Molecular and metabolic responses to immune stress in the jejunum of broiler chickens: transcriptomic and metabolomic analysis.

In the large poultry industry, where farmed chickens are fed at high density, the prevalence of pathogens and repeated vaccinations induce immune stress, which can significantly decrease the production performance and increase the mortality. This study was designed to shed light on the molecular mechanisms and metabolic pathways involved in immune stress through an in-depth analysis of transcriptomic and metabolomic changes in jejunum samples from the broilers. Two groups were established for the experiment: a control group and an LPS group. LPS group received an intraperitoneal injection of LPS solution at a dose of 250 µg per kg at 12, 14, 33, and 35 d of age, whereas the control group received a sterile saline injection. The severity of immune stress was assessed using the Disease Activity Index. A jejunal section was collected to measure the intestinal villus structure (villus length and crypt depth). RNA sequencing and metabolomics data analysis were conducted to reveal differentially expressed genes and metabolites. The results showed that the DAI index was increased and jejunal villus height/crypt depth was decreased in the LPS group. A total of 96 differentially expressed genes and 672 differentially accumulating metabolites were detected in the jejunum by LPS group compared to the control group. The comprehensive analysis of metabolomic and transcriptomic data showed that 23 pathways were enriched in the jejunum and that appetite, nutrient absorption, energy and substance metabolism disorders and ferroptosis play an important role in immune stress in broilers. Our findings provide a deeper understanding of the molecular and metabolic responses in broilers to LPS-induced immune stress, suggesting potential targets for therapeutic strategies to improve the production performance of broiler chickens.

Immunomodulatory effect of Atractylodis macrocephala Koidz. polysaccharides in vitro.

Vaccination is still the main method of preventing most infectious diseases, but there are inefficiencies and inaccuracies in immunization. Studies have reported that Atractylodis macrocephalae Koidz. polysaccharides (RAMP) have immunomodulatory effects, but the mechanisms involved in whether they can modulate the immune response in chickens are not yet clear. The aim of this study was to investigate the effect of RAMP on lymphocytes functions by analyzing cell proliferation, cell cycle, mRNA expression of cytokines and CD4 +/CD8 + ratio. To identify potential molecules involved in immune regulation, we performed a comprehensive transcriptome profiling of chicken lymphocytes. In addition, the adjuvant effect of RAMP was evaluated by detecting indicators of hemagglutination inhibition. When lymphocytes were cultured with RAMP in vitro, the proliferation rate of lymphocytes was increased (P < 0.01), more cells in S phase and G2/M phase (P < 0.01) and the mRNA expression of IFN-γ was upregulated (P < 0.05), while the mRNA expression of TGF-ß (P < 0.01) and IL-4 (P < 0.05) was downregulated and the CD4 +/CD8 + ratio was increased (P < 0.05). Transcriptomic results showed that RAMP increased the expression of HIST1H46 (P < 0.05) and CENPP (P < 0.05). Validation of qPCR showed that RAMP may play an important role in regulating cellular immunity by downregulating the Notch pathway. The results also showed that RAMP could increase the serum Newcastle disease virus antibody levels in chickens. These data suggest that RAMP could enhance immune function of lymphocytes and was a candidate vaccine adjuvant in chickens.

Biomarkers of oxidative stress in broiler chickens attacked by lipopolysaccharide: A systematic review and meta-analysis.

Oxidative stress (OS) constitutes a pivotal factor in the initiation and progression of lipopolysaccharide (LPS) challenges in broiler chickens. Increasing studies have demonstrated that Alleviation of oxidative stress seems to be a reasonable strategy to alleviate LPS-mediated afflictions in broilers. Nonetheless, the relationship between OS-related indicators and exposure to LPS remains a topic of debate. The aim of this investigation was to precisely and holistically evaluate the effect of LPS exposure on OS-associated markers. We conducted a systematic search of four electronic databases-PubMed, Web of Science, Scopus, and Cochrane for relevant studies, and a total of 31 studies were included. The overall results showed that the LPS treatment significantly increased the levels of oxygen radicals and their products, such as malondialdehydes (MDA), reactive oxygen species (ROS), and 8-hydroxy-2-deoxyguanosine (8-OHdG), while significantly reduced the levels of antioxidants, such as total antioxidative capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione (GSH), in the chickens. Intriguingly, though the observed trends in alterations were not strictly correlated with LPS concentrations, the enzyme activity levels were indeed influenced by the concentration of LPS. This observation highlights the complex relationship between LPS exposure and the body's antioxidant response. Despite some limitations, all the included studies were deemed credible. Subgroup evaluations revealed that the jejunum and duodenum has demonstrated stronger antioxidant capability compared to other tissues. Overall, our study presents compelling evidence that exposure to LPS induces significant OS in chickens. And we also found that the extent of OS was related to LPS doses, target tissues, and dietary ingredients.

Potential pharmacological mechanisms of four active compounds of Macleaya cordata extract against enteritis based on network pharmacology and molecular docking technology.

Background: Macleaya cordata extract (MCE) is effective in the treatment of enteritis, but its mechanism has not been fully elucidated. Therefore, this study combined network pharmacology and molecular docking technologies to investigate the potential pharmacological mechanism of MCE in the treatment of enteritis. Methods: The information of active compounds in MCE was accessed through the literature. Furthermore, PubChem, PharmMapper, UniProt, and GeneCards databases were used to analyze the targets of MCE and enteritis. The intersection of drug and disease targets was imported into the STRING database, and the analysis results were imported into Cytoscape 3.7.1 software to construct a protein-protein interaction (PPI) network and to screen core targets. The Metascape database was used for conducting Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. AutoDock Tools software was used for the molecular docking of active compounds with the core targets. Results: MCE has four active compounds, namely, sanguinarine, chelerythrine, protopine, and allocryptopine, and a total of 269 targets after de-duplication. Furthermore, a total of 1,237 targets were associated with enteritis, 70 of which were obtained by aiding the drug-disease intersection with the aforementioned four active compound targets of MCE. Five core targets including mitogen-activated protein kinase 1 (MAPK1) and AKT serine/threonine kinase 1 (AKT1) were obtained using the PPI network, which are considered the potential targets for the four active compounds of MCE in the treatment of enteritis. The GO enrichment analysis involved 749 biological processes, 47 cellular components, and 64 molecular functions. The KEGG pathway enrichment analysis revealed 142 pathways involved in the treatment of enteritis by the four active compounds of MCE, among which PI3K-Akt and MAPK signaling pathways were the most important pathways. The results of molecular docking showed that the four active compounds demonstrated good binding properties at the five core targets. Conclusion: The pharmacological effects of the four active compounds of MCE in the treatment of enteritis involve acting on signaling pathways such as PI3K-Akt and MAPK through key targets such as AKT1 and MAPK1, thus providing new indications for further research to verify its mechanisms.

Changed cecal microbiota involved in growth depression of broiler chickens induced by immune stress.

A previous study identified genes and metabolites associated with amino acid metabolism, glycerophospholipid metabolism, and inflammatory response in the liver of broilers with immune stress. The present research was designed to investigate the effect of immune stress on the cecal microbiome in broilers. In addition, the correlation between altered microbiota and liver gene expression, the correlation between altered microbiota and serum metabolites were compared using the Spearman correlation coefficients. Eighty broiler chicks were randomly assigned to 2 groups with 4 replicate pens per group and 10 birds per pen. The model broilers were intraperitoneally injected of 250 µg/kg LPS at 12, 14, 33, and 35 d of age to induce immunological stress. Cecal contents were taken after the experiment and kept at -80°C for 16S rDNA gene sequencing. Then the Pearson's correlation between gut microbiome and liver transcriptome, between gut microbiome and serum metabolites were calculated using R software. The results showed that immune stress significantly changed microbiota composition at different taxonomic levels. KEGG pathways analysis suggested that these gut microbiota were mainly involved in biosynthesis of ansamycins, glycan degradation, D-glutamine and D-glutamate metabolism, valine, leucine, and isoleucine biosynthesis and biosynthesis of vancomycin group antibiotics. Moreover, immune stress increased the activities of metabolism of cofactors and vitamins, as well as decreased the ability of energy metabolism and digestive system. Pearson's correlation analysis identified several bacteria were positively correlated with the gene expression while a few of bacteria were negatively correlated with the gene expression. The results identified potential microbiota involvement in growth depression mediated by immune stress and provided strategies such as supplement of probiotic for alleviating immune stress in broiler chickens.

Ginsenoside Rg1 and Re alleviates inflammatory responses and oxidative stress of broiler chicks challenged by lipopolysaccharide.

Previous study showed that ginsenoside Rg1 (Rg1) and ginsenoside Re (Re) alleviated growth inhibition of broiler chicks with immune stress. The aim of this study was to investigate the effect of Rg1 and Re on inflammatory responses, oxidative stress, and apoptosis in liver of broilers with immune stress induced by lipopolysaccharide (LPS). Forty broiler chicks were randomly divided into 4 groups, each group consisting of 10 chickens. The model group, Rg1 group, and Re group were received continuously interval injection of 250 µg/kg body weight LPS at the age of 12, 14, 33, and 35 days to induce immune stress. Control group was injected with an equivalent amount of sterile saline. Then broilers in Rg1 group and Re group were given 1mg/kg body weight Rg1 and Re intraperitoneally 2 h after the LPS challenge respectively. Blood samples were collected for the detection of hormone levels, inflammatory mediators, and antioxidant parameters. Hepatic tissues were taken for pathological observation. Total RNA was extracted from the liver for real-time quantitative polymerase chain reaction analysis. Our results showed that Rg1 or Re could alleviate histological changes of liver, reduce production of stress-related hormones, inhibit inflammatory responses, and enhance antioxidant capacity in broilers challenged by immune stress. In addition, Rg1 or Re treatment upregulated mRNA expression of antioxidant-related genes and downregulated mRNA expression of inflammation-related factors and apoptosis-related genes in the liver of immune-stressed broilers. The results suggest that the plant extracts containing Rg1 and Re can be used for ameliorating hepatic oxidative stress and inflammation and controlling immune stress in broiler chicks.

Beta-glucan enhanced immune response to Newcastle disease vaccine and changed mRNA expression of spleen in chickens.

The present study was performed to investigate the effect of oral administration of ß-glucan (G70), a product obtained from the cell wall of yeast, on Newcastle disease virus (NDV)-specific hemagglutination inhibition (HI) titers, lymphocyte proliferation, and the role of T lymphocyte subpopulations in chickens treated with live NDV vaccine. In addition, the influence of ß-glucan on splenic gene expression was investigated by transcriptome sequencing. The results revealed that the supplementation of ß-glucan boosted the titer of serum NDV HI increased the NDV stimulation index of lymphocytes in peripheral blood and intestinal tract, and promoted the differentiation of T lymphocytes into CD4+ T cells. The RNA sequencing (RNA-seq) analysis demonstrated that G70 upregulated the mRNA expressions related to G-protein coupled receptor and MHC class I polypeptide, and downregulated the mRNA expressions related to cathelicidin and beta-defensin. The immunomodulatory effect of G70 might function through mitogen-activated protein kinase signaling pathway. To sum up, G70 could boost the immunological efficacy of live NDV vaccine in chickens and could be applied as a potential adjuvant candidate in the poultry industry.

Enhancement of intestinal mucosal immunity and immune response to the foot-and-mouth disease vaccine by oral administration of danggui buxue decoction.

This study investigated the effect of Danggui Buxue decoction (DBD) on the immunity of an O-type foot-and-mouth disease (FMD) vaccine and intestinal mucosal immunity. SPF KM mice were continuously and orally administered DBD for 5 d and then inoculated with an O-type FMD vaccine. The contents of a specific IgG antibody and its isotypes IgG1, IgG2a, IgG2b, and IgG3 in serum and SIgA in duodenal mucosa were determined by ELISA at 1 and 3 W after the 2nd immunization. qRT-PCR was used to detect mRNA expression levels of IL-4, IL-10, IFN-γ, and IL-33 in the spleen, and mRNA expression levels of J-chain, pIgR, BAFF, APRIL, IL-10, IFN-γ and IL-33 in the duodenum. The results showed that compared with the control group, oral administration of DBD significantly increased levels of the anti-FMD virus (FMDV)-specific antibodies IgG, IgG1, and IgG2a in the serum of O-type FMD vaccine-immunized mice 1 W after the 2nd immunization (P < 0.05), upregulated mRNA expression levels of spleen lymphocyte cytokines IL-4 and IL-33 (P < 0.05), promoted the secretion of SIgA in duodenal mucosa (P < 0.05). The mRNA expression levels of J-chain, pIgR, BAFF, APRIL, IL-10, and IL-33 in duodenal tissues were upregulated (P < 0.05). This study indicates that DBD has a good promotion effect on the O-type FMD vaccine and the potential to be an oral immune booster.

Hepatic transcriptomics and metabolomics indicated pathways associated with immune stress of broilers induced by lipopolysaccharide.

Broilers with immune stress show decline of growth performance, causing severe economic losses. However, the molecular mechanisms underlying the immune stress still need to be elucidated. One hundred and twenty broiler chicks were randomly assigned to 2 groups with 6 repeats per group, 10 birds per repeat. The model broilers were intraperitoneally injection of 250 µg/kg LPS at 12, 14, 33, and 35 d of age to induce immunological stress. Control group was injected with an equivalent amount of sterile saline. Blood samples from chickens were collected using wing vein puncture at 35 d of age and the serum was obtained for detection of CORT and ACTH. At the end of the experiment, the liver tissues were excised and collected for omics analysis. The results showed that LPS challenge significantly inhibited growth performance, increased relative weight of liver, spleen and decreased relative weight of bursa, as well as enhanced the concentration of serum ACTH and CORT, when compared with the Control. A total of 129 DEGs and a total of 109 differential metabolites were identified between Model and Control group. Transcriptomics profiles revealed that immune stress enhanced the expression of genes related to defense function while declined the expression of genes related to oxidation-reduction process. Metabolomics further suggested that immune stress changed metabolites related to amino acid metabolism, glycerophospholipid metabolism. In addition, integrated analysis suggested that the imbalance of valine, leucine and isoleucine metabolism, glycerophospholipid metabolism, and mTOR signaling pathway played an important role in immune stress of broiler chicks.

Serum metabolomics reveal pathways associated with protective effect of ginsenoside Rg3 on immune stress.

Our previous study has demonstrated that administration of ginsenoside Rg3 ameliorates immune stress by inhibiting inflammatory responses, reducing oxidative damage and upregulating mRNA expression of mTOR, SOD-1, and HO-1. However, the specific mechanism in relation to the protective effect of ginsenoside Rg3 on stressed broilers especially the metabolites alteration remains obscure. The present study aimed to investigate the underlined mechanism in relation to the pathogenesis and protective effect of ginsenoside Rg3 on stressed broilers using liquid chromatograph-mass spectrometry profiling. Eighteen broiler chicks were randomly allocated to 3 treatments: Control, Model and Rg3. Chickens in Rg3 group received intraperitoneally administered 1 mg/kg Rg3 2 h before LPS challenge. Then the broilers were intraperitoneally injection of 250 µg/kg LPS at the age of 12, 14, 33, and 35 d to induce immune stress. Control group was injected with an equivalent amount of sterile saline. At the end of the experiment, the serum was obtained for metabolomics analysis. The changes in serum metabolic profiles were investigated with the application of metabolomics approach. Distinct changes in metabolite patterns in serum were observed by orthogonal partial least square-discriminate analysis. In total, 35 metabolites were identified, among which 17 differential metabolites were found between Control and Model group, and 18 differential metabolites were identified between Model and Rg3 group. Metabolic pathway analysis revealed potential serum metabolites involved in oxidative stress and inflammation, degradation of lipid and protein in broiler chicks with immune stress. In addition, the protective effect of Rg3 on the stressed chicks may be largely mediated by BCAA metabolism, apoptosis and mTOR signaling pathway. These results suggested the potential biomarkers involved in pathogenesis and prevention of stress induced by Escherichia coli lipopolysaccharide.

Ginsenoside Rg3 Ameliorates Stress of Broiler Chicks Induced by Escherichia coli Lipopolysaccharide.

In broiler chicks, Escherichia coli lipopolysaccharide is a prominent cause for inflammatory damage and loss of immune homeostasis in broiler chicks. Ginsenosides have been shown to have anti-inflammatory and antioxidant effects. However, it has not been demonstrated that ginsenosides protect broiler chicks against stress induced by Escherichia coli lipopolysaccharide challenge. The aim of this is to investigate the protective effect of ginsenosides Rg1, Re, and Rg3 on Escherichia coli lipopolysaccharide-induced stress. Our results showed that Rg3 ameliorated growth inhibition and fever, as well as decreased the production of stress-related hormones in broilers with stress. The protective effect of Rg3 on the stressed chicks may be largely mediated by regulating inflammatory response and oxidative damage. Moreover, real-time quantitative-polymerase chain reaction (RT-qPCR) results demonstrated that Rg3 upregulated mRNA expression of mTOR, HO-1, and SOD-1. These results suggested that ginsenoside Rg3 and ginsenoside products contains Rg3 deserve further study for the control of immunological stress and inflammation in broiler chicks.

Higher immune response induced by vaccination in Houhai acupoint relates to the lymphatic drainage of the injection site.

Houhai acupoint (HA) is a site for acupuncture stimulation, located in the fossa between the anus and tail base in animals. To evaluate HA as a potential immunization site, the immune responses were compared when HA and the conventional site nape were vaccinated in rats. The results showed that injection of a porcine epidemic diarrhea virus (PEDV) vaccine in HA induced significantly higher IgG, IgG1, IgG2, splenocyte proliferation and mRNA expression of IL-2, IL-4 and IFN-γ than in the nape. To search for the underlying mechanisms, the draining lymph nodes for HA and the nape were investigated. When rats were injected in HA with Indian ink, 11 lymph nodes including caudal mesenteric lymph node and bilateral gluteal lymph nodes, posterior inguinal lymph nodes, lumbar lymph nodes, internal iliac lymph nodes and popliteal lymph nodes were visibly stained with the ink and injection of a model antigen ovalbumin (OVA) in HA resulted in detection of OVA by western blotting while in the same lymph nodes only a pair of lymph nodes (central brachial lymph nodes) were observed when Indian ink or OVA was injected in the nape. IL-2 mRNA expression was detected in all the lymph nodes when PEDV vaccine was injected. Therefore, the enhanced immune response elicited by vaccination in HA may be attributed to more lymphocytes activated.

Protective Effect of Ginsenoside Rg1 on Oxidative Damage Induced by Hydrogen Peroxide in Chicken Splenic Lymphocytes.

Previous investigation showed that ginsenoside Rg1 (Rg1) extracted from Panax ginseng C.A. Mey has antioxidative effect on oxidative stress in chickens. The present study was designed to investigate the protective effects of Rg1 on chicken lymphocytes against hydrogen peroxide-induced oxidative stress and the potential mechanisms. Cell viability, apoptotic cells, malondialdehyde, activity of superoxide dismutase, mitochondrial membrane potential, and [Ca2+]i concentration were measured, and transcriptome analysis and quantitative real-time polymerase chain reaction were used to investigate the effect of Rg1 on gene expression of the cells. The results showed that treatment of lymphocytes with H2O2 induced oxidative stress and apoptosis. However, pretreatment of the cells with Rg1 dramatically enhanced cell viability, reduced apoptotic cells, and decreased oxidative stress induced by H2O2. In addition, Rg1 reduced these H2O2-dependent decreases in mitochondrial membrane potential and reversed [Ca2+]i overload. Transcriptome analysis showed that 323 genes were downregulated and 105 genes were upregulated in Rg1-treated cells. The differentially expressed genes were involved in Toll-like receptors, peroxisome proliferator-activated receptor signaling pathway, and cytokine-cytokine receptor interaction. The present study indicated that Rg1 may act as an antioxidative agent to protect cell damage caused by oxidative stress via regulating expression of genes such as RELT, EDA2R, and TLR4.

Protective effects of Panax notoginseng saponins on PME-Induced nephrotoxicity in mice.

Polymyxin E (PME) plays an important role in fighting against Gram-negative bacterial infections; however, it causes nephrotoxicity, which limits its clinical use. The aim of this study was to investigate the protective effects of a plant extract Panax notoginseng saponins (PNS) on PME-induced nephrotoxicity in mice. In vivo studies showed that PNS significantly reduced blood urea nitrogen (BUN), serum creatinine (CRE) and number of apoptotic cells in kidney, as well as renal histopathological damage which increased in the presence of PME, and suppressed PME-induced oxidative stress in kidney, as shown by the up-regulation of superoxide dismutase (SOD) and the down-regulation of malondialdehyde (MDA) levels. Furthermore, PNS inhibited the expression of Bax, while increased the expression of Bcl-2 compared to the PME-treated group. In vitro studies showed that PNS decreased intracellular reactive oxygen species (ROS) and MDA levels, increased glutathione (GSH) levels, and enhanced the activity of SOD and glutathione peroxidase (GSH-Px) in murine renal tubular epithelial cells (TCMK-1 cells). In addition, PNS enhanced cell viability and the expression of Bcl-2, restored the mitochondrial membrane potential, inhibited the expression of Bax, inhibited the activity of caspase-3 and caspase-9, and reduce apoptotic rate in PME-treated TCMK-1 cells. PNS could reduce PME-induced nephrotoxicity. The protective effects could result from inhibition of oxidative stress, and prevention of cell apoptosis via the mitochondrial pathway. These findings highlight the potential of PNS as a safe adjunct for ameliorating the nephrotoxicity.

Soybean oil containing ginseng saponins as adjuvants promotes production of cytokines and enhances immune responses to foot-and-mouth disease vaccine.

In the present study, the adjuvant effect of soybean oil containing ginseng root saponins (SO-GS-R) on the immune response to foot-and-mouth disease vaccine (FMDV) in mice was investigated. When immunized with FMDV antigen emulsified in an SO-GS-R formulation, mice generated remarkably higher serum antibody and cytokine responses than mice immunized with FMDV antigen alone. To elucidate the mechanisms underlying the adjuvant effect of SO-GS-R, we measured cytokines in serum and muscle tissue after intramuscular injection of SO-GS-R. The results showed that injection of SO-GS-R significantly increased the levels of IL-1ß, IL-5, IL-6, G-CSF, KC, MCP-1, MIP-1α, and MIP-1ß in both serum and muscle. These results suggested that SO-GS-R recruits neutrophils, eosinophils, T cells and macrophages, causing immune cell recruitment at the injection site, driving antigen-presenting cells to actively participate in the onset of immunity, and amplifying the immune responses. Considering its adjuvant activity and plant-derived properties, SO-GS-R should be further studied for its adjuvant effect on vaccines used in food animals.

Improved immune response to an attenuated pseudorabies virus vaccine by ginseng stem-leaf saponins (GSLS) in combination with thimerosal (TS).

Vaccination using attenuated vaccines remains an important method to control animal infectious diseases. The present study evaluated ginseng stem-leaf saponins (GSLS) and thimerosal (TS) for their adjuvant effect on an attenuated pseudorabies virus (aPrV) vaccine in mice. Compared to the group immunized with aPrV alone, the co-inoculation of GSLS and/or TS induced a higher antibody response. Particularly, when administered together with GSLS-TS, the aPrV vaccine provoked a higher serum gB-specific antibody, IgG1 and IgG2a levels, lymphocyte proliferative responses, as well as production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) from lymphocytes, and more importantly provided an enhanced cytotoxicity of NK cells and protection against virulent field pseudorabies virus challenge. Additionally, the increased expression of miR-132, miR-146a, miR-147 and miR-155 was found in murine macrophages cultured with GSLS and/or TS. These data suggest that GSLS-TS as adjuvant improve the efficacy of aPrV vaccine in mouse model and have potential for the development of attenuated viral vaccines.

Isolation and characterization of peripheral blood-derived endothelial progenitor cells from broiler chickens.

Peripheral blood-derived endothelial progenitor cells (EPCs) have been extensively studied in mammals but the isolation and characterization of EPCs in avian species have not been reported. In this study, chicken peripheral blood mononuclear cells (PBMNCs) were cultured under conditions favoring endothelial-specific differentiation for 2 weeks. One heterogeneous cell population dominated by spindle-shaped cells (early EPCs) and one homogeneous cell population exhibiting cobblestone-like morphology (endothelial outgrowth cells, EOCs) appeared sequentially. Quantitative polymerase chain reaction (PCR) showed the expression of several progenitor and endothelial cell markers such as CD133, VEGFR-2 and CD31 in both cell populations. However, CD34, another progenitor marker, was undetectable in either freshly isolated PBMNCs or cultured cells. The endothelial phenotype of the EOCs was further identified by acetylated low-density lipoprotein/lectin double staining, and in vitro tube formation. Collectively, these data demonstrate that chicken EPCs can be isolated and cultured from PBMNCs and suggest that EPCs obtained from peripheral blood may originate mainly from the CD34- subpopulation.

Involvement of matrix metalloproteinase-2 in medial hypertrophy of pulmonary arterioles in broiler chickens with pulmonary arterial hypertension.

Medial hypertrophy of pulmonary arterioles during pulmonary arterial hypertension (PAH) in humans is associated with enhanced proliferation of smooth muscle cells (SMCs). Elevated matrix metalloproteinase (MMP)-2 has been found in pulmonary artery SMCs (PA-SMCs) in humans with idiopathic PAH, leading to the hypothesis that MMP-2 contributes to the proliferation and migration of vascular SMCs in the pathogenesis of PAH. Rapidly growing meat-type (broiler) chickens provide a model of spontaneous PAH. The present study was conducted to determine whether MMP-2 is involved in the medial hypertrophy of pulmonary arterioles in this model. Cultured PA-SMCs from normal birds were used to evaluate the effect of MMPs on cell proliferation. Gelatin zymography showed that endothelin (ET)-1-induced proliferation of PA-SMCs was concomitant with increased pro- and active MMP-2 production. Reverse transcription PCR demonstrated upregulation of MMP-2 mRNA. However, PA-SMC proliferation was inhibited by the MMP inhibitors doxycycline and cis-9-octadecenoyl-N-hydroxylamide. In vivo experiments revealed a significant increase of MMP-2 expression in hypertrophied pulmonary arterioles of PAH broiler chickens, which was positively correlated with wall thickness and medial hypertrophy. MMP-2 may contribute to medial hypertrophy in pulmonary arterioles during PAH in broiler chickens by enhancing the proliferation of vascular SMCs.