The Effect of Reduced Fibrinogen on Cerebrovascular Permeability during Traumatic Brain Injury in Fibrinogen Gene Heterozygous Knockout Mice.

Vascular contribution to cognitive impairment and dementia (VCID) is a term referring to all types of cerebrovascular and cardiovascular disease-related cognitive decline, spanning many neuroinflammatory diseases including traumatic brain injury (TBI). This becomes particularly important during mild-to-moderate TBI (m-mTBI), which is characterized by short-term memory (STM) decline. Enhanced cerebrovascular permeability for proteins is typically observed during m-mTBI. We have previously shown that an increase in the blood content of fibrinogen (Fg) during m-mTBI results in enhanced cerebrovascular permeability. Primarily extravasated via a transcellular pathway, Fg can deposit into the parenchyma and exacerbate inflammatory reactions that can lead to neurodegeneration, resulting in cognitive impairment. In the current study, we investigated the effect of a chronic reduction in Fg concentration in blood on cerebrovascular permeability and the interactions of extravasated Fg with astrocytes and neurons. Cortical contusion injury (CCI) was used to generate m-mTBI in transgenic mice with a deleted Fg γ chain (Fg γ+/-), resulting in a low blood content of Fg, and in control C57BL/6J wild-type (WT) mice. Cerebrovascular permeability was tested in vivo. Interactions of Fg with astrocytes and neurons and the expression of neuronal nuclear factor-кB (NF-кB) were assessed via immunohistochemistry. The results showed that 14 days after CCI, there was less cerebrovascular permeability, lower extravascular deposition of Fg, less activation of astrocytes, less colocalization of Fg with neurons, and lower expression of neuronal pro-inflammatory NF-кB in Fg γ+/- mice compared to that found in WT mice. Combined, our data provide strong evidence that increased Fg extravasation, and its resultant extravascular deposition, triggers astrocyte activation and leads to potential interactions of Fg with neurons, resulting in the overexpression of neuronal NF-кB. These effects suggest that reduced blood levels of Fg can be beneficial in mitigating the STM reduction seen in m-mTBI.

Vascular Effects on Cerebrovascular Permeability and Neurodegeneration.

Neurons and glial cells in the brain are protected by the blood brain barrier (BBB). The local regulation of blood flow is determined by neurons and signal conducting cells called astrocytes. Although alterations in neurons and glial cells affect the function of neurons, the majority of effects are coming from other cells and organs of the body. Although it seems obvious that effects beginning in brain vasculature would play an important role in the development of various neuroinflammatory and neurodegenerative pathologies, significant interest has only been directed to the possible mechanisms involved in the development of vascular cognitive impairment and dementia (VCID) for the last decade. Presently, the National Institute of Neurological Disorders and Stroke applies considerable attention toward research related to VCID and vascular impairments during Alzheimer's disease. Thus, any changes in cerebral vessels, such as in blood flow, thrombogenesis, permeability, or others, which affect the proper vasculo-neuronal connection and interaction and result in neuronal degeneration that leads to memory decline should be considered as a subject of investigation under the VCID category. Out of several vascular effects that can trigger neurodegeneration, changes in cerebrovascular permeability seem to result in the most devastating effects. The present review emphasizes the importance of changes in the BBB and possible mechanisms primarily involving fibrinogen in the development and/or progression of neuroinflammatory and neurodegenerative diseases resulting in memory decline.

The Role of Nuclear Factor-Kappa B in Fibrinogen-Induced Inflammatory Responses in Cultured Primary Neurons.

Traumatic brain injury (TBI) is an inflammatory disease associated with a compromised blood-brain barrier (BBB) and neurodegeneration. One of the consequences of inflammation is an elevated blood level of fibrinogen (Fg), a protein that is mainly produced in the liver. The inflammation-induced changes in the BBB result in Fg extravasation into the brain parenchyma, creating the possibility of its contact with neurons. We have previously shown that interactions of Fg with the neuronal intercellular adhesion molecule-1 and cellular prion protein induced the upregulation of pro-inflammatory cytokines, oxidative damage, increased apoptosis, and cell death. However, the transcription pathway involved in this process was not defined. The association of Fg with the activation of the nuclear factor-κB (NF-κB) and the resultant expression of interleukin-6 (IL-6) and C-C chemokine ligand-2 (CCL2) were studied in cultured primary mouse brain cortex neurons. Fg-induced gene expression of CCL2 and IL-6 and the expression of NF-κB protein were increased in response to a specific interaction of Fg with neurons. These data suggest that TBI-induced neurodegeneration can involve the direct interaction of extravasated Fg with neurons, resulting in the overexpression of pro-inflammatory cytokines through the activation of transcription factor NF-κB. This may be a mechanism involved in vascular cognitive impairment during neuroinflammatory diseases.

Fibrinogen, Fibrinogen-like 1 and Fibrinogen-like 2 Proteins, and Their Effects.

Fibrinogen (Fg) and its derivatives play a considerable role in many diseases. For example, increased levels of Fg have been found in many inflammatory diseases, such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, rheumatoid arthritis, systemic lupus erythematosus, and cancer. Although associations of Fg, Fg chains, and its derivatives with various diseases have been established, their specific effects and the mechanisms of actions involved are still unclear. The present review is the first attempt to discuss the role of Fg, Fg chains, its derivatives, and other members of Fg family proteins, such as Fg-like protein 1 and 2, in inflammatory diseases and their effects in immunomodulation.

The Effects of Fibrinogen's Interactions with Its Neuronal Receptors, Intercellular Adhesion Molecule-1 and Cellular Prion Protein.

Neuroinflammatory diseases, such as Alzheimer's disease (AD) and traumatic brain injury (TBI), are associated with the extravascular deposition of the fibrinogen (Fg) derivative fibrin and are accompanied with memory impairment. We found that during the hyperfibrinogenemia that typically occurs during AD and TBI, extravasated Fg was associated with amyloid beta and astrocytic cellular prion protein (PrPC). These effects coincided with short-term memory (STM) reduction and neurodegeneration. However, the mechanisms of a direct Fg-neuron interaction and its functional role in neurodegeneration are still unclear. Cultured mouse brain neurons were treated with Fg in the presence or absence of function-blockers of its receptors, PrPC or intercellular adhesion molecule-1 (ICAM-1). Associations of Fg with neuronal PrPC and ICAM-1 were characterized. The expression of proinflammatory marker interleukin 6 (IL-6) and the generation of reactive oxygen species (ROS), mitochondrial superoxide, and nitrite in neurons were assessed. Fg-induced neuronal death was also evaluated. A strong association of Fg with neuronal PrPC and ICAM-1, accompanied with overexpression of IL-6 and enhanced generation of ROS, mitochondrial superoxide, and nitrite as well as the resulting neuronal death, was found. These effects were reduced by blocking the function of neuronal PrPC and ICAM-1, suggesting that the direct interaction of Fg with its neuronal receptors can induce overexpression of IL-6 and increase the generation of ROS, nitrite, and mitochondrial superoxide, ultimately leading to neuronal death. These effects can be a mechanism of neurodegeneration and the resultant memory reduction seen during TBI and AD.

Fibrinogen Interaction with Astrocyte ICAM-1 and PrPC Results in the Generation of ROS and Neuronal Death.

Many neuroinflammatory diseases, like traumatic brain injury (TBI), are associated with an elevated level of fibrinogen and short-term memory (STM) impairment. We found that during TBI, extravasated fibrinogen deposited in vasculo-astrocyte interfaces, which was associated with neurodegeneration and STM reduction. The mechanisms of this fibrinogen-astrocyte interaction and its functional role in neurodegeneration are still unclear. Cultured mouse brain astrocytes were treated with fibrinogen in the presence or absence of function-blocking antibody or peptide against its astrocyte receptors intercellular adhesion molecule-1 (ICAM-1) or cellular prion protein (PrPC), respectively. Fibrinogen interactions with astrocytic ICAM-1 and PrPC were characterized. The expression of pro-inflammatory markers, generations of reactive oxygen species (ROS) and nitric oxide (NO) in astrocytes, and neuronal death caused by astrocyte-conditioned medium were assessed. Data showed a strong association between fibrinogen and astrocytic ICAM-1 or PrPC, overexpression of pro-inflammatory cytokines and overproduction of ROS and NO, resulting in neuronal apoptosis and death. These effects were reduced by blocking the function of astrocytic ICAM-1 and PrPC, suggesting that fibrinogen association with its astrocytic receptors induce the release of pro-inflammatory cytokines, resulting in oxidative stress, and ultimately neuronal death. This can be a mechanism of neurodegeneration and the resultant STM reduction seen during TBI.

Effects of fibrinogen synthesis inhibition on vascular cognitive impairment during traumatic brain injury in mice.

Traumatic brain injury (TBI) is associated with increased blood content of fibrinogen (Fg), called hyperfibrinogenemia (HFg), which results in enhanced cerebrovascular permeability and leads to short-term memory (STM) reduction. Previously, we showed that extravasated Fg was deposited in the vasculo-astrocyte interface and was co-localized with cellular prion protein (PrPC) during mild-to-moderate TBI in mice. These effects were accompanied by neurodegeneration and STM reduction. However, there was no evidence presented that the described effects were the direct result of the HFg during TBI. We now present data indicating that inhibition of Fg synthesis can ameliorate TBI-induced cerebrovascular permeability and STM reduction. Cortical contusion injury (CCI) was induced in C57BL/6J mice. Then mice were treated with either Fg antisense oligonucleotide (Fg-ASO) or with control-ASO for two weeks. Cerebrovascular permeability to fluorescently labeled bovine serum albumin was assessed in cortical venules following evaluation of STM with memory assessement tests. Separately, brain samples were collected in order to define the expression of PrPC via Western blotting while deposition and co-localization of Fg and PrPC, as well as gene expression of inflammatory marker activating transcription factor 3 (ATF3), were characterized with real-time PCR. Results showed that inhibition of Fg synthesis with Fg-ASO reduced overexpression of AFT3, ameliorated enhanced cerebrovascular permeability, decreased expression of PrPC and Fg deposition, decreased formation of Fg-PrPC complexes in brain, and improved STM. These data provide direct evidence that a CCI-induced inflammation-mediated HFg could be a triggering mechanism involved in vascular cognitive impairment seen previously in our studies during mild-to-moderate TBI.

Fibrinogen and Neuroinflammation During Traumatic Brain Injury.

Many neurodegenerative diseases such as Alzheimer's disease (AD), multiple sclerosis, and traumatic brain injury (TBI) are associated with systemic inflammation. Inflammation itself results in increased blood content of fibrinogen (Fg), called hyperfibrinogenemia (HFg). Fg is not only considered an acute phase protein and a marker of inflammation, but has been shown that it can cause inflammatory responses. Fibrin deposits have been associated with memory reduction in neuroinflammatory diseases such as AD and TBI. Reduction in short-term memory has been seen during the most common form of TBI, mild-to-moderate TBI. Fibrin deposits have been found in brains of patients with mild-to-moderate TBI. The vast majority of the literature emphasizes the role of fibrin-activated microglia as the mediator in the neuroinflammation pathway. However, the recent discovery that astrocytes, which constitute approximately 30% of the cells in the mammalian central nervous system, manifest different reactive states warrants further investigations in the causative role of HFg in astrocyte-mediated neuroinflammation. Our previous study showed that Fg deposited in the vasculo-astrocyte interface-activated astrocytes. However, little is known of how Fg directly affects astrocytes and neurons. In this review, we summarize studies that show the effect of Fg on different types of cells in the vasculo-neuronal unit. We will also discuss the possible mechanism of HFg-induced neuroinflammation during TBI.

Fibrinogen-cellular prion protein complex formation on astrocytes.

Traumatic brain injury (TBI) is one of the most common neurological disorders causing memory reduction, particularly short-term memory (STM). We showed that, during TBI-induced inflammation, increased blood content of fibrinogen (Fg) enhanced vascular protein transcytosis and deposition of extravasated Fg in vasculo-astrocyte interfaces. In addition, we found that deposition of cellular prion protein (PrPC) was also increased in the vasculo-astrocyte endfeet interface. However, association of Fg and PrPC was not confirmed. Presently, we aimed to define whether Fg can associate with PrPC on astrocytes and cause their activation. Cultured mouse brain astrocytes were treated with medium alone (control), Fg (2 mg/mL or 4 mg/mL), 4 mg/mL of Fg in the presence of a function-blocking anti-PrPC peptide or anti-mouse IgG, function-blocking anti-PrPC peptide, or anti-mouse IgG alone. After treatment, either cell lysates were collected and analyzed via Western blot or coimmunoprecipitation was performed, or astrocytes were fixed and their activation was assessed with immunohistochemistry. Results showed that Fg dose-dependently activated astrocytes, increased expressions of PrPC and tyrosine (tropomyosin) receptor kinase B (TrkB), and PrP gene. Blocking the function of PrPC reduced these effects. Coimmunoprecipitation demonstrated Fg and PrPC association. Since it is known that prion protein has a greater effect on memory reduction than amyloid beta, and that activation of TrkB is involved in neurodegeneration, our findings confirming the possible formation of Fg-PrPC and Fg-induced overexpression of TrkB on astrocytes suggest a possible triggering mechanism for STM reduction that was seen previously during mild-to-moderate TBI.NEW & NOTEWORTHY For the first time we showed that fibrinogen (Fg) can associate with cellular prion protein (PrPC) on the surface of cultured mouse brain astrocytes. At high levels, Fg causes upregulation of astrocyte PrPC and astrocyte activation accompanied with overexpression of tyrosine receptor kinase B (TrkB), which results in nitric oxide (NO) production and generation of reactive oxygen species (ROS). Fg/PrPC interaction can be a triggering mechanism for TrkB-NO-ROS axis activation and the resultant astrocyte-mediated neurodegeneration.

Vascular and non-vascular contributors to memory reduction during traumatic brain injury.

Traumatic brain injury (TBI) is an increasing health problem. It is a complex, progressive disease that consists of many factors affecting memory. Studies have shown that increased blood-brain barrier (BBB) permeability initiates pathological changes in neuro-vascular network but the role of cerebrovascular dysfunction and its mediated mechanisms associated with memory reduction during TBI are still not well understood. Changes in BBB, inflammation, extravasation of blood plasma components, activation of neuroglia lead to neurodegeneration. Extravasated proteins such as amyloid-beta, fibrinogen, and cellular prion protein may form degradation resistant complexes that can lead to neuronal dysfunction and degeneration. They also have the ability to activate astrocytes, and thus, can be involved in memory impairment. Understanding the triggering mechanisms and the places they originate in vasculature or in extravascular tissue may help to identify potential therapeutic targets to ameliorate memory reduction during TBI. The goal of this review is to discuss conceptual mechanisms that lead to short-term memory reduction during non-severe TBI considering distinction between vascular and non-vascular effects on neurons. Some aspects of these mechanisms need to be confirmed further. Therefore, we hope that the discussion presented bellow may lead to experiments that may clarify the triggering mechanisms of memory reduction after head trauma.

Remodeling of Retinal Architecture in Diabetic Retinopathy: Disruption of Ocular Physiology and Visual Functions by Inflammatory Gene Products and Pyroptosis.

Diabetic patients suffer from a host of physiological abnormalities beyond just those of glucose metabolism. These abnormalities often lead to systemic inflammation via modulation of several inflammation-related genes, their respective gene products, homocysteine metabolism, and pyroptosis. The very nature of this homeostatic disruption re-sets the overall physiology of diabetics via upregulation of immune responses, enhanced retinal neovascularization, upregulation of epigenetic events, and disturbances in cells' redox regulatory system. This altered pathophysiological milieu can lead to the development of diabetic retinopathy (DR), a debilitating vision-threatening eye condition with microvascular complications. DR is the most prevalent cause of irreversible blindness in the working-age adults throughout the world as it can lead to severe structural and functional remodeling of the retina, decreasing vision and thus diminishing the quality of life. In this manuscript, we attempt to summarize recent developments and new insights to explore the very nature of this intertwined crosstalk between components of the immune system and their metabolic orchestrations to elucidate the pathophysiology of DR. Understanding the multifaceted nature of the cellular and molecular factors that are involved in DR could reveal new targets for effective diagnostics, therapeutics, prognostics, preventive tools, and finally strategies to combat the development and progression of DR in susceptible subjects.

Hyperfibrinogenemia-mediated astrocyte activation.

Fibrinogen (Fg)-containing plaques are associated with memory loss during various inflammatory neurodegenerative diseases such as Alzheimer's disease, multiple sclerosis, stroke, and traumatic brain injury. However, mechanisms of its action in neurovascular unit are not clear. As Fg is a high molecular weight blood protein and cannot translocate far from the vessel after extravasation, we hypothesized that it may interact with astrocytes first causing their activation. Cultured mouse cortical astrocytes were treated with Fg in the presence or absence of function-blocking anti-mouse intercellular adhesion molecule 1 (ICAM-1) antibody, or with medium alone (control). Expressions of ICAM-1 and tyrosine receptor kinase B (TrkB) as markers of astrocyte activation, and phosphorylation of TrkB (pTrkB) were assessed. Fg dose-dependently increased activation of astrocytes defined by their shape change, retraction of processes, and enhanced expressions of ICAM-1 and TrkB, and increased pTrkB. Blocking of ICAM-1 function ameliorated these Fg effects. Data suggest that Fg interacts with astrocytes causing overexpression of ICAM-1 and TrkB, and TrkB phosphorylation, and thus, astrocyte activation. Since TrkB is known to be involved in neurodegeneration, interaction of Fg with astrocytes and the resultant activation of TrkB can be a possible mechanism involved in memory reduction, which were observed in previous studies and were associated with formation of complexes of Fg deposited in extravascular space with proteins such as Amyloid beta or prion, the proteins involved in development of dementia.

High methionine, low folate and low vitamin B6/B12 (HM-LF-LV) diet causes neurodegeneration and subsequent short-term memory loss.

Methionine is an essential amino acid found in rich quantities in average American diet such as meats, fish and eggs. Excessive consumption of such food often exceeds the normal requirement of the methionine in our body; which found to be related to the development of neurodegenerative disorders. However, the mechanistic pathways of methionine's influence on the brain are unclear. The present study is focus on the effects of high methionine, low folate and low vitamin B6/B12 (HM-LF-LV) diet on the dysfunction of neuronal and vascular specific markers in the brain. C57BL6/J male mice (8-10 week old) were fed with HM-LF-LV diet for a 6 week period. Cognitive function of mice was determine by measuring short-term memory using a Novel Object Recognition test (NORT). Neuronal dysfunction were evaluate by measuring the levels of Neuronal nuclear antigen (NeuN), Neuron-specific-enolase (NSE) and Fluoro-jade C(FJC) fluorescence; while cerebrovascular disruption were evaluate by assessing levels of endothelial junction proteins Vascular Endothelial-Cadherin (VE-Cadherin) and Claudin-5 in harvested brain tissue. Cerebrovascular permeability was assess by evaluating microvascular leakage of fluorescently labeled albumin in vivo. Endothelial and Neuronal Nitric Oxide Synthase (eNOS, nNOS) regulation and vascular inflammation (ICAM: intercellular adhesion molecules) were also evaluate in brain tissue. All assessments were conduct at weekly intervals throughout the study duration. NORT showed a significant temporal decrease in short-term memory of mice fed on HM-LF-LV diet for 6 weeks compared to the wild-type control group. Our experimental data showed that neuronal dysfunction (decreased NeuN levels and increased FJC positive neurons in brain) was more prominent in HM-LF-LV diet fed mice compared to normal diet fed control mice. In experimental mice, cerebrovascular disruption was found to be elevated as evident from increased pial venular permeability (microvascular leakage) and decreased in VE-Cadherin expression compared to control. Slight decrease in nNOS and increase in eNOS in experimental mice suggest a trend towards the decrease in potential for neuronal development due to the long-term HM-LF-LV diet fed. Collectively, our results suggest that a diet containing high methionine, low folate and low vitamin B6/B12 results in increased neuronal degeneration and vascular dysfunction, leading to short-term memory loss. Interestingly, significant neuronal damage precedes vascular dysfunction.

Localization of Fibrinogen in the Vasculo-Astrocyte Interface after Cortical Contusion Injury in Mice.

Besides causing neuronal damage, traumatic brain injury (TBI) is involved in memory reduction, which can be a result of alterations in vasculo-neuronal interactions. Inflammation following TBI is involved in elevation of blood content of fibrinogen (Fg), which is known to enhance cerebrovascular permeability, and thus, enhance its deposition in extravascular space. However, the localization of Fg in the extravascular space and its possible interaction with nonvascular cells are not clear. The localization of Fg deposition in the extravascular space was defined in brain samples of mice after cortical contusion injury (CCI) and sham-operation (control) using immunohistochemistry and laser-scanning confocal microscopy. Memory changes were assessed with new object recognition and Y-maze tests. Data showed a greater deposition of Fg in the vascular and astrocyte endfeet interface in mice with CCI than in control animals. This effect was accompanied by enhanced neuronal degeneration and reduction in short-term memory in mice with CCI. Thus, our results suggest that CCI induces increased deposition of Fg in the vasculo-astrocyte interface, and is accompanied by neuronal degeneration, which may result in reduction of short-term memory.

Cerebrovascular disorders caused by hyperfibrinogenaemia.

KEY POINTS: Hyperfibrinogenaemia (HFg) results in vascular remodelling, and fibrinogen (Fg) and amyloid ß (Aß) complex formation is a hallmark of Alzheimer's disease. However, the interconnection of these effects, their mechanisms and implications in cerebrovascular diseases are not known. Using a mouse model of HFg, we showed that at an elevated blood level, Fg increases cerebrovascular permeability via mainly caveolar protein transcytosis. This enhances deposition of Fg in subendothelial matrix and interstitium making the immobilized Fg a readily accessible substrate for binding Aß and cellular prion protein (PrPC ), the protein that is thought to have a greater effect on memory than Aß. We showed that enhanced formation of Fg-Aß and Fg-PrPC complexes are associated with reduction in short-term memory. The present study delineates a new mechanistic pathway for vasculo-neuronal dysfunctions found in inflammatory cardiovascular and cerebrovascular diseases associated with an elevated blood level of Fg. ABSTRACT: Many cardiovascular diseases are associated with inflammation and as such are accompanied by an increased blood level of fibrinogen (Fg). Besides its well-known prothrombotic effects Fg seems to have other destructive roles in developing microvascular dysfunction that include changes in vascular reactivity and permeability. Increased permeability of brain microvessels has the most profound effects as it may lead to cerebrovascular remodelling and result in memory reduction. The goal of the present study was to define mechanisms of cerebrovascular permeability and associated reduction in memory induced by elevated blood content of Fg. Genetically modified, transgenic hyperfibrinogenic (HFg) mice were used to study cerebrovascular transcellular and paracellular permeability in vivo. The extent of caveolar formation and the role of caveolin-1 signalling were evaluated by immunohistochemistry (IHC) and Western blot (WB) analysis in brain samples from experimental animals. Formation of Fg complexes with amyloid ß (Aß) and with cellular prion protein (PrPC ) were also assessed with IHC and WB analysis. Short-term memory of mice was assessed by novel object recognition and Y-maze tests. Caveolar protein transcytosis was found to have a prevailing role in overall increased cerebrovascular permeability in HFg mice. These results were associated with enhanced formation of caveolae. Increased formation of Fg-PrPC and Fg-Aß complexes were correlated with reduction in short-term memory in HFg mice. Using the model of hyperfibrinogenaemia, the present study shows a novel mechanistic pathway of inflammation-induced and Fg-mediated reduction in short-term memory.

Hyperhomocysteinemia inhibits satellite cell regenerative capacity through p38 alpha/beta MAPK signaling.

Chronic failure in maintenance and regeneration of skeletal muscles leads to lower muscle mass (sarcopenia), muscle weakness, and poor response to injury. Evidence suggests that aberrant p38 MAPK signaling undermines the repair process after injury in aged mice. Previous studies have shown that hyperhomocysteinemia (HHcy) has been associated with muscle weakness and lower than normal body weights. However, whether or not HHcy condition also compromises skeletal muscle regenerative capabilities is not clear. In the current study, we show that CBS-/+ mice, a model for HHcy condition, exhibited compromised regenerative function and cell proliferation upon injury. However, there was no significant difference in Pax7 expression levels in the satellite cells from CBS-/+ mouse skeletal muscles. Interestingly, the satellite cells from CBS-/+ mice not only exhibited diminished in vitro proliferative capabilities, but also there was heightened oxidative stress. In addition, there was enhanced p38 MAPK activation as well as p16 and p21 expression in the CBS-/+ mouse satellite cells. Moreover, the C2C12 myoblasts also exhibited higher p38 MAPK activation and p16 expression upon treatment with homocysteine in addition to enhanced ROS presence. Tissue engraftment potential and regeneration after injury were restored to some extent upon treatment with the p38-MAPK inhibitor, SB203580, in the CBS-/+ mice. These results together suggest that HHcy-induced diminished satellite cell proliferation involves excessive oxidative stress and p38 MAPK signaling. Our study further proposes that HHcy is a potential risk factor for elderly frailty, and need to be considered as a therapeutic target while designing the alleviation interventions/postinjury rehabilitation measures for adults with HHcy.

Ablation of matrix metalloproteinase-9 gene decreases cerebrovascular permeability and fibrinogen deposition post traumatic brain injury in mice.

Traumatic brain injury (TBI) is accompanied with enhanced matrix metalloproteinase-9 (MMP-9) activity and elevated levels of plasma fibrinogen (Fg), which is a known inflammatory agent. Activation of MMP-9 and increase in blood content of Fg (i.e. hyperfibrinogenemia, HFg) both contribute to cerebrovascular disorders leading to blood brain barrier disruption. It is well-known that activation of MMP-9 contributes to vascular permeability. It has been shown that at an elevated level (i.e. HFg) Fg disrupts blood brain barrier. However, mechanisms of their actions during TBI are not known. Mild TBI was induced in wild type (WT, C57BL/6 J) and MMP-9 gene knockout (Mmp9(-/-)) homozygous, mice. Pial venular permeability to fluorescein isothiocyanate-conjugated bovine serum albumin in pericontusional area was observed 14 days after injury. Mice memory was tested with a novel object recognition test. Increased expression of Fg endothelial receptor intercellular adhesion protein-1 and formation of caveolae were associated with enhanced activity of MMP-9 causing an increase in pial venular permeability. As a result, an enhanced deposition of Fg and cellular prion protein (PrP(C)) were found in pericontusional area. These changes were attenuated in Mmp9(-/-) mice and were associated with lesser loss of short-term memory in these mice than in WT mice. Our data suggest that mild TBI-induced increased cerebrovascular permeability enhances deposition of Fg-PrP(C) and loss of memory, which is ameliorated in the absence of MMP-9 activity. Thus, targeting MMP-9 activity and blood level of Fg can be a possible therapeutic remedy to diminish vasculo-neuronal damage after TBI.

Sphingolipids affect fibrinogen-induced caveolar transcytosis and cerebrovascular permeability.

Inflammation-induced vascular endothelial dysfunction can allow plasma proteins to cross the vascular wall, causing edema. Proteins may traverse the vascular wall through two main pathways, the paracellular and transcellular transport pathways. Paracellular transport involves changes in endothelial cell junction proteins, while transcellular transport involves caveolar transcytosis. Since both processes are associated with filamentous actin formation, the two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during various pathologies causing an increase in vascular permeability. Using a newly developed dual-tracer probing method, we differentiated transcellular from paracellular transport during hyperfibrinogenemia (HFg), an increase in fibrinogen (Fg) content. Roles of cholesterol and sphingolipids in formation of functional caveolae were assessed using a cholesterol chelator, methyl-ß-cyclodextrin, and the de novo sphingolipid synthesis inhibitor myriocin. Fg-induced formation of functional caveolae was defined by association and colocalization of Na+-K+-ATPase and plasmalemmal vesicle-associated protein-1 with use of Förster resonance energy transfer and total internal reflection fluorescence microscopy, respectively. HFg increased permeability of the endothelial cell layer mainly through the transcellular pathway. While MßCD blocked Fg-increased transcellular and paracellular transport, myriocin affected only transcellular transport. Less pial venular leakage of albumin was observed in myriocin-treated HFg mice. HFg induced greater formation of functional caveolae, as indicated by colocalization of Na+-K+-ATPase with plasmalemmal vesicle-associated protein-1 by Förster resonance energy transfer and total internal reflection fluorescence microscopy. Our results suggest that elevated blood levels of Fg alter cerebrovascular permeability mainly by affecting caveolae-mediated transcytosis through modulation of de novo sphingolipid synthesis.

Ablation of MMP9 gene ameliorates paracellular permeability and fibrinogen-amyloid beta complex formation during hyperhomocysteinemia.

Increased blood level of homocysteine (Hcy), called hyperhomocysteinemia (HHcy) accompanies many cognitive disorders including Alzheimer's disease. We hypothesized that HHcy-enhanced cerebrovascular permeability occurs via activation of matrix metalloproteinase-9 (MMP9) and leads to an increased formation of fibrinogen-ß-amyloid (Fg-Aß) complex. Cerebrovascular permeability changes were assessed in C57BL/6J (wild type, WT), cystathionine-ß-synthase heterozygote (Cbs+/-, a genetic model of HHcy), MMP9 gene knockout (Mmp9-/-), and Cbs and Mmp9 double knockout (Cbs+/-/Mmp9-/-) mice using a dual-tracer probing method. Expression of vascular endothelial cadherin (VE-cadherin) and Fg-Aß complex formation was assessed in mouse brain cryosections by immunohistochemistry. Short-term memory of mice was assessed with a novel object recognition test. The cerebrovascular permeability in Cbs+/- mice was increased via mainly the paracellular transport pathway. VE-cadherin expression was the lowest and Fg-Aß complex formation was the highest along with the diminished short-term memory in Cbs+/- mice. These effects of HHcy were ameliorated in Cbs+/-/Mmp9-/- mice. Thus, HHcy causes activation of MMP9 increasing cerebrovascular permeability by downregulation of VE-cadherin resulting in an enhanced formation of Fg-Aß complex that can be associated with loss of memory. These data may lead to the identification of new targets for therapeutic intervention that can modulate HHcy-induced cerebrovascular permeability and resultant pathologies.