The gut microbiota in experimental abdominal aortic aneurysm.

Background: Abdominal aortic aneurysm (AAA) is a life-threatening disease and there are no effective treatments to inhibit aneurysm progression and rupture. The gut microbiota has been increasingly recognized, as a new therapeutic target, because of its role in host homeostasis. However, the role of the gut microbiota in AAA has not been clarified. Therefore, we performed 16S rRNA analysis to determine and compare the composition of the gut microbiota between AAA and control groups. Methods: We used the classical angiotensin-II induced AAA mouse model to investigate the role of gut microbiota and abdominal aortic aneurysm. The mice were randomly assigned to 2 groups: the control (n = 7) group received saline (vehicle), while the AAA (n = 13) group received solutions of Ang II. Aortic tissue and fecal samples were harvested 28 days after infusion. Fecal samples were analyzed by 16S rRNA sequencing. Results: The levels of Oscillospira, Coprococcus, Faecalibacterium prausnitzii, Alistipes massiliensis, and Ruminococcus gnavus were increased in the AAA group, while those of Akkermansia muciniphila, Allobaculum, and Barnesiella intestinihominis were increased in the control group. Furthermore, network analysis and ZiPi score assessment highlighted species in the phylum Bacteroidetes as the keystone species. PICRUSt2 analysis revealed that PWY-6629 (a super pathway of L-tryptophan biosynthesis), PWY-7446 (sulfoglycolysis), and PWY-6165 [chorismate biosynthesis II (archaea)] may-be involved in the metabolic pathways that contribute to AAA formation, and E. coli/Shigella may be the key bacteria that influence those three pathways. Conclusion: Alterations in the gut microbiota may be associated with the formation of AAA. Akkermansia and Lactobacillus were significantly decreased in the AAA group, but the keystone species in the phylum Bacteroidetes and the metabolic products of these bacteria should be given more attention in AAA formation research.

4-Octyl Itaconate Alleviates Lipopolysaccharide-Induced Acute Lung Injury in Mice by Inhibiting Oxidative Stress and Inflammation.

BACKGROUND: Acute lung injury (ALI) is a fatal disease in the absence of pharmacological treatment. Oxidative stress and inflammation are closely related to ALI. Innate immune cells are the main source of reactive oxygen species (ROS). Macrophages play an extremely important role in ALI through the activation of inflammation and oxidative stress. Itaconate, a metabolite of tricarboxylic acid, has been reported to have strong antioxidant and anti-inflammatory effects. However, the role of itaconate in ALI is unclear. Herein, we use 4-octyl itaconate (OI), the cellular permeable derivate of itaconate, to study the effects of itaconate in vivo and in vitro. METHODS: We used OI to pretreat C57BL/6 mice and LPS-induced ALI models to illustrate the role of itaconate in acute lung injury. The mice were randomly divided into four groups: control group, OI (100 mg/kg) group, ALI Group, ALI + OI (50 mg/kg) group, and ALI + OI (100 mg/kg) group. RAW264.7 cells were used to further prove the role and mechanism of itaconate in vitro. RESULTS: According to the H&E staining of the lung, OI was observed to significantly reduce lung inflammation. The active oxygen content of tissues was also significantly reduced (P<0.05). OI reduced the accumulation of neutrophils and secretion of inflammatory factors in LPS-induced ALI (P<0.05). At the cellular level, OI also reduced oxidative stress and inflammation. Intervention with OI was also observed to upregulate the expression of nuclear factor erythroid 2-related factor-2 (Nrf-2) and Nrf-2 target genes in the lung tissue and RAW264.7 cells. CONCLUSION: OI alleviates LPS-induced ALI. Moreover, the antioxidant and anti-inflammatory effects of OI might depend on the activation of Nrf-2. Therefore, OI might have therapeutic potential for the treatment of ALI.

Modulating Neuro-Immune-Induced Macrophage Polarization With Topiramate Attenuates Experimental Abdominal Aortic Aneurysm.

The development of abdominal aortic aneurysm (AAA) is attributed to psychological and physical factors. Topiramate, which is an agonist of the GABAA receptor, makes contributions to neuronal disease and is partially involved in immune regulation, may be effective upon abdominal aortic aneurysm progression. We used experimental abdominal aortic aneurysm models: Angiotensin II (Ang II)-induced ApoE-/- male mice (Ang II/APOE model) in our study. In the Ang II/APOE model, all mice (n=64) were divided into four groups: sham group (PBS treatment), control group (Ang II treatment), low-dose group (Ang II + low-dose topiramate, 3 mg/day per mouse), and high-dose group (Ang II + high-dose topiramate, 6 mg/day per mouse). All treatments began on the day after surgery. Moreover, collected tissues and cultured cell were used for histology and biochemical examination. In vitro, the effects of topiramate on bone marrow-derived macrophage stimulated by LPS were investigated. Our data implied that topiramate treatment significantly promoted macrophages preservation and conversion of M1 to M2 macrophage phenotypes in vivo and in vitro. Accordingly, proinflammatory activities mediated by the M1 macrophages were decreased and the repair process mediated by M2 macrophages was enhanced. The low-dose and high-dose groups had abdominal aortic aneurysm incidences of 50% and 37.5%, respectively, compared with 75% in the control group. Topiramate, a promising drug for the psychological disease, that target neuro-immune-induced macrophage polarization may attenuate experimental abdominal aortic aneurysm progression.

All-trans retinoic acid attenuates the progression of Ang II-induced abdominal aortic aneurysms in ApoE-/-mice.

BACKGROUND: To determine whether all-trans retinoic acid (ATRA) can influence the development of Angiotensin II (Ang II) induced experimental abdominal aortic aneurysms (AAAs). METHODS: Apolipoprotein E knock-out (ApoE-/-) mice were randomly assigned to 4 groups. Mice in the AAA and ATRA groups underwent continuous subcutaneous Ang II infusion for 28 days to induce AAA, while the Sham and Control groups were infused with saline. Systolic blood pressure was measured by the tail-cuff technique. The Control and ATRA groups received ATRA treatment. Aortic tissue samples were obtained at 28 days after surgery and evaluated by aortic diameter measurement, Western blotting, immunohistochemistry, and hematoxylin-eosin (H&E) and Verhoeff-Van Gieson (EVG) staining. RESULTS: The abdominal aortic diameter was significantly reduced in the ATRA group compared with the AAA group (3 of 12 (25%) vs 9 of 12 (75%), P < 0.05), and the ATRA group exhibited reduced blood pressure on days 7, 14, and 28. Low expression of angiotensin II receptor type 1 (AT1), matrix metalloproteinase 2 (MMP2), and matrix metalloproteinase 9 (MMP9) and EVG staining revealed a significant reduction in the disruption of elastic fibers in the abdominal aortic tissue of the ATRA group compared to the AAA group. Western blot analysis indicated that protein levels of retinoic acid receptor α (RARα), MMP2, MMP9, and AT1 were dramatically affected by ATRA treatment. CONCLUSIONS: In conclusion, ATRA attenuates the progression of Ang II-induced AAAs, possibly by downregulating MMP2, MMP9, and AT-1 expression.

Experimental abdominal aortic aneurysm growth is inhibited by blocking the JAK2/STAT3 pathway.

BACKGROUND: The JAK/STAT pathway is a vital transcription signaling pathway that regulates gene expression and cellular activity. Our recently published study highlighted the role of IL-17A in abdominal aortic aneurysm (AAA) formation and rupture. IL-17A has been proven to upregulate vascular endothelial growth factor (VEGF) expression in some diseases. However, no study has demonstrated the relationships among JAK2/STAT3, IL-17A and VEGF. Therefore, we hypothesized that IL-17A may up-regulate VEGF expression via the JAK2/STAT3 signaling pathway to amplify the inflammatory response, exacerbate neovascularization, and accelerate AAA progression. METHODS: To fully verify our hypothesis, two separate studies were performed: i) a study investigating the influence of JAK2/STAT3 on AAA formation and progression. ii) a study evaluating the relationship among IL-17A, JAK2/STAT3 and VEGF. Human tissues were collected from 7 AAA patients who underwent open surgery and 7 liver transplantation donors. All human aortic tissues were examined by histological and immunohistochemical staining, and Western blotting. Furthermore, mouse aortic tissues were also examined by histological and immunohistochemical staining and Western blotting, and the mouse aortic diameters were assessed by high-resolution Vevo 2100 microimaging system. RESULTS: Among human aortic tissues, JAK2/STAT3, IL-17A and VEGF expression levels were higher in AAA tissues than in control tissues. Group treated with WP1066 (a selective JAK2/STAT3 pathway inhibitor), IL-17A, and VEGF groups had AAA incidences of 25%, 40%, and 65%, respectively, while the control group had an incidence of 75%. Histopathological analysis revealed that the IL-17A- and VEGF-related inflammatory responses were attenuated by WP1066. Thus, blocking the JAK2/STAT3 pathway with WP1066 attenuated experimental AAA progression. In addition, in study ii, we found that IL-17A siRNA seemed to attenuate the expression of IL-17A and VEGF in vivo study; treatment with VEGF siRNA decreased the expression of VEGF, while IL-17A expression remained high. In an in vitro study, rhIL-17A treatment increased JAK2/STAT3 and VEGF expression in macrophages in a dose-dependent manner. CONCLUSION: Blocking the JAK2/STAT3 pathway with WP1066 (a JAK2/STAT3 specific inhibitor) attenuates experimental AAA progression. During AAA progression, IL-17A may influence the expression of VEGF via the JAK2/STAT3 signaling pathway. This potential mechanism may suggest a novel strategy for nonsurgical AAA treatment.

MicroRNA­124 suppresses cell proliferation and invasion of triple negative breast cancer cells by targeting STAT3.

MicroRNAs (miRNAs) are pivotal regulators of the progression of carcinogenesis and negatively regulate the expression of tumour­associated genes. Downregulation of miR­124 expression has been demonstrated in various human cancer tissues, wherein miR­124 serves as a tumour suppressor by targeting oncogenes. However, its function and underlying mechanism of action remain unclear in breast cancer. In the present study, the tissue­specific expression of miR­124 was detected in 10 paired triple­negative breast cancer and normal tissues, and its inhibitory effects on cell growth and invasion were evaluated in vitro and in vivo. Bioinformatics analysis identified signal transducer and activator of transcription 3 (STAT3), a well­known oncogene in breast cancer, as the potential target. Upregulation of miR­124 expression decreased STAT3 mRNA and protein levels in breast cancer cells and the relative luciferase activity. Rescue experiments revealed that the transfection of a STAT3 expression plasmid reversed the inhibitory effect of miR­124 on the proliferation and invasion of MDA­MB­468 cells. These data demonstrate that miR­124 serves vital roles in the suppression of triple­negative breast cancer via inhibition of cell proliferation and invasion through STAT3. These results highlight the potential role of miR­124 as a diagnostic or therapeutic target in patients with breast cancer.

Preferred Revascularization Strategies in Patients with Ischemic Heart Failure: A Meta-Analysis.

Clinically, coronary artery bypass grafting (CABG) or percutaneous coronary intervention (PCI) is generally used to treat patients with ischemic heart failure. However, the optimal treatment strategy remains unknown. This study examined the efficacy of the two coronary revascularization strategies for severe ischemic heart failure by using a meta-analysis. Studies comparing the efficacy of CABG and PCI were obtained from PubMed, EMBASE, Google Scholar and Cochrane Central Register of Controlled Trials (CENTRAL). The quality of each eligible article was evaluated by Newcastle-Ottawa Quality Assessment Scale (NOS), and the meta-analysis was performed using Stata version 12.0 software. Eventually, 12 studies involving 9248 patients (n=4872 in CABG group; n=4376 in PCI group) were subject to the meta-analysis for subsequent pooling calculation. The pooled hazard ratio (HR) [HR=0.83, 95% CI (0.76, 0.90), P<0.001; heterogeneity, P=0.218, I2=22.9%] of CABG compared with that of PCI revealed a statistical superiority of CABG to PCI in terms of the long-term mortality. Furthermore, CABG showed more advantages over PCI with respect to the incidence of myocardial infarction [HR=0.51, 95% CI (0.39, 0.67), P<0.001; heterogeneity, P=0.707, I2=0%] and repeat revascularization [HR=0.40, 95% CI (0.27, 0.59), P<0.001; heterogeneity, P<0.001, I2=80.1%]. It was concluded that CABG appears to be more advantageous than PCI for the treatment of ischemic heart failure in the given clinical setting.

Targeting the class IA PI3K isoforms p110α/δ attenuates heart allograft rejection in mice by suppressing the CD4+ T lymphocyte response.

Acute rejection is the most important factor causing allograft loss, which remains a challenge for patients undergoing organ transplantation. There is considerable evidence indicating that the activity of PI3K and its downstream positive and negative regulators plays a major role in regulating the activation of different subsets of effector CD4+ T cells. Thus, we investigated whether class A PI3Ks are involved in the development of acute allograft rejection, we found that p110α protein expression levels in the allograft group were significantly up-regulated on day 7 post-transplantation, while p110ß and p110δ expression was significantly increased on days 5 and 7 post-transplantation. Treatment with PIK and IC but not TGX significantly prolonged allograft survival and altered pathological grades. The percentages of Th1 and Th2, Th17 and Tfh cells/monocytes in the spleens from the IC treatment group were all down-regulated. In contrast, the percentage of Treg cells in the spleens from IC treatment group was remarkably increased. IL-17A and IL-21 and IFN-γ expression levels were significantly decreased in the IC group. Moreover, IC significantly reduced P70 S6 Kinase ß and 4E-BP1 protein expression. In conclusion, small-molecule inhibitors of p110δ and p110α suppress acute heart allograft rejection in mice. These inhibitors may play a role in anti-rejection by impacting the phosphorylation and expression of proteins in the AKT/mTOR pathway to modulate CD4+ T cell subsets levels in recipients, reduce proinflammatory factor expression and increase anti-inflammatory cytokine expression. These findings indicate that some small-molecule inhibitors of p110 can serve as novel targets in acute allograft rejection treatment.

Organ transplantation and gut microbiota: current reviews and future challenges.

Organ transplantation is often the only effective treatment for patients with end-stage diseases, such as heart, liver, kidney and small bowel failure and is carried out frequently worldwide. Still the post-transplantation complications remain health- and life-threatening outcome that needed to be resolved. With the rapid development of molecular technologies in recent years, more and more researchers realize that the gut microbiota may play a critical role in human diseases. The intestinal microbiome has been proved to provide a lot of functions to the host, such as digesting food, modulating metabolism, promoting angiogenesis and regulating the immune system. Several studies have investigated the alteration of intestinal microbiota in post-transplantation patients and observed significant changes in the intestinal microbiome compared to the pre-transplant condition. Due to the abovementioned features that the gut microbiota may be used in the prognosis of clinical outcome of organ transplantation. In addition, the FMT (fecal microbiota transplantation), probiotics and prebiotics as the newest therapy methods, effectiveness of which has been verified in some diseases, such as Clostridium difficile infection, inflammatory bowel disease and other chronic disorders, might be used as the prognosis tool in organ transplantation as well. The purpose of this present review is to elucidate the relationship between gut microbiota and organ transplantation as well as the potential use of new therapies like fecal microbiota transplantation, probiotic and prebiotic administration after the transplantation, and provide some ideas for future researches in field of organ transplantation.

Inhibiting the Th17/IL-17A-related inflammatory responses with digoxin confers protection against experimental abdominal aortic aneurysm.

OBJECTIVE: T helper 17 cells and interleukin-17A have been implicated in the progression of abdominal aortic aneurysm (AAA). Retinoic acid-related orphan receptor gamma thymus, the master transcription factor of T helper 17 cell differentiation, is selectively antagonized by digoxin. However, the effect of antagonizing retinoic acid-related orphan receptor gamma thymus on AAA has not been investigated. APPROACH AND RESULTS: We used human aortic sample analysis and 2 different experimental AAA models: (a) Angiotensin II (Ang II)-induced ApoE(-/-) male mice (Ang II/APOE model) and (b) porcine pancreatic elastase perfusion C57BL/6 mice (porcine pancreatic elastase/C57 model). In the Ang II/APOE model, all mice (n=80) were divided into 4 groups: sham group (saline+0.5% dimethyl sulfoxide treatment), control group (Ang II+0.5% dimethyl sulfoxide treatment), low-dose group (Ang II+low-dose digoxin, 20 µg/d per mouse), and high-dose group (Ang II+high-dose digoxin, 40 µg/d per mouse). All treatments began on day 0 after surgery. Efficacy was determined via aortic diameter and systolic blood pressure measurements, histopathology and protein expression, and flow cytometry analysis when euthenized. Human aortic tissue analysis showed that both interleukin-17A and retinoic acid-related orphan receptor gamma thymus increased in AAA tissues. The low-dose and high-dose groups had AAA incidences of 60% and 35%, respectively, compared with 70% in the control group. The T helper 17- and interleukin-17A-related inflammatory responses were dose-dependently attenuated by digoxin treatment. Digoxin was also highly effective in the porcine pancreatic elastase/C57 model. CONCLUSIONS: Digoxin attenuates experimental AAA progression in a model-independent manner. Antagonizing retinoic acid-related orphan receptor gamma thymus activity by digoxin may become a novel strategy for nonsurgical AAA treatment.

Inhibitory effects of suppressor of cytokine signaling 3 on inflammatory cytokine expression and migration and proliferation of IL-6/IFN-γ-induced vascular smooth muscle cells.

The main pathogenesis of saphenous vein graft neointimal hyperplasia after coronary artery bypass grafting (CABG) is inflammation-caused migration and proliferation of vascular smooth muscle cells (VSMCs). Janus kinase 2/signal transducer and activators of transcription 3 (JAK2/STAT3) pathway is an important signaling pathway through which VSMCs phenotype conversion occurs. Suppressor of cytokine signaling 3 (SOCS3) is the classic negative feedback inhibitor of JAK2/STAT3 pathway. Growing studies show that SOCS3 plays an important anti-inflammatory role in numerous autoimmune diseases, inflammatory diseases and inflammation-related tumors. However, the effect and mechanism of SOCS3 on vein graft disease is unclear. The purpose of this study was to investigate the effects of SOCS3 on the inflammation, migration and proliferation of VSMCs in vitro and the mechanism. The small interference RNA plasmid targeting rat SOCS3 (SiRNA-rSOCS3) and the recombinant adenovirus vector carrying rat SOCS3 gene (pYrAd-rSOCS3) were constructed, and the empty plamid (SiRNA-control) and vector (pYrAd-GFP) only carrying GFP reported gene were constructed as control. The rat VSMCs were cultured. There were two large groups of A (SOCS3 up-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+pYrAd-rSOCS3 group, IL-6/IFN-γ(+)pYrAd-GFP group; and B (SOCS3 down-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+SiRNA-rSOCS3 group and IL-6/ IFN -γ+SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs induced by IL-6/IFN-γ. After 24 h, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of SOCS3, STAT3 (only by Western blotting), P-STAT3 (only by Western blotting), IL-1ß, IL-6, TNF-α, MCP-1 and ICAM-1. The MTT, Transwell assay and flow cytometry were used to examine VSMCs proliferation, migration and cell cycle progression, respectively. As compared with control group, the mRNA and protein expression of SOCS3, STAT3, P-STAT3, IL-1ß, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly up-regulated in VSMCs stimulated by IL-6/IFN-γ. However, in VSMCs transfected with pYrAd-rSOCS3 before stimulation with IL-6/IFN-γ, the expression of SOCS3 mRNA and protein was further up-regulated, and that of STAT3, P-STAT3, IL-1ß, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly down-regulated as compared with IL-6/IFN-γ group and IL-6/IFN-γ+pYrAd-GFP group. The expression of those related-cytokines in IL-6/IFN-γ+SiRNA-rSOCS3 group was markedly increased as compared with IL-6/IFN-γ group and IL-6/IFN-γ+SiRNA-control group. The absorbance (A) values, the number of cells migrating to the lower chamber, and percentage of cells in the G2/M+S phase were increased in VSMCs stimulated by IL-6/IFN-γ. In VSMCs incubated with pYrAd-rSOCS3 or SiRNA-rSOCS3 before IL-6/IFN-γ stimulation, the A values, the number of cells migrating to the lower chamber, and the percentage of cells in the G2/M+S phase were significantly decreased, and increased respectively. These results imply that IL-6/IFN-γ, strong inflammatory stimulators, can promote transformation of VSMCs phenotype form a quiescent contractile state to a synthetic state by activating JAK2/STAT3 pathway. Over-expresssed SOCS3 might inhibit pro-inflammatory effect, migration and growth of VSMCs by blocking STAT3 activation and phosphorylation. These data in vitro confirm that SOCS3 may play a negatively regulatory role in development and progression of vein graft failure. These conclusions can provide a novel strategy for clinical treatment of vein graft diseases and a new theoretic clue for related drug development.