Dysregulated VEGF/VEGFR-2 Signaling and Plexogenic Lesions in the Embryonic Lungs of Chickens Predisposed to Pulmonary Arterial Hypertension.

Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.

Vertical variation in prokaryotic community composition and co-occurrence patterns in sediments of the Three Gorges Reservoir, China.

Archaea and bacteria are distributed throughout the sediment; however, our understanding of their biodiversity patterns, community composition, and interactions is primarily limited to the surface horizons (0-20 cm). In this research, sediment samples were collected from three vertical sediment profiles (depths of 0-295 cm) in the Three Gorges Reservoir (TGR), one of the largest reservoirs in the world. Through 16S rRNA sequencing, it was shown that sediment microbial diversity did not significantly vary across the sediment. Nevertheless, a decline in the similarity of archaeal and bacterial communities over distance along sediment vertical profiles was noted. Nonmetric multidimensional scaling (NMDS) analysis revealed that archaeal and bacterial communities could be clearly separated into two groups, located in the upper sediments (0-135 cm) and deep sediments (155-295 cm). Meanwhile, at the fine-scale of the vertical section, noteworthy variations were observed in the relative abundance of prominent archaea (e.g., Euryarchaeota) and bacteria (e.g., Proteobacteria). The linear discriminant analysis effect size (LEfSe) demonstrated that twenty-four bacterial and twenty-six archaeal biomarker microbes exist in the upper and deep sediment layers. Each layer exhibited distinctive microbial divisions, suggesting that microbes with diverse biological functions are capable of thriving and propagating along the sediment profile. Co-occurrence network analysis further indicated that the microbial network in the upper sediments was more complex than that in the deep sediments. Additionally, the newly discovered anaerobic methanotrophic archaeon Candidatus Methanoperedens was identified as the most abundant keystone archaeal taxon in both sediment layers, highlighting the significance of methane oxidation in material cycling within the TGR ecosystem. In summary, our study examined the biodiversity and coexistence patterns of benthic microbial communities throughout the vertical sediment profile, providing detailed insights into the vertical geography of archaeal and bacterial communities in typical deep-water reservoir ecosystems.

Potential contribution of early endothelial progenitor cell (eEPC)-to-macrophage switching in the development of pulmonary plexogenic lesion.

BACKGROUND: Plexiform lesions, which have a dynamic appearance in structure and cellular composition, are the histological hallmark of severe pulmonary arterial hypertension in humans. The pathogenesis of the lesion development remains largely unknown, although it may be related to local inflammation and dysfunction in early progenitor endothelial cells (eEPCs). We tested the hypothesis that eEPCs contribute to the development of plexiform lesions by differentiating into macrophages in the setting of chronic inflammation. METHODS: The eEPC markers CD133 and VEGFR-2, macrophage lineage marker mannose receptor C-type 1 (MRC1), TNFα and nuclear factor erythroid 2-related factor 2 (Nrf2) in plexiform lesions in a broiler model were determined by immunohistochemistry. eEPCs derived from peripheral blood mononuclear cells were exposed to TNFα, and macrophage differentiation and angiogenic capacity of the cells were evaluated by phagocytotic and Matrigel plug assays, respectively. The role of Nrf2 in eEPC-to-macrophage transition as well as in MRC1 expression was also evaluated. Intratracheal installation of TNFα was conducted to determine the effect of local inflammation on the formation of plexiform lesions. RESULTS: Cells composed of the early lesions have a typical eEPC phenotype whereas those in more mature lesions display molecular and morphological characteristics of macrophages. Increased TNFα production in plexiform lesions was observed with lesion progression. In vitro studies showed that chronic TNFα challenge directed eEPCs to macrophage differentiation accompanied by hyperactivation of Nrf2, a stress-responsive transcription factor. Nrf2 activation (Keap1 knockdown) caused a marked downregulation in CD133 but upregulation in MRC1 mRNA. Dual luciferase reporter assay demonstrated that Nrf2 binds to the promoter of MRC1 to trigger its expression. In good agreement with the in vitro observation, TNFα exposure induced macrophage differentiation of eEPCs in Matrigel plugs, resulting in reduced neovascularization of the plugs. Intratracheal installation of TNFα resulted in a significant increase in plexiform lesion density. CONCLUSIONS: This work provides evidence suggesting that macrophage differentiation of eEPCs resulting from chronic inflammatory stimulation contributes to the development of plexiform lesions. Given the key role of Nrf2 in the phenotypic switching of eEPCs to macrophages, targeting this molecular might be beneficial for intervention of plexiform lesions.

A UPLC-MS/MS method for quantification of perindopril and perindoprilat and applied in a bioequivalence study for their pharmacokinetic parameter measurement
.

OBJECTIVES: To investigate the pharmacokinetic parameters of perindopril and perindoprilat in healthy volunteers, a simple and sensitive UPLC-MS/MS method with isotope-labeled internal standards of perindopril-d4 and perindoprilat-d4 was established and further applied in a bioequivalence study. MATERIALS AND METHODS: A simple and sensitive UPLC-MS/MS method with isotope-labeled internal standards of perindopril-d4 and perindoprilat-d4 was validated and applied in a single-center, randomized, cross-over, and two-period bioequivalence study. 20 healthy Chinese subjects (16 males and 4 females) were enrolled and had their plasma concentrations of perindopril and perindoprilat quantified and calculated for the pharmacokinetic parameters. After acetonitrile precipitation, the analytes and internal standards were gradient eluted with methanol-acetonitrile-ammonium acetate on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column. Detection was carried out in a multireaction monitoring mode using positive ionization electrospray mass spectrometry. RESULTS: The total chromatographic run time was 4 minutes with retention time for perindopril and perindopril-d4 of ~ 1.86 minutes, whereas perindoprilat and perindoprilat-d4 was ~ 1.79 minutes. The calibration curves of perindopril and perindoprilat were linear over 0.4 - 80 ng/mL and 0.2 - 40 ng/mL, respectively. The method was fully validated to meet the requirement for bioassay in accuracy (89.6 - 112.4%), precision (coefficient of variation (CV) ≤ 13.8%), recovery (79.65 - 97.83%), matrix effect (CV ≤ 5.9%), and stability (CV ≤ 10.0%). The 90% confidence intervals (CIs) for the geometric mean ratios of Cmax, AUC0-tlast, and AUC0-∞ of perindopril and perindoprilat all fell within the bioequivalence acceptance criteria (80 - 125%). There were no significant differences between the two formulations in terms of tmax and T1/2 of perindopril and perindoprilat. There was no adverse event in this clinical study. Interestingly, it was found that the pharmacokinetics of perindoprilat in 1 subject were significantly different from that of the others which may be associated with genetic diversity. CONCLUSION: This method was successfully applied to the bioequivalence test of two perindopril tert-butylamine tablets. The two one-sided t-tests showed that these two products were bioequivalent.

Metabonomics Profiling Reveals Biochemical Pathways Associated with Pulmonary Arterial Hypertension in Broiler Chickens.

Pulmonary arterial hypertension (PAH) is the major cause of death in fast growing meat-type chickens (broiler chickens). At present, the underlying mechanisms that give rise to PAH are not fully understood. To identify the metabonomics profiles characterizing the process, we conducted a comprehensive gas chromatography-mass spectrometry (GC-MS)-based metabolic profiling of lung tissues from PAH broilers and age-matched controls. PAH was induced by excess salt in drinking water. Medial hypertrophy of pulmonary arteries was present in PAH birds as compared with controls. The metabonomics profiles of lung tissues well distinguished PAH broilers from control subjects. Significant changes in the levels of 41 metabolites were detected in PAH vs normal birds. Aside from the metabolic alterations indicating a status of oxidative stress and inflammation, evidence of reduced cellular uptake of arginine due to increased lysine biosynthesis and of a shift of arginine metabolism to arginase pathway were observed. In addition, PAH birds showed increased biosynthesis of fatty acids, which may be associated with excessive proliferation of vascular cells during pulmonary vascular remodeling. Furthermore, we observed significant changes in pentose phosphate pathway and increased aminomalonic acid in PAH broilers. These results provide additional biochemical insights into the pathogenesis of the PAH. Our data may lead to the development of new strategies to control PAH in broilers.

Synergistic effect of organic matter-floc size-bound water and multifactorial quantitative model of optimal reagent demand in sewage sludge conditioning process prior to dewatering.

The high amount of densely hydrated organic substance present in sewage sludge impedes its filterability, thus restricting sludge disposal. Although chemical conditioning can facilitate filtration, the diverse sludge properties complicate the quantitative control of conditioning process. Investigating how to accurately quantify the optimal reagent demand (ORD) based on the critical physicochemical properties of the target sludge is an effective way to address the current issue. This study focused on the sewage and stockpiled sludge with varying properties, and their ORD under different chemical conditioning. The results showed that organic content, floc size, and bound water synergistically influenced conditioning process. The quantitative models were established between their coupling indicators and ORD, with coupling indicators including the ratio of organic content to floc size, the ratio of flow viscosity to floc size, and the ratio of the product of organic content and bound water to floc size. The linear correlation of the coupling indicator with ORD was higher than that of the traditional single-factor indicator. Furthermore, the inherent filterability of the sludge was somewhat separate from the adjustability of its filtration. A "dual-system" impact model was proposed to characterized the conditioning and filtration processes. These results provide theoretical guidance for the quantitative regulation of conditioning and filtration processes of sludge with complex characteristics.

Migrasome, a novel organelle, differs from exosomes.

Migrasomes, a newly discovered organelle produced by migrating cells, are vesicles with membranous structure that form on the tips and intersections of retraction fibers (RFs). These structures are released into the extracellular environment or taken up by surrounding cells, mediating the release of cytoplasmic contents and intercellular communication. Retractosomes, a new type of small extracellular vesicles generated from broken-off RFs, are closely related to migrasomes in their physical location and origin, but were defined later. Despite their widespread existence in cells and biological organisms, little is known about the regulatory mechanisms underlying their formation and potential function. In this review, we provide an overview of the discovery, biogenesis, distribution, and functions of migrasomes and retractosomes, as well as their differences from exosomes.

Sediment depth-related variations of comammox Nitrospira: Evidence in the Three Gorges Reservoir, China.

The recent discovery of comammox Nitrospira as complete ammonia-oxidizing microorganism has fundamentally revolutionized our understanding of nitrogen cycling in sediment environments. However, knowledge regarding their abundance, biodiversity, community structure, and interactions is predominantly limited to the upper layers (0-20 cm). To address this gap, we collected sediment samples along profiles ranging from 0 to 300 cm in depth at three locations within the middle segment of the Three Gorges Reservoir (TGR), China. Quantitative real-time PCR (qPCR) analyses suggested that comammox bacteria were not only ubiquitous in deep sediments but also more abundant than ammonia-oxidizing bacteria (AOB). Ammonia monooxygenases subunit A (amoA) gene amplicon sequencing illuminated that comammox bacteria were more sensitive to sedimental depth compared to AOB and ammonia-oxidizing archaea (AOA), as evidenced by a more significant decline in community diversity and similarity over distance along sediment vertical profiles. Notably, we discovered that the amoA gene abundance, alpha- and beta-diversity of comammox bacteria exerted an essential contribution to potential nitrification rates according to random forest model. Phylogenetic analysis indicted that most comammox bacteria within sediment samples belonged to clade A.2. Intriguingly, the average relative abundance of comammox clade A.2 displayed a noteworthy rise with sediment depth, whereas clade A.1 demonstrated a converse pattern, unveiling distinct ecological niche adaptations of these two clades along the sediment profile. Ecological network analysis further revealed closer interactions between comammox bacteria and canonical ammonia oxidizers in the superficial layer (0-40 cm), with the network structure gradually simplifying from superficial to deep sediment (200-300 cm). Overall, these findings broaden the current recognition of the geographic distribution and niche segregation of comammox bacteria at the fine scale of the sediments ecosystems and provide insights into sediment depth-related variations of their coexistence network patterns in large freshwater reservoirs.

MSC Transplantation Attenuates Inflammation, Prevents Endothelial Damage and Enhances the Angiogenic Potency of Endogenous MSCs in a Model of Pulmonary Arterial Hypertension.

Purpose: Pulmonary arterial hypertension (PAH) is a progressive and fatal pulmonary vascular disease initiated by endothelial dysfunction. Mesenchymal stromal cells (MSCs) have been shown to ameliorate PAH in various rodent models; however, these models do not recapitulate all the histopathological alterations observed in human PAH. Broiler chickens (Gallus gallus) can develop PAH spontaneously with neointimal and plexogenic arteriopathy strikingly similar to that in human patients. Herein, we examined the protective effects of MSC transplantation on the development of PAH in this avian model. Methods: Mixed-sex broilers at 15 d of age were received 2×106 MSCs or PBS intravenously. One day later, birds were exposed to cool temperature with excessive salt in their drinking water to induce PAH. Cumulative morbidity from PAH and right-to-left ventricle ratio were recorded. Lung histologic features were evaluated for the presence of endothelial damage, endothelial proliferation and plexiform lesions. Expression of proinflammatory mediators and angiogenic factors in the lung was detected. Matrigel tube formation assay was performed to determine the angiogenic potential of endogenous MSCs. Results: MSC administration reduced cumulative PAH morbidity and attenuated endothelial damage, plexiform lesions and production of inflammatory mediators in the lungs. No significant difference in the expression of paracrine angiogenic factors including VEGF-A and TGF-ß was determined between groups, suggesting that they are not essential for the beneficial effect of MSC transplantation. Interestingly, the endogenous MSCs from birds receiving MSC transplantation demonstrated endothelial differentiatial capacity in vitro whereas those from the mock birds did not. Conclusion: Our results support the therapeutic use of MSC transplantation for PAH treatment and suggest that exogenous MSCs produce beneficial effects through modulating inflammation and endogenous MSC-mediated vascular repair.

Pathological observations of an experimental infection of geese with goose circovirus.

There is so far no report describing the pathogenicity of goose circovirus (GoCV) following experimental infection. We report here an experimental inoculation of 21-day-old geese with a GoCV polymerase chain reaction (PCR)-positive sample of bursa of Fabricius (BF) homogenate, and the results of clinicopathological changes. Forty geese were randomly assigned to two groups: the inoculated group (n=30) receiving tissue homogenate and the uninoculated group (n=10) receiving a placebo (physiological saline). Tissue samples were collected for histopathological examination and GoCV detection. The main clinical signs in the GoCV-inoculated geese included diarrhoea at 17 to 30 days post inoculation, marked differences of growth rate and feather disorders in three of the geese. Viraemia was detected by PCR only at 14 days post inoculation. The BF, thymus, spleen, duodenum and liver of some inoculated geese were also PCR-positive. Real-time PCR showed that there was relatively more viral load in the BF. Histological examination revealed lymphocytic depletion and histiocytosis in the BF and inflammation characterized by lymphatic infiltration in the lung, liver and kidney. Immunohistochemistry applied to PCR-positive samples showed that in only three BF samples was GoCV antigen detected in lymphocytes in both the cortex and medulla. In situ apoptosis detection of all BF samples indicated that GoCV inoculation was associated with an increase in bursal lymphocytic apoptotic events.

Annexin A2-Mediated Internalization of Staphylococcus aureus into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

BACKGROUND: Staphylococcus aureus is a leading cause of contagious mastitis in dairy cattle. Internalization of S. aureus by bovine mammary gland epithelial cells is thought to be responsible for persistent and chronic intramammary infection, but the underlying mechanisms are not fully understood. METHODS: In the present study, we evaluated the role of Annexin A2 (AnxA2), a membrane-binding protein, in S. aureus invasion into bovine mammary epithelial cell line (MAC-T). In vitro binding assays were performed to co-immunoprecipitate the binding proteins of AnxA2 in the lysates of S. aureus. RESULTS: AnxA2 mediated the internalization but not adherence of S. aureus. Engagement of AnxA2 stimulated an integrin-linked protein kinase (ILK)/p38 MAPK cascade to induce S. aureus invasion. One of the AnxA2-precipitated proteins was identified as S. aureus clumping factor B (ClfB) through use of mass spectrometry. Direct binding of ClfB to AnxA2 was further confirmed by using a pull-down assay. Pre-incubation with recombinant ClfB protein enhanced S. aureus internalization, an effect that was specially blocked by anti-AnxA2 antibody. CONCLUSION: Our results demonstrate that binding of ClfB to AnxA2 has a function in promoting S. aureus internalization. Targeting the interaction of ClfB and AnxA2 may confer protection against S. aureus mastitis.

New methods for quantification of Fenton's reagent addition based on aged sludge indicators to improve filterability.

Fenton oxidation can effectively improve the dewaterability of aged sludge. Quantification of the addition of optimal reagents is central to the conditioning and dewatering of aged sludge. Improving the accuracy of quantification is significant to promote cost effectiveness. The effects of reagent addition and the mechanism governing the improved filterability of the aged sludge need to be understood uniformly. In this study, the optimal reagent additions have been determined using the response surface method (RSM) for five out of the eight aged sludges that were investigated. The physicochemical characteristics of eight aged sludges, including the extracellular polymer substance, undissolved organic matter, and suspension structure network, were investigated. Meanwhile, a comprehensive correlation analysis of critical indicators was conducted to investigate the interactions among the properties of the aged sludge. The effects of these interactions on the conditioning and filtration processes were examined, and a unified understanding of the combination of factors affecting the optimal reagent addition was obtained. The key factors were aggregate size, dewatering extent, yield stress, and organic substance content. Based on these results, a new reagent addition quantification method was developed along with an empirical model of the relationship between physicochemical properties and the economically optimal reagent addition.

Escherichia coli and Staphylococcus aureus Differentially Regulate Nrf2 Pathway in Bovine Mammary Epithelial Cells: Relation to Distinct Innate Immune Response.

Escherichia coli and Staphylococcus aureus are major mastitis causing pathogens in dairy cattle but elicit distinct immune and an inflammatory response in the udder. However, the host determinants responsible for this difference remains largely unknown. Our initial studies focused on the global transcriptomic response of primary bovine mammary epithelial cells (pbMECs) to heat-killed E. coli and S. aureus. RNA-sequencing transcriptome analysis demonstrates a significant difference in expression profiles induced by E. coli compared with S. aureus. A major differential response was the activation of innate immune response by E. coli, but not by S. aureus. Interestingly, E. coli stimulation increased transcript abundance of several genes downstream of Nrf2 (nuclear factor erythroid 2-related factor 2) that were enriched in gene sets with a focus on metabolism and immune system. However, none of these genes was dysregulated by S. aureus. Western blot analysis confirms that S. aureus impairs Nrf2 activation as compared to E. coli. Using Nrf2-knockdown cells we demonstrate that Nrf2 is necessary for bpMECs to mount an effective innate defensive response. In support of this notion, nuclear Nrf2 overexpression augmented S. aureus-stimulated inflammatory response. We also show that, unlike E. coli, S. aureus disrupts the non-canonical p62/SQSTM1-Keap1 pathway responsible for Nrf2 activation through inhibiting p62/SQSTM1 phosphorylation at S349. Collectively, our findings provide important insights into the contribution of the Nrf2 pathway to the pathogen-species specific immune response in bovine mammary epithelial cells and raise a possibility that impairment of Nrf2 activation contributes to, at least in part, the weak inflammatory response in S. aureus mastitis.

Strength and microstructure properties of solidified sewage sludge with two types of cement-based binders.

Solidification treatment with cementitious binder is an effective way to reduce environmental hazards of sewage sludge. Two cementitious binders, i.e., ordinary Portland cement (OPC) and sulfo-aluminate cement (SAC), were compared in this study to treat the sewage sludge. The strength of solidified sewage sludge (SSS) and changes in microscopic characteristics before and after treatment were analyzed through microscopic analysis methods. The effect of organic matter in sludge on the strength of SSS were also discussed. The results showed that the strength of SSS were lower than that of the solidified clay with no organic matter, and the filtrate extracted from the sludge can also weaken the cementation of the two cements significantly. The solidification effect of the OPC on the sludge was lower than that of the SAC evidently. The organic matter in the sewage sludge caused the surface of the soil particles to carry a large negative potential, which interfered with the hydration of the binder and reduced the amount of cementation skeleton formed by the binder hydration products. This resulted in a porous structure with low mechanical strength. The amount of early hydration product formed in the SAC-based solidified samples was higher than that of the OPC-based samples. This was favorable for filling the pores of the solidified samples and increasing their density. SAC had a better compatibility with soft soil containing high organic matter than OPC, and the which provides an effective alternative binder for dealing with sewage sludge.

Enhancing effect of FSH on follicular development through yolk formation and deposition in the low-yield laying chickens.

Healthy and efficient development of ovarian follicles largely determines poultry laying performance. In low-yield laying chickens, retarded follicle progression resulted in decreased prehierarchical follicles. In this study the extenuating effect of follicle-stimulating hormone (FSH) on delayed follicular development was investigated in the low-yield chickens. Results showed that FSH administration in vivo accelerated development of prehierarchical follicles, with increased expression of steroidogenic enzymes and follicular angiogenesis through elevating plasma levels of 17ß-estradiol, progesterone, luteinizing hormone and the expression of vascular endothelial growth factor and its receptor as well as angiopoietins. Furthermore, treatment with FSH raised expression of lipid uptake and adipogenesis-related proteins and decreased tight junctions between granulosa cells. Meanwhile, the results of the in vivo studies were confirmed by the in vitro studies as FSH promoted development of the cultured prehierarchical follicles with increased angiogenesis, cell proliferation, steroid hormones synthesis and yolk deposition. These results indicated FSH enhanced follicular development in the low-yield laying chickens involving increased follicular angiogenesis.

Reduced PKCalpha expression in pulmonary arterioles of broiler chickens is associated with early feed restriction.

OBJECTIVE: The present study was conducted to investigate the effect of early feed restriction on protein kinase Calpha (PKCalpha) expression in pulmonary arterioles, which has been revealed to promote pulmonary vascular remodeling in pulmonary hypertensive broilers. METHODS: A total of 270day-old mixed sex commercial broilers were randomly distributed to a normal temperature control group (NT), a low temperature control group (LT) and a low temperature plus feed restriction group (LR). The PHS incidence, the right/total ventricular weight ratio (RV/TV), the vessel wall area/vessel total area ratio (WA/TA), the mean media thickness in pulmonary arterioles (mMTPA) and the expression of PKCalpha in the pulmonary arterioles were measured weekly. RESULTS: Low temperature treatment significantly increased the PHS mortality. The RV/TV, WA/TA and mMTPA values of group LT were significantly elevated compared with those of group NT on d 35 and 42. The LT chickens had increased PKCalpha expression compared with their NT counterparts on d 28 and afterwards. Feed restriction reduced the PHS mortality, RV/TV, WA/TA and mMTPA in cold-exposed broilers. The LR chickens had much lower PKCalpha expression in pulmonary arterioles than the LT chickens. CONCLUSION: Early time feed restriction inhibited pulmonary vascular remodeling in broilers, which might be partly attributed to reduced PKCalpha expression in pulmonary arterioles.

The injury effect of oxygen free radicals in vitro on cultured pulmonary artery endothelial cells from broilers.

Pulmonary artery endothelial cells (PAEC) were isolated from broilers by the method of tissue explantation. The cells were identified using morphological features and immunocytochemical staining using a specific antiserum against factor VIII related antigen. Xanthine/Xanthine oxidase (X/XO) served as the oxygen free radical (OFR) generating system. In vitro model of oxidative injury of PAEC was established based on the X/XO system. The effect of OFR on the growth and viability of PAEC was determined with methylthiazol tetrazolium (MTT) colorimetric assay. Malondialdehyde (MDA, a product of lipid peroxidation) in culture medium of PAEC was detected by a thiobarbituric acid colorimetric assay. The results showed that PAEC survive in vitro and can be subcultured for 5-6 passages. Morphological and immunocytochemical observations of cultured cells demonstrated specific characteristics of endothelial cells. PAECs were severely damaged by OFR. The viability of cells was reduced by the X/XO system, and a dose-dependent decrease in cell viability was found with increasing XO dosages. OFR promoted lipid peroxidation of PAEC and increased the MDA concentration in culture media. These results suggest that OFR can injure the endothelial cells from broiler pulmonary arteries in vitro, which confirms previous results obtained in vivo. Oxidative injury may play an important role in the pathogenesis of pulmonary hypertension syndrome in broiler.

L-arginine prevents reduced expression of endothelial nitric oxide synthase (NOS) in pulmonary arterioles of broilers exposed to cool temperatures.

This study investigated nitric oxide synthase (NOS) expression in the endothelium of pulmonary arterioles of broilers during the development of pulmonary hypertension syndrome (PHS). PHS was triggered by exposing broilers to sub-thermoneutral (cool) temperatures and an additional 1.0% L-arginine was added to the basal diet to evaluate the effects of supplemental L-arginine on nitric oxide (NO) production, endothelial NOS expression, and the incidence of PHS. Cumulative mortality from PHS, right/total ventricle weight ratios (RV/TV), and body weights were recorded. Plasma NO concentration and NOS expression in the endothelium of pulmonary arterioles with an outer diameter ranging from 100 to 200 microm were determined. Birds exposed to cool temperatures had increased pulmonary hypertension and PHS mortality and diminished endothelial NOS expression. Supplemental dietary L-arginine reduced PHS mortality and elicited higher NOS expression within the pulmonary endothelium coincident with elevated NO production. The results demonstrated that broilers developing PHS exhibited diminished NOS expression in the endothelium of their pulmonary arterioles. Supplemental L-arginine prevented the reduced expression of NOS in the pulmonary endothelium, which might contribute to the increased production of NO by the pulmonary vasculature.

Involvement of endothelial progenitor cells in the formation of plexiform lesions in broiler chickens: possible role of local immune/inflammatory response.

Plexiform lesions (PLs), which are often accompanied by perivascular infiltrates of mononuclear cells, represent the hallmark lesions of pulmonary arteries in humans suffering from severe pulmonary arterial hypertension (PAH). Endothelial progenitor cells (EPCs) have been recently implicated in the formation of PLs in human patients. PLs rarely develop in rodent animal models of PAH but can develop spontaneously in broiler chickens. The aim of the present study was to confirm the presence of EPCs in the PLs in broilers. The immune mechanisms involved in EPC dysfunction were also evaluated. Lungs were collected from commercial broilers at 1 to 4 weeks of age. The right/total ventricle ratios indicated normal pulmonary arterial pressures for all sampled birds. Immunohistochemistry was performed to determine the expressions of EPC markers (CD133 and VEGFR-2) and proangiogenic molecule hepatocyte growth factor (HGF) in the lung samples. An EPC/lymphocyte co-culture system was used to investigate the functional changes of EPCs under the challenge of immune cells. PLs with different cellular composition were detected in the lungs of broilers regardless of age, and they were commonly surrounded by moderate to dense perivascular mononuclear cell infiltrates. Immunohistochemical analyses revealed the presence of CD133+ and VEGFR-2+ cells in PLs. These structures also exhibited a strong expression of HGF. Lymphocyte co-culture enhanced EPC apoptosis and completely blocked HGF-stimulated EPC survival and in vitro tube formation. Taken together, this work provides evidence for the involvement of EPCs in the development of PLs in broilers. It is suggested that the local immune cell infiltrate might serve as a contributor to EPC dysfunction by inducing EPC death and limiting their response to angiogenic stimuli. Broiler chickens may be valuable for investigating reversibility of plexogenic arteriopathy using gene-modified inflammation-resistant EPCs.

Role of the NOD1/NF-κB pathway on bovine neutrophil responses to crude lipopolysaccharide.

Cytosolic nucleotide oligomerisation domain (NOD)-like receptors play an important role in host defence against infection. Reduced NOD1 expression has been observed in dysfunctional neutrophils derived from periparturient cattle known to be most susceptible to coliform mastitis. However, whether impairment of NOD1 suppresses the immune responses of bovine neutrophils during bacterial infections remains unknown. Crude (phenol extracted) lipopolysaccharide (cLPS), which often contains other immunostimulatory molecules, including NOD1 agonist, is known to induce almost the whole bacterial response. This study was conducted to explore the role of NOD1/nuclear factor (NF)-κB pathway in the cytokine and functional responses of bovine neutrophils challenged with Escherichia coli-derived cLPS. Freshly isolated blood neutrophils from healthy heifers were pre-incubated for 2 h with ML130, a selective inhibitor of NOD1/NF-κB pathway. Cells were then exposed to cLPS for additional 4 h. Inhibition of the NOD1/NF-κB pathway resulted in a decrease in cLPS-induced phosphorylation of the inhibitor of NF-κBα (IκBα) in neutrophils. Impairment of the NOD1/NF-κB pathway tended to down-regulate mRNA levels of pro-inflammatory cytokines interleukin (IL)-1ß and tumour necrosis factor (TNF)-α, chemokines IL-8 and C-X-C motif ligand 2 (CXCL2), and adhesion molecules CD11b and CD62L, in cLPS-challenged cells. Functional analyses showed that blocking the NOD1/NF-κB pathway inhibited neutrophil migration and phagocytic killing capacity, and promoted neutrophil death upon cLPS stimulation. The data presented here demonstrate that activation of NOD1/NF-κB pathway contributes to the functional responses of neutrophils to cLPS.