoxia-induced vascular disruption, though it was much less under normoxic conditions. Interestingly, under both normoxic and hypoxic conditions, ß1 integrin antibody-induced BBB disruption was greater in young mice. Enhanced BBB breakdown was associated with increased levels of the leaky BBB marker MECA-32 and with greater loss of endothelial tight junction proteins and the adherens protein VE-cadherin. Surprisingly, ß1 integrin blockade did not reduce hypoxia-induced endothelial proliferation, nor did it prevent the hypoxia-associated increase in vascularity. Commensurate with the increased vascular disruption, ß1 integrin blockade enhanced microglial activation both in young and aged brain, though the impact was much greater in young brain. In vitro studies revealed that ß1 integrin blockade also reduced the integrity of a brain endothelial monolayer and triggered disruptions in tight junction proteins. CONCLUSIONS: These data demonstrate that ß1 integrin plays an essential role in maintaining BBB integrity, both under stable normoxic conditions and during hypoxia-induced vascular remodeling. As ß1 integrin blockade had a greater disruptive effect in young brain, effectively shifting the BBB phenotype of young brain towards that of the aged, we speculate that enhancing ß1 integrin function at the aged BBB may hold therapeutic potential by reverting the deteriorating BBB phenotype back towards that of the young.

Astrocytic tissue inhibitor of metalloproteinase-1 (TIMP-1) promotes oligodendrocyte differentiation and enhances CNS myelination.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an extracellular protein and endogenous regulator of matrix metalloproteinases (MMPs) secreted by astrocytes in response to CNS myelin injury. We have previously reported that adult TIMP-1 knock-out (KO) mice exhibit poor myelin repair following demyelinating injury. This observation led us to hypothesize a role for TIMP-1 in oligodendrogenesis and CNS myelination. Herein, we demonstrate that compact myelin formation is significantly delayed in TIMP-1 KO mice, a situation that coincided with dramatically reduced numbers of white matter astrocytes in the developing CNS. Analysis of differentiation in CNS progenitor cells (neurosphere) cultures from TIMP-1 KO mice revealed a specific deficit of NG2(+) oligodendrocyte progenitor cells. Application of recombinant murine TIMP-1 (rmTIMP-1) to TIMP-1 KO neurosphere cultures evoked a dose-dependent increase in NG2(+) cell numbers, while treatment with GM6001, a potent broad-spectrum MMP inhibitor did not. Similarly, administration of rmTIMP-1 to A2B5(+) immunopanned oligodendrocyte progenitors significantly increased the number of differentiated O1(+) oligodendrocytes, while antisera to TIMP-1 reduced oligodendrocyte numbers. We also determined that A2B5(+) oligodendrocyte progenitors grown in conditioned media derived from TIMP-1 KO primary glial cultures resulted in reduced differentiation of mature O1(+) oligodendrocytes. Finally, we report that addition of rmTIMP-1 to primary glial cultures resulted in a dose-dependent proliferative response of astrocytes. Together, these findings describe a previously uncharacterized role for TIMP-1 in the regulation of oligodendrocytes and astrocytes during development and provide a novel function for TIMP-1 on myelination in the developing CNS.

Coxsackievirus preferentially replicates and induces cytopathic effects in undifferentiated neural progenitor cells.

Enteroviruses, including coxsackieviruses, exhibit significant tropism for the central nervous system, and these viruses are commonly associated with viral meningitis and encephalitis. Previously, we described the ability of coxsackievirus B3 (CVB3) to infect proliferating neuronal progenitor cells located in the neonatal subventricular zone and persist in the adult murine central nervous system (CNS). Here, we demonstrate that cultured murine neurospheres, which comprise neural stem cells and their progeny at different stages of development, were highly susceptible to CVB3 infection. Neurospheres, or neural progenitor and stem cells (NPSCs), isolated from neonatal C57BL/6 mice, supported high levels of infectious virus production and high viral protein expression levels following infection with a recombinant CVB3 expressing enhanced green fluorescent protein (eGFP) protein. Similarly, NPSCs isolated from neonatal actin-promoter-GFP transgenic mice (actin-GFP NPSCs) were highly susceptible to infection with a recombinant CVB3 expressing DsRed (Discosoma sp. red fluorescent protein). Both nestin-positive and NG2(+) progenitor cells within neurospheres were shown to preferentially express high levels of viral protein as soon as 24 h postinfection (p.i.). By day 3 p.i., viral protein expression and viral titers increased dramatically in NPSCs with resultant cytopathic effects (CPE) and eventual cell death. In contrast, reduced viral replication, lower levels of CPE, and diminished viral protein expression levels were observed in NPSCs differentiated for 5 or 16 days in the presence of fetal bovine serum (FBS). Despite the presence of CPE and high levels of cell death following early CVB3 infection, surviving neurospheres were readily observed and continued to express detectable levels of viral protein as long as 37 days after initial infection. Also, CVB3 infection of actin-GFP NPSCs increased the percentage of cells expressing neuronal class III ß-tubulin following their differentiation in the presence of FBS. These results suggest that neural stem cells may be preferentially targeted by CVB3 and that neurogenic regions of the CNS may support persistent viral replication in the surviving host. In addition, normal progenitor cell differentiation may be altered in the host following infection.