Decreased readmission rates following use of modified trauma-specific frailty index in older trauma patients: A follow-up study.

INTRODUCTION: Post-discharge readmission rates using modified Trauma-Specific Frailty Index (mTSFI) compared to the Emergency Severity Index (ESI) are unknown. In our pilot study, we demonstrated that mTSFI usage more accurately triages older trauma patients. In the current study, we hypothesized that adult trauma patients triaged using mTSFI would have lower readmission rates at the 30-day interval post discharge. METHODS: Retrospective review of readmission rates for 96 trauma patients ≥ 50 years old was performed. The two study groups were categorized as mTSFI-concordant and ESI-concordant. Fisher's exact test was performed. RESULTS: Mean ages for ESI and mTSFI groups were 63.8 (SD 10.6) and 65.2 (SD 10.8) years. The 30-day readmission rate was 0% (0/32) in the mTSFI group vs 11% (7/64) in the ESI group (p = 0.104). CONCLUSIONS: Utilization of mTSFI for adult trauma patients may lead to lower 30-day readmission rates compared to using ESI, despite our sample sizes being too small to demonstrate a statistically significant difference.

A Comparative Analysis of Hospital Triage Systems in the Geriatric Adult Trauma Patients: A Quality Improvement Pilot Study.

BACKGROUND: The objective of our study is to compare the predicted hospital admission disposition based on the level of risk as determined by the modified Trauma-Specific Frailty Index (mTSFI) score with those determined by arbitrary decisions made based on the Emergency Severity Index (ESI) severity level. METHODS: We surveyed 100 trauma patients ages 50 and older, admitted to a level 1 trauma center between April 2019 and July 2019. We retrospectively reviewed the hospital admission disposition of each patient under the ESI, which was then compared to the mTSFI-predicted hospital admission disposition. The mTSFI scores were calculated by surveying each patient. Statistical analysis was performed to identify any statistical significance of concordance and discordance when comparing the mTSFI and ESI. RESULTS: The average age was 57.6 ± 4.2 years old in the non-geriatric group vs 76.3 ± 7.3 years old in the geriatric group. There was a male predominance in both groups (61% vs 69.5%). The mTSFI identified a higher percentage of triage discordance in the non-geriatric group (73%) compared to the geriatric cohort (53%) (95% difference CI, [39.6-40], P = .05). DISCUSSION: Non-geriatric patients have higher recorded rate of frailty than previously recognized and screening should begin at age 50, not 65. The mTSFI may be an effective tool to appropriately triage adult trauma patients at increased risk due to frailty and may reduce in-hospital complications.

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.

Unassisted N-acetyl-phenylalanine-amide transport across membrane with varying lipid size and composition: kinetic measurements and atomistic molecular dynamics simulation.

Biological membranes are essential to preserve structural integrity and regulate functional properties through the permeability of nutrients, pharmaceutical drugs, and neurotransmitters of a living cell. The movement of acetylated and amidated phenylalanine (NAFA) across 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane bilayers is investigated to probe physical transport. The rate of transport is measured experimentally applying parallel artificial membrane permeation assay (PAMPA). At the physiological temperature, 310 K, the measured time constants in the neutral pH were ∼6 h in DOPC and ∼3 h in POPC, while in a more acidic condition, at a pH 4.8, the time constants were ∼8 h in both lipids. Computationally, we have expanded our transport study of three aromatic dipeptides across a bilayer composed of DOPC18. In this study, we have examined the effects of lipid composition and bilayer size on the passive transport of NAFA by simulating the dipeptide in three different bilayers, with 50 DOPC lipids, 50 POPC lipids, and 40 POPC molecules. Specifically, atomistic molecular dynamics simulations with umbrella sampling were used to calculate the potential of mean force for the passive permeation of NAFA across the bilayers. Diffusion constants were then calculated by numerically solving the Smoluchowski equation. Permeability coefficients and mean first passage times were then calculated. Structural properties - Ramachandran plots, sidechain torsions, peptide insertion angles, radial distribution functions, and proximal peptide water molecules - were also examined to determine the effect of system size and lipid type. In terms of systems size, we observed a small decrease in the highest barrier of the potential of mean force and fewer sampled sidechain dihedral angle conformations with 40 versus 50 POPC lipids due to weaker membrane deformations within a smaller lipid bilayer. In terms of lipid type, DOPC contains two monounsaturated acyl chains compared to only one such acyl chain in POPC; therefore, DOPC bilayers are less ordered and more easily deformed, as seen by a much broader potential of mean force profile. The NAFA in DOPC lipid also transitioned to an internally hydrogen-bonded backbone conformation at lower membrane depths than in POPC. Similarly, as for other aromatic dipeptides, NAFA tends to insert into the membrane sidechain-first, remains mostly desolvated in the membrane center, and exhibits slow reorientations within the bilayer in both DOPC and POPC. With a joint experimental and computational study we have gained a new insight into the rate of transport and the underlying microscopic mechanism in different lipid bilayer conditions of the simplest hydrophobic aromatic dipeptide.Communicated by Ramaswamy H. Sarma.

Observing reorientation dynamics with Time-Resolved fluorescence and molecular dynamics in varying periodic boundary conditions.

This work presents a combined study of time-resolved fluorescence spectroscopy and all-atom molecular dynamics simulation to investigate periodic boundary conditions' and water models' influence on the orientation dynamics and translational and rotational diffusion of peptides in solution. We have characterized the effects of solvent box size and water model choice on the dynamics of two peptide systems, NATA and WK5. Computationally, translational, and rotational diffusion and internal fluctuations are investigated through all-atom molecular dynamics simulation with two water models and different box sizes. These results are compared with time-resolved fluorescence anisotropy decay (FAD) measurements. The associated time constant and orientation dynamics from FAD measurement along the 1Lb axis provided baseline data to validate molecular dynamics simulation. The modeling results show that diffusion rates vary roughly in inverse proportion to water model viscosity, as one would expect. Corrections for finite box size are significant for translational diffusion and insignificant for rotational diffusion. This study also finds that internal dynamics described by autocorrelation functions and kinetic network models are relatively insensitive to both box size and water model properties. Our observation suggests that different peptide properties respond differently to a change in simulation conditions.Communicated by Ramaswamy H. Sarma.

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.

Probing the Internal Dynamics and Shape of Simple Peptides in Urea, Guanidinium Hydrochloride, and Proline Solutions with Time-Resolved Fluorescence Anisotropy and Atomistic Cosolvent Simulations.

Picosecond time-resolved fluorescence anisotropy was used to measure the effect of denaturants and osmolytes on the reorientation dynamics of the simplest dipeptide. The solvent denaturants guanidinium hydrochloride (gdm), urea, and the osmolyte proline were used at several concentrations. Analysis of the concentration dependence of denaturants at a fixed temperature showed faster and slower reorientation time in two different denaturants at a nearly identical solvent viscosity (η). The reorientation time τ significantly deviates from Kramers' theory (τ ∝ η1) in the high friction limit for guanidinium and urea with r ≈ 0.4 and r ≈ 0.6 at pH 7.2, respectively. In proline, τ is nearly proportional to η. Atomistic molecular dynamics simulations of the dipeptide in identical cosolvents showed excellent agreement with the measured rotational orientation time. The dipeptide dihedral (ϕ, ψ) isomerization times in water and 6 M urea are almost identical and significantly slower in guanidinium. If a faster and slower reorientation time can be associated with the compact and expanded shapes, the fractional viscosity dependence for guanidinium and urea may result from the fact that internal dynamics of peptides in these cosolvents involve higher and lower internal friction within the dynamic elements.

Dissecting Multiple Pathways in the Relaxation Dynamics of Helix <==> Coil Transitions with Optimum Dimensionality Reduction.

Fast kinetic experiments with dramatically improved time resolution have contributed significantly to understanding the fundamental processes in protein folding pathways involving the formation of a-helices and b-hairpin, contact formation, and overall collapse of the peptide chain. Interpretation of experimental results through application of a simple statistical mechanical model was key to this understanding. Atomistic description of all events observed in the experimental findings was challenging. Recent advancements in theory, more sophisticated algorithms, and a true long-term trajectory made way for an atomically detailed description of kinetics, examining folding pathways, validating experimental results, and reporting new findings for a wide range of molecular processes in biophysical chemistry. This review describes how optimum dimensionality reduction theory can construct a simplified coarse-grained model with low dimensionality involving a kinetic matrix that captures novel insights into folding pathways. A set of metastable states derived from molecular dynamics analysis generate an optimally reduced dimensionality rate matrix following transition pathway analysis. Analysis of the actual long-term simulation trajectory extracts a relaxation time directly comparable to the experimental results and confirms the validity of the combined approach. The application of the theory is discussed and illustrated using several examples of helix <==> coil transition pathways. This paper focuses primarily on a combined approach of time-resolved experiments and long-term molecular dynamics simulation from our ongoing work.

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.

Kinetic pathway analysis of an α-helix in two protonation states: Direct observation and optimal dimensionality reduction.

Thermodynamically stable conformers of secondary structural elements make a stable tertiary/quaternary structure that performs its proper biological function efficiently. Formation mechanisms of secondary and tertiary/quaternary structural elements from the primary structure are driven by the kinetic properties of the respective systems. Here we have carried out thermodynamic and kinetic characterization of an alpha helical heteropeptide in two protonation states, created with the addition and removal of a proton involving a single histidine residue in the primary structure. Applying far-UV circular dichroism spectroscopy, the alpha helix is observed to be significantly more stable in the deprotonated state. Nanosecond laser temperature jump spectroscopy monitoring time-resolved tryptophan fluorescence on the protonated conformer is carried out to measure the kinetics of this system. The measured relaxation rates at a final temperature between 296K and 314 K generated a faster component of 20 ns-11 ns and a slower component of 314 ns-198 ns. Atomically detailed characterization of the helix-coil kinetic pathways is performed based on all-atom molecular dynamics trajectories of the two conformers. Application of clustering and kinetic coarse-graining with optimum dimensionality reduction produced description of the trajectories in terms of kinetic models with two to five states. These models include aggregate states corresponding to helix, coil, and intermediates. The "coil" state involves the largest number of conformations, consistent with the expected high entropy of this structural ensemble. The "helix" aggregate states are found to be mixed with the full helix and partially folded forms. The experimentally observed higher helix stability in the deprotonated form of the alpha helical heteropeptide is reflected in the nature of the "helix" aggregate state arising from the kinetic model. In the protonated form, the "coil" state exhibits the lowest free energy and longest lifetime, while in the deprotonated form, it is the "helix" that is found to be most stable. Overall, the coarse grained models suggest that the protonation of a single histidine residue in the primary structure induces significant changes in the free energy landscape and kinetic network of the studied helix-forming heteropeptide.

A Rat Model of Hemorrhagic Shock for Studying Vascular Hyperpermeability.

Vascular hyperpermeability is one of the known detrimental effects of hemorrhagic shock, which we continually try to understand, minimize, and reverse. Here, we describe induction of hemorrhagic shock in a rat and studying of its effects on vascular permeability, using intravital microscopy. In this protocol, hemorrhagic shock will be induced by withdrawing blood to reduce the mean arterial pressure (MAP) to 40 mmHg for 60 min followed by resuscitation for 60 min. To study the changes in vascular permeability following hemorrhagic shock, the rats will be given FITC-albumin, a fluorescent tracer, intravenously. Following this, the FITC-albumin flux across the vessel will be measured in mesenteric postcapillary venules by determining fluorescent intensity intravascularly and extravascularly under intravital microscopy.

Vitamin D3 Suppresses Class II Invariant Chain Peptide Expression on Activated B-Lymphocytes: A Plausible Mechanism for Downregulation of Acute Inflammatory Conditions.

Class II invariant chain peptide (CLIP) expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP(+) B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI) based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol) had the most dramatic reduction in CLIP density at an MFI of 257.3 [baseline of 701.1 (P value = 0.01)]. Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings.

Glycogen synthase kinase 3 inhibitor protects against microvascular hyperpermeability following hemorrhagic shock.

BACKGROUND: Hemorrhagic shock (HS)-induced microvascular hyperpermeability involves disruption of endothelial cell adherens junctions leading to increase in paracellular permeability. ß-Catenin, an integral component of the adherens junctional complex and Wnt pathway, and caspase 3 via its apoptotic signaling regulate endothelial cell barrier integrity. We have hypothesized that inhibiting phosphorylation of ß-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 would attenuate microvascular hyperpermeability following HS. METHODS: In Sprague-Dawley rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mm Hg for 60 minutes followed by resuscitation. Rats were given SB216763 (600 µg/kg) intravenously 10 minutes before shock. To study microvascular permeability, the rats were intravenously injected with fluorescein isothiocyanate (FITC)-albumin (50 mg/kg), and its flux across the mesenteric postcapillary venules was determined using intravital microscopy. In cell culture studies, rat lung microvascular endothelial cell monolayers grown on Transwell plates were pretreated with SB216763 (5 µM) followed by BAK (5 µg/mL) and caspase 3 (5 µg/mL) protein transfection. FITC-albumin (5 mg/mL) flux across cell monolayers indicates change in monolayer permeability. Activity of canonical Wnt pathway was determined by luciferase assay. Caspase 3 enzyme activity was assayed fluorometrically. RESULTS: The HS group showed significant increase in FITC-albumin extravasation (p < 0.05) compared with sham. SB216763 significantly decrease HS-induced FITC-albumin extravasation (p < 0.05). Pretreatment with SB216763 protected against a BAK-induced increase in rat lung microvascular endothelial cell monolayer permeability and caspase 3 activity but failed to show similar results with a caspase 3-induced increase in monolayer permeability. Wnt3a treatment showed an increase in ß-catenin-dependent T-cell factor-mediated transcription. CONCLUSION: Inhibiting phosphorylation of ß-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 help regulates HS-induced microvascular hyperpermeability.

Collecting lymphatic vessel permeability facilitates adipose tissue inflammation and distribution of antigen to lymph node-homing adipose tissue dendritic cells.

Collecting lymphatic vessels (CLVs), surrounded by fat and endowed with contractile muscle and valves, transport lymph from tissues after it is absorbed into lymphatic capillaries. CLVs are not known to participate in immune responses. In this study, we observed that the inherent permeability of CLVs allowed broad distribution of lymph components within surrounding fat for uptake by adjacent macrophages and dendritic cells (DCs) that actively interacted with CLVs. Endocytosis of lymph-derived Ags by these cells supported recall T cell responses in the fat and also generated Ag-bearing DCs for emigration into adjacent lymph nodes (LNs). Enhanced recruitment of DCs to inflammation-reactive LNs significantly relied on adipose tissue DCs to maintain sufficient numbers of Ag-bearing DCs as the LN expanded. Thus, CLVs coordinate inflammation and immunity within adipose depots and foster the generation of an unexpected pool of APCs for Ag transport into the adjacent LN.

RECOMBINANT BCL-XL ATTENUATES VASCULAR HYPERPERMEABILITY IN A RAT MODEL OF HEMORRHAGIC SHOCK.

Following hemorrhagic shock (HS), vascular hyperpermeability i.e. the leakage of fluid, nutrients and proteins into the extravascular space occurs primarily due to the disruption of the endothelial cell-cell adherens junctional complex. Studies from our laboratory demonstrate that activation of the mitochondria mediated 'intrinsic' apoptotic signaling cascade has a significant role in modulating HS-induced hyperpermeability. Here we report the novel use of recombinant Bcl-xL, an anti-apoptotic protein, to control HS-induced vascular hyperpermeability. Our results corroborate involvement of vascular hyperpermeability and apoptotic signaling. Hemorrhagic shock (HS) (mean arterial pressure [MAP] was reduced to 40 mmHg for 60 minutes followed by resuscitation to 90 mmHg for 60 minutes) in rats resulted in vascular hyperpermeability as determined by intra-vital microscopy. Treatment of Bcl-xL (2.5ug/ml of rat blood in non-lipid cationic polymer, i.v.) before, during and even after HS attenuated or reversed HS-induced vascular hyperpermeability significantly (p<0.05). Conversely, treatment using Bcl-xL inhibitors, 2-methoxy antimycin (2-MeOAA) and ABT 737, significantly increased vascular hyperpermeability compared to sham (p<0.05). Bcl-xL treatment also decreased the amount of fluid volume required to maintain a MAP of 90 mmHg during resuscitation (p<0.05). HS resulted in increased mitochondrial ROS formation, reduction of ΔΨm, mitochondrial release of cytochrome c and significant activation of caspase-3 (p<0.05). All of these effects were significantly inhibited by Bcl-xL pre-treatment (p<0.05). Our results show that recombinant Bcl-xL is effective against HS-induced vascular hyperpermeability that appears to be mediated through preservation of ΔΨm and subsequent prevention of caspase-3 activation.

Tumor necrosis factor-alpha-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling

Tumor necrosis factor-alfa (TNF-alfa), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-alfa induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-alfa (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 μM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using β-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 μM) attenuated TNF-alfa-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-alfa (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability

Tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling.

Tumor necrosis factor-α (TNF-α), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-α induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-α (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 µM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using ß-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 µM) attenuated TNF-α-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-α (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability.

The role of intrinsic apoptotic signaling in hemorrhagic shock-induced microvascular endothelial cell barrier dysfunction.

Hemorrhagic shock leads to endothelial cell barrier dysfunction resulting in microvascular hyperpermeability. Hemorrhagic shock-induced microvascular hyperpermeability is associated with worse clinical outcomes in patients with traumatic injuries. The results from our laboratory have illustrated a possible pathophysiological mechanism showing involvement of mitochondria-mediated "intrinsic" apoptotic signaling in regulating hemorrhagic shock-induced microvascular hyperpermeability. Hemorrhagic shock results in overexpression of Bcl-2 family of pro-apoptotic protein, BAK, in the microvascular endothelial cells. The increase in BAK initiates "intrinsic" apoptotic signaling cascade with the release of mitochondrial cytochrome c in the cytoplasm and activation of downstream effector caspase-3, leading to loss of endothelial cell barrier integrity. Thus, this review article offers a brief overview of important findings from our past and present research work along with new leads for future research. The summary of our research work will provide information leading to different avenues in developing novel strategies against microvascular hyperpermeability following hemorrhagic shock.

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.

Doxycycline attenuates burn-induced microvascular hyperpermeability.

BACKGROUND: Burns induce systemic microvascular hyperpermeability resulting in shock, and if untreated, cardiovascular collapse. Damage to the endothelial cell adherens junctional complex plays an integral role in the pathophysiology of microvascular hyperpermeability. We hypothesized that doxycycline, a known inhibitor of matrix metalloproteinases (MMPs), could attenuate burn-induced adherens junction damage and microvascular hyperpermeability. METHODS: Male Sprague-Dawley rats were divided into sham, burn, and burn + doxycycline (n = 5). The experimental groups underwent a 30% total body surface area full-thickness burn. Fluorescein isothiocyanate-albumin was administered intravenously. Mesenteric postcapillary venules were examined with intravital microscopy to determine flux of albumin from the intravascular space to the interstitium. Fluorescence intensity was compared between the intravascular space to the interstitium at 30, 60, 80, 100, 120, 140, 160, and 180 minutes after burn. Parallel experiments were performed in which rat lung microvascular endothelial cells were treated with sera from sham or burn animals as well as separate groups pretreated with either doxycycline or a specific inhibitor of MMP-9. Monolayer permeability was determined by fluorescein isothiocyanate albumin-flux across Transwell plates and immunofluorescense staining for the adherens junction protein ß-catenin was performed. Western blot and gelatin zymography were performed to assess MMP-9 level and activity. RESULTS: MMP-9 levels were increased after burn. Monolayer permeability was significantly increased with burn serum treatment; this was attenuated with doxycycline as well as the specific MMP-9 inhibitor (p < 0.05). Damage of the endothelial cell adherens junction complex was induced by serum from burned rats, and doxycycline restored the integrity of the adherens junction similar to the MMP-9 inhibitor. Intravital microscopy revealed microvascular hyperpermeability after burn; this was attenuated with doxycycline (p < 0.05). CONCLUSION: Burns induce microvascular hyperpermeability via endothelial adherens junction disruption associated with MMP-9, and this is attenuated with doxycycline.