Protective effects of FK 506 against haemorrhagic shock-induced microvascular hyperpermeability.

Microvascular hyperpermeability, the excessive leakage of fluid and proteins from the intravascular space to the interstitium, is a devastating clinical concern in haemorrhagic shock (HS), sepsis, burn and so forth. Previous studies have shown that HS-induced microvascular hyperpermeability is associated with activation of the mitochondria-mediated 'intrinsic' apoptotic signalling cascade and caspase-3 mediated disruption of the endothelial cell barrier. In this study, our objective was to test if FK506, an immunomodulator that is also known to protect mitochondria, would protect barrier functions and decrease vascular hyperpermeability following HS by acting on this pathway. FK506 (25 µM) was given 10 minutes before the shock period in a rat model of HS. The HS model was a non-traumatic/fixed pressure model of hypovolemic shock developed by withdrawing blood to reduce the mean arterial pressure to 40 mm Hg for 60 minutes. The mesenteric post-capillary venules were monitored for changes in permeability using intravital microscopic imaging. The changes in mitochondrial transmembrane potential (MTP) were determined using the cationic dye 5,5',6,6' tetrachoro-1,1',3,3' tetraethyl benzimidazolyl carbocyanine iodide (JC-1), that was superfused on the mesenteric vasculature followed by intravital imaging. The mesenteric caspase-3 activity was measured fluorometrically. Haemorrhagic shock induced a significant increase in hyperpermeability compared to the sham-control group and FK506 treatment decreased HS-induced hyperpermeability significantly (P < .05). FK506 dampened HS-induced loss of MTP and elevation of caspase-3 activity significantly (P < .05). FK506 has protective effects against HS-induced microvascular hyperpermeability. The maintenance of the MTP and protection against caspase-3 mediated endothelial cell barrier disruption are possible mechanisms by which FK506 attenuates HS-induced hyperpermeability. FK506, currently used in clinical settings as an immunomodulator, needs to be explored further for its therapeutic usefulness against HS-induced vascular hyperpermeability and associated complications.

Attenuation of Blood-Brain Barrier Breakdown and Hyperpermeability by Calpain Inhibition.

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1ß (IL-1ß) that is up-regulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1ß-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1ß-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1ß treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1ß-induced calpain activity significantly (p < 0.05). IL-1ß had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1ß-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1ß-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.

Blood-brain barrier dysfunction following traumatic brain injury.

Traumatic brain injury is a serious cause of morbidity and mortality worldwide. After traumatic brain injury, the blood-brain barrier, the protective barrier between the brain and the intravascular compartment, becomes dysfunctional, leading to leakage of proteins, fluid, and transmigration of immune cells. As this leakage has profound clinical implications, including edema formation, elevated intracranial pressure and decreased perfusion pressure, much interest has been paid to better understanding the mechanisms responsible for these events. Various molecular pathways and numerous mediators have been found to be involved in the intricate process of regulating blood-brain barrier permeability following traumatic brain injury. This review provides an update to the existing knowledge about the various pathophysiological pathways and advancements in the field of blood-brain barrier dysfunction and hyperpermeability following traumatic brain injury, including the role of various tight junction proteins involved in blood-brain barrier integrity and regulation. We also address pitfalls of existing systems and propose strategies to improve the various debilitating functional deficits caused by this progressive epidemic.

The effects of inflammatory cytokines on lymphatic endothelial barrier function.

Proper lymphatic function is necessary for the transport of fluids, macromolecules, antigens and immune cells out of the interstitium. The lymphatic endothelium plays important roles in the modulation of lymphatic contractile activity and lymph transport, but it's role as a barrier between the lymph and interstitial compartments is less well understood. Alterations in lymphatic function have long been associated with edema and inflammation although the integrity of the lymphatic endothelial barrier during inflammation is not well-defined. In this paper we evaluated the integrity of the lymphatic barrier in response to inflammatory stimuli commonly associated with increased blood endothelial permeability. We utilized in vitro assays of lymphatic endothelial cell (LEC) monolayer barrier function after treatment with different inflammatory cytokines and signaling molecules including TNF-α, IL-6, IL-1ß, IFN-γ and LPS. Moderate increases in an index of monolayer barrier dysfunction were noted with all treatments (20-60 % increase) except IFN-γ which caused a greater than 2.5-fold increase. Cytokine-induced barrier dysfunction was blocked or reduced by the addition of LNAME, except for IL-1ß and LPS treatments, suggesting a regulatory role for nitric oxide. The decreased LEC barrier was associated with modulation of both intercellular adhesion and intracellular cytoskeletal activation. Cytokine treatments reduced the expression of VE-cadherin and increased scavenging of ß-catenin in the LECs and this was partially reversed by LNAME. Likewise the phosphorylation of myosin light chain 20 at the regulatory serine 19 site, which accompanied the elevated monolayer barrier dysfunction in response to cytokine treatment, was also blunted by LNAME application. This suggests that the lymphatic barrier is regulated during inflammation and that certain inflammatory signals may induce large increases in permeability.

Reactive oxygen species-caspase-3 relationship in mediating blood-brain barrier endothelial cell hyperpermeability following oxygen-glucose deprivation and reoxygenation.

OBJECTIVE: Microvascular hyperpermeability that occurs due to breakdown of the BBB is a major contributor of brain vasogenic edema, following IR injury. In microvascular endothelial cells, increased ROS formation leads to caspase-3 activation following IR injury. The specific mechanisms, by which ROS mediates microvascular hyperpermeability following IR, are not clearly known. We utilized an OGD-R in vitro model of IR injury to study this. METHODS: RBMEC were subjected to OGD-R in presence of a caspase-3 inhibitor Z-DEVD, caspase-3 siRNA or an ROS inhibitor L-AA. Cytochrome c levels were measured by ELISA and caspase-3 activity was measured fluorometrically. TJ integrity and cytoskeletal assembly were studied using ZO-1 immunofluorescence and rhodamine phalloidin staining for f-actin, respectively. RESULTS: OGD-R significantly increased monolayer permeability, ROS formation, cytochrome c levels, and caspase-3 activity (p < 0.05) and induced TJ disruption and actin stress fiber formation. Z-DEVD, L-AA and caspase-3 siRNA significantly attenuated OGD-R-induced hyperpermeability (p < 0.05) while only L-AA decreased cytochrome c levels. Z-DEVD and L-AA protected TJ integrity and actin cytoskeletal assembly. CONCLUSIONS: These results suggest that OGD-R-induced hyperpermeability is ROS and caspase-3 dependent and can be regulated by their inhibitors.

Matrix Metalloproteinase-9 inhibitors as therapeutic drugs for traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality among young adults and the elderly. In the United States, TBI is responsible for around 30 percent of all injuries brought on by injuries in general. Vasogenic cerebral edema due to blood-brain barrier (BBB) dysfunction and the associated elevation of intracranial pressure (ICP) are some of the major causes of secondary injuries following traumatic brain injury. Matrix metalloproteinase-9 (MMP-9) is a therapeutic target for being an enzyme that degrades the proteins that make up a part of the microvascular basal lamina as well as inter-endothelial tight junctions of the blood-brain barrier. MMP-9-mediated BBB dysfunctions and the compromise of the BBB is a major pathway that leads the development of vasogenic cerebral edema, elevation of ICP, poor cerebral perfusion and brain herniation following traumatic brain injury. That makes MMP-9 an effective therapeutic target and endogenous or exogenous MMP-9 inhibitors as therapeutic drugs for preventing secondary brain damage after traumatic brain injury. Although our understanding of the mechanisms that underlie the primary and secondary stages of damage following a TBI has significantly improved in recent years, such information has not yet resulted in the successful development of novel pharmacological treatment options for traumatic brain injury. Recent pre-clinical and/or clinical studies have demonstrated that there are several compounds with specific or non-specific MMP-9 inhibitory properties either directly binding and inhibiting MMP-9 or by indirectly inhibiting MMP-9, with potential as therapeutic agents for traumatic brain injury. This article reviews the efficacy of several such medications and potential agents that include endogenous and exogeneous compounds that are at various levels of research and development. MMP-9-based therapeutic drug development has enormous potential in the pharmacological treatment of cerebral edema and/or neuronal injury resulting from traumatic brain injury.

Regulation of tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability by recombinant B-cell lymphoma-extra large.

BACKGROUND: Tumor necrosis factor-α (TNF-α), a cytotoxic cytokine, induces endothelial cell barrier dysfunction and microvascular hyperpermeability, leading to tissue edema, a hallmark of traumatic injuries. The objective of the present study was to determine whether B-cell lymphoma-extra large (Bcl-xL), an antiapoptotic protein, would regulate and protect against TNF-α-mediated endothelial cell barrier dysfunction and microvascular hyperpermeability. METHODS: Rat lung microvascular endothelial cells were grown as monolayers on Transwell membranes, and fluorescein isothiocyanate-bovine albumin flux (5 mg/mL) across the monolayer was measured fluorometrically to indicate changes in monolayer permeability. The rat lung microvascular endothelial cell adherens junctional integrity and actin cytoskeleton was studied using ß-catenin immunofluorescence and rhodamine phalloidin dye, respectively. Pretreatment of caspase-8 inhibitor (Z-IETD-FMK, 100 µM) for 1 hour and transfection of Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA (10 µM) for 48 hours were performed to study their respective effects on TNF-α-induced (10 ng/mL; 1-hour treatment) monolayer permeability. Recombinant Bcl-xL protein (2.5 µg/ml) was transfected in rat lung microvascular endothelial cells for 1 hour, and its effect on permeability was demonstrated using a permeability assay. Caspase-3 activity was assayed fluorometrically. RESULTS: Z-IETD-FMK pretreatment protected the adherens junctions and decreased TNF-α-induced monolayer hyperpermeability. Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA transfection attenuated the TNF-α-induced increase in monolayer permeability. Recombinant Bcl-xL protein showed protection against TNF-α-induced actin stress fiber formation, an increase in caspase-3 activity, and monolayer hyperpermeability. CONCLUSIONS: Our results have demonstrated the protective effects of recombinant Bcl-xL protein against TNF-α-induced endothelial cell adherens junction damage and microvascular endothelial cell hyperpermeability. These findings support the potential for Bcl-xL-based drug development against microvascular hyperpermeability and tissue edema.

Marrow stimulation improves meniscal healing at early endpoints in a rabbit meniscal injury model.

PURPOSE: To critically evaluate the effect of marrow stimulation (MS) on the extent of healing and the local biological environment after meniscal injury in ligamentously stable knees in a rabbit model. METHODS: A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in 18 New Zealand White rabbits (36 knees). In right knees (MS knees), a 2.4-mm Steinman pin was drilled into the apex of the femoral intercondylar notch and marrow contents were observed spilling into the joint. Left knees served as controls. Rabbits were killed in 3 groups (n = 6 rabbits each) at 1, 4, and 12 weeks with meniscal harvest and blinded histomorphometric and histologic evaluation using an established 3-component tissue quality score (range, 0 to 6). One-week specimens were also evaluated for the presence of proregenerative cytokines using immunohistochemistry. RESULTS: The mean proportion of the avascular zone defect bridged by reparative tissue was greater in MS knees than in controls at each endpoint (1 week, 55% v 30%, P = .02; 4 weeks, 71% v 53%, P = .047; 12 weeks, 96% v 77%, P = .16). Similarly, there was a consistent trend toward superior tissue quality scores in knees treated with MS compared with controls (1 week, 1.8 v 0.3, P = .03; 4 weeks, 4.3 v 2.8, P = .08; 12 weeks, 5.9 v 4.5, P = .21). No statistically significant differences, however, were observed at the 12-week endpoint. Increased staining for insulin-like growth factor I, transforming growth factor-ß, and platelet-derived growth factor was observed in regenerated tissue, compared with native meniscal tissue, in all specimens at 1 week. Staining density for all growth factors was similar, however, in reparative tissue of MS and control knees. CONCLUSIONS: The results of this study suggest that marrow stimulation leads to modest improvements in quality and quantity of reparative tissue bridging a meniscal defect, particularly during the early recovery period. CLINICAL RELEVANCE: Clinical evaluation of marrow stimulation techniques designed to enhance healing in isolated meniscus repair surgery may be indicated.

Racial/Ethnic Differences in Traumatic Brain Injury: Pathophysiology, Outcomes, and Future Directions.

Traumatic brain injury (TBI) is a major cause of death and disability in the United States, exacting a debilitating physical, social, and financial strain. Therefore, it is crucial to examine the impact of TBI on medically underserved communities in the U.S. The purpose of the current study was to review the literature on TBI for evidence of racial/ethnic differences in the U.S. Results of the review showed significant racial/ethnic disparities in TBI outcome and several notable differences in other TBI variables. American Indian/Alaska Natives have the highest rate and number of TBI-related deaths compared with all other racial/ethnic groups; Blacks/African Americans are significantly more likely to incur a TBI from violence when compared with Non-Hispanic Whites; and minorities are significantly more likely to have worse functional outcome compared with Non-Hispanic Whites, particularly among measures of community integration. We were unable to identify any studies that looked directly at underlying racial/ethnic biological variations associated with different TBI outcomes. In the absence of studies on racial/ethnic differences in TBI pathobiology, taking an indirect approach, we looked for studies examining racial/ethnic differences in oxidative stress and inflammation outside the scope of TBI as they are known to heavily influence TBI pathobiology. The literature indicates that Blacks/African Americans have greater inflammation and oxidative stress compared with Non-Hispanic Whites. We propose that future studies investigate the possibility of racial/ethnic differences in inflammation and oxidative stress within the context of TBI to determine whether there is any relationship or impact on TBI outcome.

ETS-Related Gene Activation Preserves Adherens Junctions and Permeability in Microvascular Endothelial Cells.

ABSTRACT: ERG (ETS-related gene) is a member of the ETS (Erythroblast-transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. A wide variety of diseases and clinical conditions including trauma-hemorrhagic shock and burn injury are associated with microvascular dysfunctions, which causes excessive microvascular permeability, tissue edema and eventually, multiple organ dysfunction and death. The main purpose of this study was to determine the specific role of ERG in regulating microvascular permeability in human lung microvascular endothelial cells (HLMEC) and to evaluate if exogenous ERG will protect the barrier. The HLMECs were grown on Transwell inserts as monolayers and were transfected with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid, recombinant ERG protein or their respective controls. Recombinant vascular endothelial growth factor (VEGF) was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Changes in barrier integrity and permeability were studied using monolayer permeability assay and immunofluorescence of adherens junction proteins (VE-cadherin and ß-catenin) respectively. CRISPR/cas9-based ERG knockdown as well as VEGF treatment induced monolayer hyperpermeability, VE-cadherin, and ß-catenin junctional relocation and cytoskeletal F-actin stress fiber formation. CRISPR based ERG activation and recombinant ERG transfection attenuated VEGF-induced monolayer hyperpermeability. ERG activation preserved the adherens junctions and cytoskeleton. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability in human lung microvascular endothelial cells and endogenously or exogenously enhancing ERG provides protection against barrier dysfunction and hyperpermeability.

Doxycycline prevents blood-brain barrier dysfunction and microvascular hyperpermeability after traumatic brain injury.

The main objective of this study was to determine the cellular and molecular effects of doxycycline on the blood-brain barrier (BBB) and protection against secondary injuries following traumatic brain injury (TBI). Microvascular hyperpermeability and cerebral edema resulting from BBB dysfunction after TBI leads to elevation of intracranial pressure, secondary brain ischemia, herniation, and brain death. There are currently no effective therapies to modulate the underlying pathophysiology responsible for TBI-induced BBB dysfunction and hyperpermeability. The loss of BBB integrity by the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) is critical to TBI-induced BBB hyperpermeability, and doxycycline possesses anti-MMP-9 effect. In this study, the effect of doxycycline on BBB hyperpermeability was studied utilizing molecular modeling (using Glide) in silico, cell culture-based models in vitro, and a mouse model of TBI in vivo. Brain microvascular endothelial cell assays of tight junction protein immunofluorescence and barrier permeability were performed. Adult C57BL/6 mice were subjected to sham versus TBI with or without doxycycline treatment and immediate intravital microscopic analysis for evaluating BBB integrity. Postmortem mouse brain tissue was collected to measure MMP-9 enzyme activity. It was found that doxycycline binding to the MMP-9 active sites have binding affinity of -7.07 kcal/mol. Doxycycline treated cell monolayers were protected from microvascular hyperpermeability and retained tight junction integrity (p < 0.05). Doxycycline treatment decreased BBB hyperpermeability following TBI in mice by 25% (p < 0.05). MMP-9 enzyme activity in brain tissue decreased with doxycycline treatment following TBI (p < 0.05). Doxycycline preserves BBB tight junction integrity following TBI via inhibiting MMP-9 activity. When established in human subjects, doxycycline, may provide readily accessible medical treatment after TBI to attenuate secondary injury.

Inhibition of VE-cadherin proteasomal degradation attenuates microvascular hyperpermeability.

OBJECTIVE: VE-cadherin, an integral component of the adherens junction complex, is processed through the endosome-lysosome pathway and proteasome system for degradation. Our objective was to determine if inhibition of this pathway would protect against microvascular hyperpermeability. METHODS: To induce VE-cadherin degradation, we utilized a mutant VE-cadherin protein that lacks the extracellular domain (rVE-cad CPD). Intravital microscopy was employed to study the changes in microvascular permeability in rat mesenteric postcapillary venules. Rat lung microvascular endothelial cell (RLMEC) monolayers were utilized in parallel studies. The adherens junction integrity was determined using VE-cadherin and ß-catenin immunofluorescence. TOPflash/FOPflash transfection and luciferase reporter assay were performed to study ß-catenin-mediated transcriptional activation. RESULTS: rVE-cad CPD (2.5 µg/mL of blood volume) increased hyperpermeability significantly (p < 0.05). The VE-cadherin siRNA as well as rVE-cad CPD induced significant increase in monolayer hyperpermeability (p < 0.05). Transfection of rVE-cad CPD disrupted adherens junctions evidenced by discontinuity in ß-catenin and VE-cadherin immunofluorescence (p < 0.05). Proteasome inhibitor MG132 attenuated rVE-cad CPD induced monolayer hyperpermeability and adherens junction damage. CONCLUSIONS: VE-cadherin disruption in animals results in hyperpermeability. Parallel studies in RLMEC demonstrated similar results. In addition, inhibition of proteasomal degradation attenuated microvascular hyperpermeability. These findings have significance in understanding the role of VE-cadherin in regulating vascular hyperpermeability.

Role of ß-catenin in regulating microvascular endothelial cell hyperpermeability.

BACKGROUND: Paracellular microvascular hyperpermeability occurs mainly because of the disruption of the endothelial adherens junction complex. Vascular endothelial-cadherin that consists of an extracellular and intracellular domain to confer cell-cell contact is linked to the actin cytoskeletal assembly through ß-catenin. Our objective was to determine the functional role of ß-catenin during paracellular hyperpermeability and to evaluate whether exogenous ß-catenin would protect against vascular leak. METHODS: ß-Catenin siRNA (2.5 µg/mL) was administered to Sprague-Dawley rats through tail vein. FITC-albumin extravasation of the mesenteric postcapillary venules was evaluated after 48 hours using intravital microscopy. Parallel studies using rat lung microvascular endothelial cell monolayers were transfected with ß-catenin siRNA, and hyperpermeability was determined using monolayers after 48 hours. The effectiveness of ß-catenin siRNA was tested using immunofluorescence and Western blot. To study the protective effect of ß-catenin, rat lung microvascular endothelial cell monolayers were transfected with a ß-catenin gene expression construct for 48 hours or a recombinant ß-catenin protein (1 µg/mL) for 2 hours, followed by transfection with proapoptotic BAK peptide (5 µg/mL), a known inducer hyperpermeability. RESULTS: ß-Catenin siRNA induced a significant increase in vascular hyperpermeability in vivo (p<0.05) and monolayer permeability (in vitro; p<0.05). ß-Catenin siRNA significantly altered the adherens junction complex and decreased ß-catenin protein levels. ß-Catenin gene expression construct or recombinant ß-catenin protein attenuated BAK-induced monolayer hyperpermeability significantly (p<0.05). CONCLUSION: Posttranscriptional gene silencing of ß-catenin leads to vascular hyperpermeability in vivo and monolayer hyperpermeability in vitro. The enhancement of ß-catenin gene expression at the adherens junction or exogenous introduction of ß-catenin protein shows protection against vascular hyperpermeability.

Detecting Three-Dimensional Vascular Networks in the Mouse Embryonic Hindbrain.

Blood vessel formation is a fine-regulated process and interfering with blood vessel formation causes embryonic lethality as well as associated with many diseases in the adult, including inflammatory, ischemic, and cancer metastatic diseases. Brain contains abundant blood vessels and has some unique physiological functions, such as blood-brain barrier. Due to the thickness and opaque characters of the tissues, it is a challenge to visualize the three-dimensional structures of the brain blood vessels in the mouse. Therefore, establishing a protocol to display the three-dimensional structures in the brain is required for exploring the regulatory molecular mechanisms in brain blood vessel formation. In this manuscript, we introduced a whole-mount and a vibratome thick section of mouse embryonic hindbrain to display the three-dimensional structures of brain vascular system.

Curcumin inhibits reactive oxygen species formation and vascular hyperpermeability following haemorrhagic shock.

1. Oxidative stress induced by reactive oxygen species (ROS) is a key mediator of haemorrhagic shock (HS)-induced vascular hyperpermeability. In the present study, curcumin, a natural anti-oxidant obtained from turmeric (Curcuma longa), was tested against HS-induced hyperpermeability and associated ROS formation in rat mesenteric post-capillary venules in vivo and in rat lung microvascular endothelial cells (RLMEC) in vitro. 2. In rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mmHg for 60 min, followed by resuscitation for 60 min. To investigate vascular permeability, rats were given fluorescein isothiocyanate (FITC)-albumin (50 mg/kg, i.v.). The FITC-albumin flux was measured in mesenteric post-capillary venules by determining optical intensity intra- and extravascularly under intravital microscopy. Mitochondrial ROS formation was determined using dihydrorhodamine 123 in vivo. Parallel studies were conducted in vitro using serum collected after HS. The serum was tested on rat lung microvascular endothelial cell RLMEC monolayers. 3. In rats, HS induced a significant increase in vascular hyperpermeability and ROS formation in vivo (P < 0.05). Treatment with curcumin (20 micromol/L) attenuated both these effects (P < 0.05). In RLMEC in vitro, HS serum induced monolayer permeability and ROS formation. Curcumin (10 micromol/L) attenuated HS serum-induced monolayer hyperpermeability and ROS formation. Curcumin (2-100 micromol/L) scavenged 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro, indicating its potential as a free radical scavenger. 4. The present study demonstrates that curcumin is an inhibitor of vascular hyperpermeability following HS, with its protective effects mediated through its anti-oxidant properties.

Exploring blood-brain barrier hyperpermeability and potential biomarkers in traumatic brain injury.

Blood-brain barrier breakdown and associated vascular hyperpermeability leads to vasogenic edema in traumatic brain injury (TBI). Tight junctions maintain blood-brain barrier integrity; their disruption in TBI holds significant promise for diagnosis and treatment. A controlled cortical impactor was used for TBI in mouse studies. Blood was collected 1 h after injury and sent for antibody microarray analysis. Twenty human subjects with radiographic evidence of TBI were enrolled and blood collected within 48 h of admission. Control subjects were individuals with nontrauma diagnoses. The subjects were matched by age and gender. Enzyme-linked immunosorbent assays were performed on each TBI and control sample for tight junction-associated proteins (TJPs), inflammatory markers, and S100ß. Plasma was used to conduct in vitro monolayer permeability studies with human brain endothelial cells. S100ß and the TJP occludin were significantly elevated in TBI plasma in both the murine and human studies. Monolayer permeability studies showed increased hyperpermeability in TBI groups. Plasma from TBI subjects increases microvascular hyperpermeability in vitro. TJPs in the blood may be a potential biomarker for TBI.

Doxycycline improves traumatic brain injury outcomes in a murine survival model.

BACKGROUND: Traumatic brain injury (TBI) has significant morbidity and cost implications. Primary treatment modalities aim to decrease intracranial pressure; however, therapies targeting the underlying pathophysiology of a TBI are limited. The TBI-induced microvascular leak and secondary injury are largely due to proteolysis of the blood-brain barrier (BBB) by matrix metalloproteinase-9. We previously observed doxycycline's inhibitory affinity on matrix metalloproteinase-9 resulting in preserved BBB integrity in nonsurvival murine studies. This study sought to determine the effect of doxycycline on functional motor and behavioral outcomes in the setting of a TBI murine survival model. METHODS: C57BL/6J mice were assigned to a sham, TBI, or TBI with doxycycline arm. A moderate TBI was induced utilizing a controlled cortical impactor. The TBI with doxycycline cohort received a dose of doxycycline (20 mg/kg) 2 hours after injury and every 12 hours until postoperative day (POD) 6. All mice underwent preoperative testing for weight, modified neurological severity score, wire grip, and ataxia analysis (DigiGait). Postoperative testing was performed on POD 1, POD 3, and POD 6 for the same measures. SAS 9.4 was used for comparative analysis. RESULTS: Fifteen sham mice, 15 TBI mice, and 10 TBI with doxycycline mice were studied. Mice treated with doxycycline had significantly improved modified neurological severity score and wire grip scores at POD 1 (all p < 0.05). Mice treated with doxycycline had significantly improved ataxia scores by POD 3 and POD 6 (all p < 0.05). There was no significant difference in rate of weight change between the three groups. CONCLUSION: Mice treated with doxycycline following TBI demonstrated improved behavioral and motor function suggesting doxycycline's role in preserving murine BBB integrity. Examining the role of doxycycline in human TBIs is warranted given the relative universal accessibility, affordability, and safety profile of doxycycline.

Vascular leak in a rat model of preeclampsia.

BACKGROUND/AIMS: Preeclampsia is a hypertensive disorder which develops de novo in women during pregnancy. The urinary excretion of the cardiotonic steroid, marinobufagenin (MBG), is increased prior to the development of hypertension. Preeclamptic patients are volume expanded but much of the excess salt and water appears to be located primarily in the interstitial space. Therefore, 'capillary leak' syndrome has been postulated in this disorder. METHODS: We evaluated the vascular leakage in normal rats following MBG injection and in a rat model of human preeclampsia. We measured the changes in light intensity comparing that in the intravascular to the extravascular space by assessing 'leak' of fluorescein-labeled albumin (FITC-albumin) from mesenteric postcapillary venules. RESULTS: FITC-albumin extravasation continued to increase in a time-dependent fashion after MBG infusion and was significant (p < 0.05) at 60 min of observation when compared to sham rats. We also observed a significant difference in 'vascular leakage' in preeclamptic rats compared to control non-pregnant and normal pregnant groups starting at 20 min after the FITC-albumin infusion. CONCLUSION: We propose that MBG is involved in the production of a 'vascular leak' in our rat model of preeclampsia.

Cyclosporine A prevents vascular hyperpermeability after hemorrhagic shock by inhibiting apoptotic signaling.

BACKGROUND: Hemorrhagic shock (HS) is associated with the activation of caspase-dependent or -independent apoptotic signaling pathways, disruption of endothelial cell adherens junctions, and vascular hyperpermeability. Recent studies have suggested that the vascular hyperpermeability observed after HS is associated with activation of the intrinsic apoptotic signaling cascade resulting in caspase-mediated cleavage of endothelial cell adherens proteins and subsequent cell-cell detachment. We hypothesized that cyclosporine A (CsA) would attenuate vascular hyperpermeability after HS by protecting mitochondrial transition pores and thereby preventing the activation of caspase-mediated apoptotic signaling. The objective of this study was to determine the effect of CsA on, HS-induced hyperpermeability, mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and caspase 3 activation. METHODS: HS was induced in Sprague-Dawley rats by withdrawing blood to reduce the mean arterial pressure to 40 mm Hg for 60 minutes. CsA (10 microL/mL) was given 10 minutes before the shock period. The mesenteric postcapillary venules of the proximal ileum were monitored for permeability changes using intravital microscopy. The changes in mitochondrial transmembrane potential were determined using the cationic dye JC-1. Mitochondrial release of cytochrome c in to the cytosol was detected using ELISA. Caspase-3 activity was measured using a fluorometric assay. RESULTS: HS induced vascular hyperpermeability, release of cytochrome c, and activation of caspase-3 (p < 0.05). CsA (10 microL/mL) attenuated HS-induced hyperpermeability (p < 0.05) and prevented HS-induced decrease in mitochondrial transmembrane potential. CsA treatment decreased the HS-induced rise in cytosolic cytochrome c levels and caspase-3 activity (p < 0.05). CONCLUSIONS: These findings demonstrate that CsA protects mitochondrial permeability transition pores to prevent HS-induced release of cytochrome c and caspase-3 activation.

Centella asiatica extract selectively decreases amyloid beta levels in hippocampus of Alzheimer's disease animal model.

PSAPP mice expressing the 'Swedish' amyloid precursor protein and the M146L presenilin 1 mutations are a well-characterized model for spontaneous amyloid beta plaque formation. Centella asiatica has a long history of use in India as a memory enhancing drug in Ayurvedic literature. The study investigated whether Centella asiatica extract (CaE) can alter the amyloid pathology in PSAPP mice by administering CaE (2.5 or 5.0 g/kg/day) starting at 2 months of age prior to the onset of detectable amyloid deposition and continued for either 2 months or 8 months. A significant decrease in amyloid beta 1-40 and 1-42 was detectable by ELISA following an 8 month treatment with 2.5 mg/kg of CaE. A reduction in Congo Red stained fibrillar amyloid plaques was detected with the 5.0 mg/kg CaE dose and long-term treatment regimen. It was also confirmed that CaE functions as an antioxidant in vitro, scavenging free radicals, reducing lipid peroxidation and protecting against DNA damage. The data indicate that CaE can impact the amyloid cascade altering amyloid beta pathology in the brains of PSAPP mice and modulating components of the oxidative stress response that has been implicated in the neurodegenerative changes that occur with Alzheimer's disease.