Dynamics of Gill Responses to a Natural Infection with Neoparamoeba perurans in Farmed Tasmanian Atlantic Salmon.

Gill health has become a significant global challenge for Atlantic salmon (Salmo salar) aquaculture, particularly during the marine phase of farming. The increasing prevalence of gill pathologies has been linked to rising seawater temperatures, underscoring the need to evaluate existing tools for monitoring gill health and to develop novel approaches for early detection. In this study, we investigated the gill responses of commercially farmed Atlantic salmon to natural infection with Neoparamoeba perurans during an outbreak of amoebic gill disease (AGD) in Tasmania. Our focus spanned the low AGD prevalence, high AGD prevalence, and post-freshwater treatment stages of the outbreak. Evaluations of gill tissue included assessments of the gross AGD score, histopathological score, abundance of N. perurans (measured by 18S rRNA gene expression), and expression levels of inflammation-related transcripts. We demonstrated a strong correlation between different measures of AGD-related gill pathology and significant differences between distinct stages of the N. perurans outbreak. Post-treatment, fish exhibited considerable variability in their responses to the freshwater bath, highlighting the necessity for personalized management strategies that consider genetic, environmental, and health status factors. The expression patterns of angiogenin-1 (ANG1) and complement C1q tumour necrosis factor-related protein 3-like (C1QTNF3) emphasize their potential as biomarkers for early detection of gill damage in salmon aquaculture worldwide.

Synbiotic feed supplementation significantly improves lipid utilization and shows discrete effects on disease resistance in rainbow trout (Oncorhynchus mykiss).

Enteric redmouth disease caused by the bacterial pathogen Yersinia ruckeri is the main reason for antimicrobial prescription, and a cause of substantial economic losses and decreased animal welfare in aquaculture. Given the importance of the intestinal microbiota in digestion and disease, our aim was to investigate whether synbiotic feed supplementation strategies could improve feed performance and disease resistance. Four experimental synbiotic feeds formulated with pre- and probiotics were tested against a commercially available probiotic control feed. Each experimental feed was evaluated for feed performance, effects on gross as well as intestinal morphometrics, and finally their effect on resistance against a waterborne experimental infection with Yersinia ruckeri serotype O1, biotype 2. While co-supplementing Pediococcus acidilactici with citrus flavonoids or bacterial paraprobiotics significantly improved utilization of feed lipid content relative to the control group, a decrease in lipid utilization was observed for feeds that combined P. acidilactici with yeast paraprobiotics. No significant improvements on disease resistance were observed. Still, synbiotic formulations including P. acidilactici led to reduced risks relative to that of the control group, while an increased relative risk was observed for a Bacillus-based formulation. In conclusion, two of the synbiotic supplements significantly improved lipid utilization and contributed to minor increases in disease resistance.

Citrus flavonoids, ß-Glucan and organic acid feed additives decrease relative risk during Yersinia ruckeri O1 biotype 2 infection of rainbow trout (Oncorhynchus mykiss).

Whether through direct supplementation of bacteria or by prebiotic supplementation thought to favour subsets of bacteria, modulation of gut microbiota constitutes an important and promising alternative to the use of prophylactic and growth promoting antibiotics in worldwide aquaculture. We fed a commercial base feed, alone or supplemented with either proprietary ß-glucan, ß-glucan and organic acids, citrus flavonoid or yeast cell wall supplements, to rainbow trout over a period of four weeks. Fish from each feed group were then subjected to experimental, waterborne infection with Yersinia ruckeri O1 biotype 2. Following experimental feeding, the ß-glucan and organic acids supplemented group showed significantly improved feed conversion and lipid efficiency ratios. Furthermore, the ß-glucan, ß-glucan and organic acids and citrus flavonoid supplements proved to significantly reduce the risk of mortality in rainbow trout during experimental infection as shown by hazard ratio analysis. Resulting in 33.2%, 30.6% and 30.5% reduction in risk relative to the non-supplemented base feed, respectively, these three supplements show a promising potential either as stand-alone feed supplements, or as components in complex feed formulations.

Influence of Dietary Supplementation of Probiotic Pediococcus acidilactici MA18/5M During the Transition From Freshwater to Seawater on Intestinal Health and Microbiota of Atlantic Salmon (Salmo salar L.).

The aim of this study was to assess the effect of the transfer from freshwater to seawater on the distal intestinal bacterial communities of Atlantic salmon (Salmo salar L.) and to evaluate the effect of dietary inclusion of Pediococcus acidilactici MA18/5M (at 1.19 × 106 CFU/g). In this context, fish health and antiviral response were also investigated. A 12-week feeding trial was conducted in a flow-through rearing system involving 6 weeks in freshwater and 6 weeks in seawater. Fish received a control and probiotic diet. The composition of the salmon gut bacterial communities was determined by high-throughput sequencing of digesta and mucosa samples from both the freshwater and seawater stage. The main phyla detected during both freshwater and seawater stages were Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria. Significant differences were observed between the intestinal microbiota in the digesta and the mucosa. Both probiotic supplementation and the seawater transfer (SWT) had a substantial impact on the microbial communities, with most pronounced changes detected in the mucosal communities after SWT. This last finding together with a significantly higher antiviral response (mx-1 and tlr3 gene expression) in the distal intestine of fish fed the probiotic diet suggest a causal link between the microbiota modulation and activation of antiviral response. Feeding probiotics during the freshwater stage did not significantly increase survival after infectious pancreatic necrosis virus (IPNV) challenge after SWT, although higher survival was observed in one out of two replicate challenge tanks. In conclusion, this study demonstrated that both dietary probiotic supplementation and transfer from freshwater to seawater have an important role in modulating the bacterial communities in the distal intestine of Atlantic salmon. Furthermore, supplementation of the diet with P. acidilactici MA18/5M can modulate antiviral response.

The compositional and metabolic responses of gilthead seabream (Sparus aurata) to a gradient of dietary fish oil and associated n-3 long-chain PUFA content.

The replacement of fish oil (FO) with vegetable oil (VO) in feed formulations reduces the availability of n-3 long-chain PUFA (LC-PUFA) to marine fish such as gilthead seabream. The aim of this study was to examine compositional and physiological responses to a dietary gradient of n-3 LC-PUFA. Six iso-energetic and iso-nitrogenous diets (D1-D6) were fed to seabream, with the added oil being a blend of FO and VO to achieve a dietary gradient of n-3 LC-PUFA. Fish were sampled after 4 months feeding, to determine biochemical composition, tissue fatty acid concentrations and lipid metabolic gene expression. The results indicated a disturbance to lipid metabolism, with fat in the liver increased and fat deposits in the viscera reduced. Tissue fatty acid profiles were altered towards the fatty acid compositions of the diets. There was evidence of endogenous modification of dietary PUFA in the liver which correlated with the expression of fatty acid desaturase 2 (fads2). Expression of sterol regulatory element binding protein 1 (srebp1), fads2 and fatty acid synthase increased in the liver, whereas PPARα1 pathways appeared to be supressed by dietary VO in a concentration-dependent manner. The effects in lipogenic genes appear to become measurable in D1-D3, which agrees with the weight gain data suggesting that disturbances to energy metabolism and lipogenesis may be related to performance differences. These findings suggested that suppression of ß-oxidation and stimulation of srebp1-mediated lipogenesis may play a role in contributing toward steatosis in fish fed n-3 LC-PUFA deficient diets.

Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein.

In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

Alternative Protein Sources in the Diet Modulate Microbiota and Functionality in the Distal Intestine of Atlantic Salmon (Salmo salar).

The present study aimed to investigate whether alternative dietary protein sources modulate the microbial communities in the distal intestine (DI) of Atlantic salmon, and whether alterations in microbiota profiles are reflected in modifications in host intestinal function and health status. A 48-day feeding trial was conducted, in which groups of fish received one of five diets: a reference diet in which fishmeal (diet FM) was the only protein source and four experimental diets with commercially relevant compositions containing alternative ingredients as partial replacements of fishmeal, i.e., poultry meal (diet PM), a mix of soybean meal and wheat gluten (diet SBMWG), a mix of soy protein concentrate and poultry meal (diet SPCPM), and guar meal and wheat gluten (diet GMWG). Samples were taken of DI digesta and mucosa for microbial profiling using high-throughput sequencing and from DI whole tissue for immunohistochemistry and expression profiling of marker genes for gut health. Regardless of diet, there were significant differences between the microbial populations in the digesta and the mucosa in the salmon DI. Microbial richness was higher in the digesta than the mucosa. The digesta-associated bacterial communities were more affected by the diet than the mucosa-associated microbiota. Interestingly, both legume-based diets (SBMWG and GMWG) presented high relative abundance of lactic acid bacteria in addition to alteration in the expression of a salmon gene related to cell proliferation (pcna). It was, however, not possible to ascertain the cause-effect relationship between changes in bacterial communities and the host's intestinal responses to the diets.IMPORTANCE The intestine of cultivated Atlantic salmon shows symptoms of compromised function, which are most likely caused by imbalances related to the use of new feed ingredients. Intestinal microbiota profiling may become in the future a valuable endpoint measurement in order to assess fish intestinal health status and effects of diet. The present study aimed to gain information about whether alternative dietary protein sources modulate the microbial communities in the Atlantic salmon intestine and whether alterations in microbiota profiles are reflected in alterations in host intestinal function and health status. We demonstrate here that there are substantial differences between the intestinal digesta and mucosa in the presence and abundance of bacteria. The digesta-associated microbiota showed clear dependence on the diet composition, whereas mucosa-associated microbiota appeared to be less affected by diet composition. Most important, the study identified bacterial groups associated with diet-induced gut dysfunction that may be utilized as microbial markers of gut health status in fish.

Intestinal Fluid Permeability in Atlantic Salmon (Salmo salar L.) Is Affected by Dietary Protein Source.

In Atlantic salmon (Salmo salar L.), and also in other fish species, certain plant protein ingredients can increase fecal water content creating a diarrhea-like condition which may impair gut function and reduce fish growth. The present study aimed to strengthen understanding of the underlying mechanisms by observing effects of various alternative plant protein sources when replacing fish meal on expression of genes encoding proteins playing key roles in regulation of water transport across the mucosa of the distal intestine (DI). A 48-day feeding trial was conducted with five diets: A reference diet (FM) in which fish meal (72%) was the only protein source; Diet SBMWG with a mix of soybean meal (30%) and wheat gluten (22%); Diet SPCPM with a mix of soy protein concentrate (30%) and poultry meal (6%); Diet GMWG with guar meal (30%) and wheat gluten (14.5%); Diet PM with 58% poultry meal. Compared to fish fed the FM reference diet, fish fed the soybean meal containing diet (SBMWG) showed signs of enteritis in the DI, increased fecal water content of DI chyme and higher plasma osmolality. Altered DI expression of a battery of genes encoding aquaporins, ion transporters, tight junction and adherens junction proteins suggested reduced transcellular transport of water as well as a tightening of the junction barrier in fish fed the SBMWG diet, which may explain the observed higher fecal water content and plasma osmolality. DI structure was not altered for fish fed the other experimental diets but alterations in target gene expression and fecal water content were observed, indicating that alterations in water transport components may take place without clear effects on intestinal structure.

Regulatory factors controlling muscle mass: Competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon.

Atrogin-1 is a conserved ubiquitin E3 ligase that is central to the early stages of skeletal and cardiac muscle wasting and degradation following starvation and inflammatory diseases. The control of protein turnover is different between endothermic and ectothermic animals reflecting the body energy requirements. Here we have characterised the promoter of the atrogin-1 gene in a phylogenetically diverse group of vertebrates and show conserved FOXO elements are present in all species examined. We have examined the gene expression responses in primary muscle cell culture to key immune modulators (IL-1ß, interferon type 1 and interferon γ) and to the anabolic hormone insulin like growth factor (IGF-1). We show that the IL-1ß and interferon type 1 increased atrogin-1 mRNA expression whereas IGF-1 suppressed atrogin-1 expression. The proximal promoter of salmon atrogin-1 was used to transfect primary muscle cell cultures and we found all three cytokines increased promoter activity whereas there was a decrease caused by IGF-1 exposure. We hypothesise that the main drivers for atrogin-1 expression are via the conserved FOXO site, but other transcription binding sites such as NFκB, STAT and IRFs may also be involved in a synergistic manner following immune stimulation when free amino acids need to be released for muscle protein reserves.

Antiviral and metabolic gene expression responses to viral infection in Atlantic salmon (Salmo salar).

Salmonid alphavirus (SAV), the aetiological agent of pancreas disease, is recognized as a serious pathogen of farmed Atlantic salmon. This disease results in loss of weight followed by poor growth of surviving fish, as such it is viewed as a wasting disease. SAV and other chronic disease causing viruses affect the heart and skeletal muscle tissues, at present the mechanisms by which pathology occurs is unknown. The relationship between antiviral activity and other physiological parameters especially in skeletal muscle are currently not examined in depth in fish. An experimental SAV (isotype 3) infection was carried out using a cohabitation approach, from which samples were collected at 0, 4, 8 & 12 week post challenge. Maximum viral load in the muscle tissue was 4 weeks post infection which was reduced at 8 weeks and undetectable by 12 weeks. Histopathology score peaked at 4 weeks post infection in pancreas and heart whereas there was maximum damage in skeletal muscle at 8 weeks. The peak expression of antiviral immune genes coincided with the viral load. Several genes involved in protein degradation were increased following infection including atrogin-1 and cathepsin D, at 4 weeks post challenge suggesting reallocation of amino acid reserves. Taken together, these observations increase our understanding of salmon poor growth during viral infection, and will serve as a basis to develop strategies to manage this viral wasting disease.

ß-Catenin dynamics in the regulation of microvascular endothelial cell hyperpermeability.

ß-Catenin, a key regulator of barrier integrity, is an important component of the adherens junctional complex. Although the roles of ß-catenin in maintaining the adherens junctions and Wnt signaling are known, the dynamics of ß-catenin following insult and its potential role in vascular recovery/repair remain unclear. Our objective was to define ß-catenin's dynamics following disruption of the adherens junctional complex and subsequent recovery. Rat lung microvascular endothelial cells were treated with active caspase 3 enzyme, by protein transference method, as an inducer of junctional damage and permeability. The disruption and subsequent recovery of ß-catenin to the adherens junctions were studied via immunofluorescence. Rat lung microvascular endothelial cell monolayers were used to measure hyperpermeability. To understand the role of ß-catenin on nuclear translocation/transcriptional regulation in relationship to the recovery of the adherens junctions, Tcf-mediated transcriptional activity was determined. Active caspase 3 induced a loss of ß-catenin at the adherens junctions at 1 and 2 h followed by its recovery at 3 h. Transference of Bak peptide, an inducer of endogenous caspase 3 activation, induced hyperpermeability at 1 h followed by a significant decrease at 2 h. Inhibition of GSK-3ß and the transfection of ß-catenin vector increased Tcf-mediated transcription significantly (P < 0.05). The dissociated adherens junctional protein ß-catenin translocates into the cytoplasm, resulting in microvascular hyperpermeability followed by a time-dependent recovery and relocation to the cell membrane. Our data suggest a recycling pathway for ß-catenin to the cell junction.

Interleukin-10 reduces inflammation, endothelial dysfunction, and blood pressure in hypertensive pregnant rats.

Hypertensive disorders of pregnancy are characterized by systemic and placental inflammation; however, treatment for these conditions has remained elusive. We tested whether administration of the anti-inflammatory cytokine interleukin-10 (IL-10) during pregnancy would attenuate the hypertension, endothelial dysfunction, proteinuria, and inflammation seen in pregnant DOCA/saline-treated (PDS) rats. Normal pregnant (NP) rats and PDS were given daily intraperitoneal injections of recombinant IL-10 from gestational day 13 until death on day 20. Systolic blood pressure, aortic endothelium-dependent relaxation responses, and urinary protein excretion were measured on days 13 and 20 of gestation. Fetal number and development, plasma endothelin-1 levels, serum and placental levels of IFNgamma and IL-10, and aortic and placental levels of platelet endothelial cell adhesion molecule (PECAM) were assessed on gestational day 20. Systolic blood pressure, aortic endothelial dysfunction, and urinary protein excretion were significantly increased at gestational day 13 in PDS rats. However, all of these were restored to NP levels following IL-10 treatment in PDS rats. IL-10 treatment also significantly increased the number of pups per litter in PDS rats and did not further affect fetal development. The beneficial effects of IL-10 in PDS rats were likely mediated by the decreased plasma levels of endothelin-1, decreased levels of circulating and placental IFNgamma, as well as decreased aortic and placental expression of PECAM. These data demonstrate that exogenous IL-10 can normalize blood pressure and endothelial function in pregnancy-induced hypertensive rats and may be beneficial in women with hypertensive disorders of pregnancy.

Toll-like receptor 3 activation during pregnancy elicits preeclampsia-like symptoms in rats.

BACKGROUND: Preeclampsia (PE), a pregnancy-specific hypertensive syndrome, is one of the leading causes of premature births as well as fetal and maternal death. There is strong evidence that maternal immune system activation, of which Toll-like receptors (TLRs) play a major role, contributes to the development of PE. Viral infections, sensed by TLR3, are associated with hypertensive disorders of pregnancy. We tested the hypothesis that TLR3 activation during pregnancy would cause hypertension, endothelial dysfunction, proteinuria, and intrauterine growth restriction in normal pregnant rats. METHODS: We treated pregnant and nonpregnant rats with the viral mimetic polyinosinic:polycytidylic acid (poly I:C) or vehicle every other day beginning at day 10 of gestation and measured systolic blood pressure, aortic vasodilation, urinary protein concentration, fetal growth, and serum and placental cytokine levels. RESULTS: Pregnant rats treated with poly I:C displayed significantly elevated systolic blood pressures compared to pregnant rats and nonpregnant rats treated with poly I:C on day 18 of gestation. Poly I:C-treated pregnant rats also exhibited significantly decreased aortic vasodilation, significantly increased urinary protein concentrations, and had more malformed pups/litter. Additionally, poly I:C-treated rats exhibited a significant increase in placental TLR3 expression and proinflammatory/anti-inflammatory cytokine ratio compared to vehicle-treated rats. Poly I:C treatment of nonpregnant control rats had no effect on systolic blood pressure, aortic vasodilation, or urinary protein concentrations. CONCLUSION: These findings demonstrate that sustained maternal immune system activation via TLR3 during pregnancy causes PE-like symptoms in rats and suggest that viral infection during pregnancy may contribute to the development of PE.

Immunosuppression improves blood pressure and endothelial function in a rat model of pregnancy-induced hypertension.

BACKGROUND: Hypertensive disorders of pregnancy, including preeclampsia (PE), affect approximately 7-10% of pregnancies in the US. Clinical and experimental studies strongly suggest that the maternal immune system plays a role in the development of these disorders; however, few therapeutic options exist aside from delivery. METHODS: Using a deoxycorticosterone acetate (DOCA)/salt-low renin rat model, which exhibits hypertension, proteinuria, endothelial dysfunction, and intrauterine growth restriction (IUGR), we measured serum cytokine levels as an indication of immune system activation. In addition, we suppressed the immune system with either azathioprine (Aza) or mycophenolate mofetil (MMF) during the second half of pregnancy to determine whether the these symptoms could be ameliorated. RESULTS: Our results demonstrate that serum T helper-1 (Th1)-type inflammatory cytokines interleukin (IL)-2, IL-12, interferon-gamma (IFNgamma), and RANTES were significantly elevated in hypertensive pregnant rats while the Th2-type cytokine IL-4 was elevated in normal pregnant animals. Either Aza or MMF significantly attenuated the hypertension, proteinuria, and endothelial dysfunction as well as the increased proinflammatory Th1 cytokine profile in pregnant rats treated with DOCA/salt, and had no effect on these parameters in normal pregnant rats. CONCLUSION: These data strongly suggest that maternal immune system activation plays a role in the development of pregnancy-induced hypertension (PIH).

PKC and MLCK-dependent, cytokine-induced rat coronary endothelial dysfunction.

BACKGROUND: Heart disease is one of the leading causes of death in the United States, killing nearly one million people every year. Inflammatory mediators or cytokines are released following myocardial infarction and ischemia/reperfusion injury. These cytokines, of which interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha (TNF-alpha) are among the most important, propagate the activation of a multitude of signaling pathways, such as the protein kinase C (PKC) and myosin light chain kinase (MLCK) pathways, which lead to deleterious changes in the structure and function of the coronary microvascular endothelium. METHODS: The effects of cytokines on rat heart microvascular endothelial cell monolayer integrity, PKC activity, and adherens junction protein alteration were examined. Further, an in vivo rat coronary ischemia/reperfusion injury model was used to determine vascular leakage and TNF-alpha release. RESULTS: Administration of the above mentioned cytokines to cell monolayers resulted in significant increases in PKC activation, gap formation, and hyperpermeability across the monolayer and beta-catenin phosphorylation/reorganization. Inhibition of conventional PKC and MLCK attenuated permeability increases. Ischemia/reperfusion injury to the left ventricle resulted in TNF-alpha release as well as conventional PKC- and MLCK-dependent protein extravasation from the circulation to the heart tissue. CONCLUSION: These results identify the conventional PKC and MLCK pathways as important factors in coronary endothelial dysfunction elicited by IR injury and cytokine release. Further examination of these molecular signaling cascades has the potential of identifying targets for therapeutic intervention following ischemic events in the heart.

Angiopoietin-1 inhibits intrinsic apoptotic signaling and vascular hyperpermeability following hemorrhagic shock.

Studies from our laboratory demonstrated the involvement of intrinsic apoptotic signaling in hyperpermeability following hemorrhagic shock (HS). Angiopoietin 1 (Ang-1), a potent inhibitor of hyperpermeability, was recently shown to inhibit apoptosis. The purpose of our study was to determine the effectiveness of Ang-1 in attenuating HS-induced hyperpermeability and its relationship to apoptotic signaling. HS was induced in rats by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 h, followed by reperfusion. Mesenteric postcapillary venules were examined for changes in hyperpermeability by intravital microscopy. Mitochondrial release of second mitochondrial derived activator of caspases (smac) and cytochrome c were determined by Western blot and ELISA, respectively. Caspase-3 activity was determined by fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cell (RLMEC) monolayers, utilizing HS serum and the proapoptotic Bcl-2 homologous antagonist/killer [BAK (BH3)] peptide as inducers of hyperpermeability. In rats, Ang-1 (200 ng/ml) attenuated HS-induced hyperpermeability versus the HS group (P < 0.05). Ang-1 prevented HS-induced collapse of mitochondrial transmembrane potential (DeltaPsi(m)), smac and cytochrome c release, and caspase-3 activity (P < 0.05). In RLMEC monolayers, HS serum and BAK (BH3) peptide both induced hyperpermeability that was inhibited by Ang-1 (P < 0.05). Ang-1 attenuated HS and BAK (BH3) peptide-induced collapse of DeltaPsi(m), smac release, cytochrome c release, activation of caspase-3, and vascular hyperpermeability. In vivo, BAK (BH3) induced vascular hyperpermeability that was attenuated by Ang-1 (P < 0.05). These findings suggest that Ang-1's role in maintaining microvascular endothelial barrier integrity involves the intrinsic apoptotic signaling cascade.

Apoptotic signaling induces hyperpermeability following hemorrhagic shock.

Hemorrhagic shock (HS) disrupts the endothelial cell barrier, resulting in microvascular hyperpermeability. Recent studies have also demonstrated that activation of the apoptotic signaling cascade is involved in endothelial dysfunction, which may result in hyperpermeability. Here we report involvement of the mitochondrial "intrinsic" pathway in microvascular hyperpermeability following HS in rats. HS resulted in the activation of the mitochondrial intrinsic pathway, as is evident from an increase in the proapoptotic Bcl-2 family member BAK, release of mitochondrial cytochrome c into the cytoplasm, and activation of caspase-3. This, along with the in vivo transfection of the proapoptotic peptide BAK (BH3), resulted in hyperpermeability (as visualized by intravital microscopy), release of mitochondrial cytochrome c into the cytoplasm, and activation of caspase-3. Conversely, transfection of the BAK (BH3) mutant had no effect on hyperpermeability. Together, these results demonstrate involvement of the mitochondrial intrinsic apoptotic pathway in HS-induced hyperpermeability and that the attenuation of this pathway may provide an alternative strategy in preserving vascular barrier integrity.

PKC-dependent, burn-induced adherens junction reorganization and barrier dysfunction in pulmonary microvascular endothelial cells.

Rat lung microvascular endothelial cell monolayers were exposed to donor plasma from burned rats (25% total body surface area) at 1:3 dilution for 30 min. Immunofluorescence analysis revealed that concomitant with gap formation alterations were seen in the adherens junction (AJ) proteins beta-catenin and vascular endothelial-cadherin. Both of these components were shown to exist in a smooth, uniform arrangement at the cell periphery in untreated cells. However, upon exposure to burn plasma, this uniformity was lost, and the AJ proteins showed a disrupted, zipper-like pattern at the cells' edge. In addition, these proteins were absent from areas of gap formation between the cells, and an increase in punctate staining throughout the cells suggests they were internalized in response to burn plasma. Measurements of both transendothelial electrical resistance and FITC-albumin flux across the cell monolayer were used to assess barrier integrity. Our study found that exposure to burn plasma rapidly caused the electrical resistance across confluent monolayers to decrease and albumin flux to increase, phenomena associated with barrier dysfunction. Furthermore, all the above responses to burn plasma were attenuated when cells were pretreated with the PKC inhibitor bisindolylmaleimide, suggesting that PKC is required for burn-induced pulmonary endothelial dysfunction.

Isoform-specific knockout of endothelial myosin light chain kinase: closing the gap on inflammatory lung disease.

Inflammatory lung diseases result in high rates of morbidity and mortality. Central to the pathogenesis of these diseases is disruption of endothelial barrier function. Activation of myosin light chain kinase (MLCK) is a key regulatory step in the modulation of endothelial permeability. Recent studies show that mice with selective knockout of the endothelial MLCK are less susceptible to endotoxin-induced acute lung injury and that a new small-molecule inhibitor of MLCK also protects against lung injury.