Background Unmanaged bone exposed wounds that don't receive proper treatment can potentially lead to additional complications. Human umbilical cord mesenchymal stem cell (HUC-MSC)-derived exosomes (EXO) have a significant effect in promoting wound healing. Methods HUC-MSCs and HUC-MSC-derived EXOs were added to a CS to prepare HUC-MSC + CS and HUC-MSC EXO + CS complexes, respectively. The CS and prepared CS complexes were observed by scanning electron microscopy and applied to bone-exposed large-area skin wounds in rabbits. Then, wound tissues were collected and analyzed. Hematoxylin and eosin and Masson staining were performed to observe histopathological features in wound tissues. Immunohistochemistry and immunofluorescence were performed to observe neovascularization and neural fiber density. The expressions of growth factors (platelet-derived growth factor receptor β [PDGFR-β], vascular endothelial growth factor receptor 2 [VEGFR2], and epidermal growth factor receptor [EGFR]) in wound tissues were detected by western blotting. Results On healing days 3, 7, 10, 14, and 16, compared with the control group, wounds in the CS group healed well. Coverage by collagen fiber was evident, angiogenic factor CD34 expression and neural fiber density increased, along with high EGFR, PDGFR-β, and VEGFR2 levels. Compared with the CS group, the HUC-MSC + CS and HUC-MSC EXO + CS groups had a better neovascularization and wound healing. In particular, the HUC-MSC EXO + CS group showed abundant collagen. Conclusion In the treatment of bone-exposed wounds, HUC-MSCs and HUC-MSC-derived EXOs loaded in CS were more efficient than CS alone, contributing to neovascularization in wounds.

BACKGROUND:Antiapoptosis is a major factor in the resistance of tumor cells to chemotherapy and radiotherapy. Thus, activation of cell pyroptosis may be an effective option to deal with antiapoptotic cancers such as esophageal adenocarcinoma (EAC). METHODS:Differential expression of ubiquitin-like versus PHD and ring finger structural domain 1 (UHRF1) in EAC and near normal tissues was analyzed, as well as the prognostic impact on survival in EAC. Also, the same study was done for globular adiponectin (gAD). Simultaneously, the mRNA expression of UHRF1 was observed in different EAC cell lines. Real time cellular analysis (RTCA) was used to detect cell proliferation, and flow cytometry and inverted fluorescence microscopy were used to detect pyroptosis. Biocredit analysis was conducted to observe the correlation between UHRF1 and key pyroptosis proteins. OD values and CCK8 assay were used to determine the effect of miR-378a-3p on EAC cells. Quantitative real-time polymerase chain reaction and Western blot were used to detect the correlation between UHRF1, gAD, and miR-378a-3p in EAC cells. Moreover, in vivo and in vitro experiments were performed to detect the relevant effects on tumor migration and invasion after inhibiting UHRF1 expression. RESULTS:UHRF1 was negatively correlated with the survival of patients with EAC, while miR-378a-3p showed the opposite effect. Additionally, gAD promoted EAC cell pyroptosis, upregulated miR-378a-3p, and significantly inhibited the proliferation of EAC cells. gAD directly reduced UHRF1 expression in EAC cells by upregulating miR-378a-3p. In cell migration and invasion assays, inhibition of UHRF1 expression significantly suppressed EAC cell metastasis. In animal experiments, we again demonstrated that gAD induced pyroptosis in EAC cells by inhibiting the expression of UHRF1. CONCLUSION:gAD-induced upregulation of miR-378a-3p significantly inhibited the proliferation of EAC by targeting UHRF1. Therefore, gAD may serve as an alternative therapy for chemotherapy- and radiation-refractory EAC or other cancers with the same mechanism of pyroptosis action.

Extracellular matrix (ECM) stiffness is closely related to the progress of diabetic cardiomyopathy (DCM) and the response of treatment of DCM to anti-diabetic drugs. Dapagliflozin (Dapa) has been proven to have cardio-protective efficacy for diabetes and listed as the first-line drug to treat heart failure. But the regulatory relationship between ECM stiffness and treatment efficacy of Dapa remains elusive. This work investigated the effect of ECM stiffness on DCM progression and Dapa efficacy using both in vivo DCM rat model and in vitro myocardial cell model with high glucose injury. First, through DCM rat models with various levels of myocardial injury and administration with Dapa treatment for four weeks, the levels of myocardial injury, myocardial oxidative stress, expressions of AT1R (a mechanical signal protein) and the stiffness of myocardial tissues were obtained. Then for mimicking the stiffness of myocardial tissues at early and late stages of DCM, we constructed cell models through culturing H9c2 myocardial cells on the polyacrylamide gels with two stiffness and exposed to a high glucose level and without/with Dapa intervention. The cell viability, reactive oxygen species (ROS) levels and expressions of mechanical signal sensitive proteins were obtained. The DCM progression is accompanied by the increased myocardial tissue stiffness, which can synergistically exacerbate myocardial cell injury with high glucose. Dapa can improve the ECM stiffness-induced DCM progression and its efficacy on DCM is more pronounced on the soft ECM, which is related to the regulation pathway of AT1R-FAK-NOX2. Besides, Dapa can inhibit the expression of the ECM-induced integrin β1, but without significant impact on piezo 1. Our study found the regulation and effect of biomechanics in the DCM progression and on the Dapa efficacy on DCM, providing the new insights for the DCM treatment. Additionally, our work showed the better clinical prognosis of DCM under early Dapa intervention.

Multiple circular RNAs (circRNAs) were proven to regulate the development of pancreatic cancer. However, the action of circ_0018909 in pancreatic cancer was still unclear. The expression of circ_0018909, microRNA-545-3p (miR-545-3p), and fatty acid synthase (FASN) was measured using quantitative reverse-transcriptase PCR (qRT-PCR). Cell growth, cell cycle arrest, apoptotic cells, metastasis, and epithelial to mesenchymal transition (EMT) were determined using EdU assay, flow cytometry, wound-healing assay, transwell invasion, and western blotting, respectively. The expression of the macrophage markers, including CD80, MCP-1, iNOS, and IL-6 (M1 markers), as well as CD206 and CD163 (M2 markers), was analyzed using qRT-PCR. Circ_0018909 knockdown dramatically depressed cell growth, migration, invasion, EMT, and elevated the number of apoptotic cells in pancreatic cancer cells, and repressed tumor growth in mice. Moreover, we proved that the absence of miR-545-3p rescued the action of circ_0018909 downregulation on cell growth, metastasis, apoptosis, and EMT in pancreatic cancer cells. MiR-545-3p bound to FASN and FASN overexpression hindered the impacts of miR-545-3p on the progression of pancreatic cancer. Besides this, our data demonstrated that circ_0018909 induced polarization from M0 macrophages to M2 macrophages. Circ_0018909 knockdown retarded the development of pancreatic cancer by modulating miR-545-3p to regulate FASN expression.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for skin and soft tissue infections with multi-resistance to many antibiotics. It is thus imperative to explore alternative antimicrobial treatments to ensure future treatment options. Nisin (NIS), an antibacterial peptide produced by Lactococcus lactis, was selected to combine with Oxacillin (OX), to evaluate the antimicrobial effect and potential mechanism against MRSA. The synergistic antimicrobial effect of OX and NIS was verified by Minimal Inhibitory Concentration (MIC) assays, checkerboard analysis, time-kill curve, biofilm producing ability, and mice skin infection model in vivo. For the potential synergistic antimicrobial mechanism, the microstructure and integrity change of MRSA cells were determined by Scanning and Transmission Electron Microscope (SEM and TEM), intracellular alkaline phosphatase activity and propidium iodide staining were assayed; And transcription of mecA, main gene of MRSA resistant to OX, were detected by qRT-PCR. The results showed NIS could restore the sensitivity of MRSA to OX and inhibit biofilm production; OX + NIS can make MRSA cell deform; NIS may recover OX sensitivity by inhibiting the transcription of mecA. In vivo, mice skin infection models indicate that OX + NIS can substantially alleviate MRSA infections. As a safe commercially available biological compound, NIS and the combination of antibiotics are worth developing as new anti-MRSA biomaterials.

WWP2 is a HECT-type E3 ubiquitin ligase that regulates various physiological and pathological activities by binding to different substrates,but its role in atherosclerosis(AS)remains largely unknown.The objective of the present study is to investigate the role and underlying molecular mechanisms of WWP2 in endothelial injury.We found that WWP2 expression is significantly decreased in Apolipoprotein E(ApoE)-/-mice.Overexpression of WWP2 attenu-ates oxidative stress and inflammation in AS mice,while knockdown of WWP2 has opposite effects.WWP2 over-expression alleviates oxidized low-density lipoprotein(ox-LDL)-induced human umbilical vein endothelial cell(HUVEC)injury,evidenced by the decreased oxidative stress levels and the secretion of inflammatory cytokines.Programmed cell death 4(PDCD4)is identified as a potential substrate of WWP2.Co-immunoprecipitation(Co-IP)further demonstrates that WWP2 interacts with PDCD4,which is enhanced by ox-LDL treatment.Furthermore,the level of PDCD4 ubiquitination is significantly increased by WWP2 overexpression under the condition of MG132 treatment,while WWP2 knockdown shows opposite results.Subsequently,rescue experiments demonstrate that WWP2 knockdown further aggravates oxidative stress and inflammation in ox-LDL-treated HUVECs,while knock-down of PDCD4 alleviates this effect.Moreover,the use of sn-protoporphyrin(SnPP),an inhibitor of HO-1 pathway,confirms that PDCD4 enhances endothelial injury induced by ox-LDL through inhibiting HO-1 pathway.In conclu-sion,our results suggest that WWP2 protects against atherosclerosis progression via the PDCD4/HO-1 pathway,which may provide a novel treatment strategy for atherosclerosis.

Objectives The lung injury is often secondary to severe trauma. In the model of crush syndrome, there may be secondary lung injury. We hypothesize that high-mobility group box 1 (HMGB1), released from muscle tissue, mediates the apoptosis of alveolar epithelial cells (AEC) via HMGB1/Receptor of advanced glycation end-products (RAGE)/c-Jun N-terminal kinase (JNK) pathway. The study aimed to investigate how HMGB1 mediated the apoptosis of AEC in the rat model. Methods Seventy-five SD male rats were randomly divided into five groups: CS, CS + vehicle, CS + Ethyl pyruvate (EP), CS + FPS-ZM1 group, and CS + SP600125 groups. When the rats CS model were completed after 24 h, the rats were sacrificed. We collected the serum and the whole lung tissues. Inflammatory cytokines were measured in serum samples. Western blot and RT-qPCR were used to quantify the protein and mRNA. Lastly, apoptotic cells were detected by TUNEL. We used SPSS 25.0 for statistical analyses. Results Nine rats died during the experiments. Dead rats were excluded from further analysis. Compared to the CS group, levels of HMGB1 and inflammatory cytokines in serum were downregulated in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups. Western blot and RT-qPCR analysis revealed a significant downregulation of HMGB1, RAGE, and phosphorylated-JNK in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups, compared with the CS groups, excluding total-JNK mRNA. Apoptosis of AEC was used TUNEL to assess. We found the TUNEL-positive cells were downregulated in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups. Conclusion The remote lung injury begins early after crush injuries. The HMGB1/RAGE/JNK signaling axis is an attractive target to abrogate the apoptosis of AEC after crush injuries.

Bone marrow stromal stem cells (BMSCs) can be used to treat bone defects but BMSCs are damaged under oxidative stress. The neuropeptide substance P (SP) involves various cellular activities. However, SP’s role in BMSCs differentiation under oxidative stress is unknown. Rat BMSCs were isolated and assigned into control group; oxidative stress group treated with 200 μM H 2 O 2 ; and SP group, in which 10 mM SP was added under oxidative stress followed by analysis of SP secretion by ELISA, cell proliferation by MTT method, Caspase3 activity, Bax and Bcl-2 level by Real time PCR, ALP activity ROS and SOD content as well as NF-κB level by Western blot. Under oxidative stress, SP secretion was significantly decreased, BMSCs proliferation was inhibited, Caspase3 activity and Bax expression increased, Bcl-2 and ALP activity was decreased along with increased ROS activity and NF-κB level and reduced SOD activity ( P <0.05), adding SP to BMSCs under oxidative stress can significantly promote SP secretion and cell proliferation, reduce Caspase3 activity and Bax expression, increase Bcl-2 expression and ALP activity, decreased ROS activity and NF-κB level, and elevated SOD activity ( P <0.05). SP secretion from BMSCs cells was reduced under oxidative stress. Up-regulation of SP in BMSCs cells under oxidative stress can inhibit BMSCs apoptosis and promote cell proliferation and osteogenesis by regulating NF-κB.

Objective: Ischemic stroke leads to cellular death and tissue damage by depriving the areas of glucose and oxygen supplies. The effective treatment of stroke remains a challenge for modern medicine. This study used an oxygen-glucose deprivation (OGD) model of human umbilical vein endothelial cells (HUVECs) to mimic ischemic injuries and explored the role and mechanism of intelectin-1. Methods: Intelectin-1 was transduced into the HUVECs using a lentiviral vector. The PI3K/Akt signaling was examined in intelectin-induced eNOS phosphorylation. The PI3K inhibitor LY294002 was dealed in HUVECs. Results: Our results demonstrated an increase in capillary density, decrease in apoptotic cells, and increase in HIF-1α protein expression following intelectin-1 treatment. Real-time PCR and Western blotting revealed the increased intelectin-1 expression alongside eNOS and Akt phosphorylation with enhanced bcl-2 expression under OGD. Capillary density decreased significantly after LY294002 treatment. Conclusion: These results suggest intelectin-1 promotes angiogenesis, inhibits oxidative stress and reduces apoptosis by stimulating the Akt-eNOS signaling pathway in response to ischemia in vitro.

Study Design. Retrospective single-center study. Objective. We want to know whether interleukin (IL)-10-secreting regulatory T cells (Treg) promote the new bone formation (NBF) through suppressing Th17 in ankylosing spondylitis (AS). Summary of Background Data. NBF in AS is unknown. Since there are balances of bone remodeling in human body and proinflammatory helper T cells Th17 promoted bone resorption. Methods. Eighteen AS patients with or without NBF (both nine cases) and nine healthy individuals were selected and the demographic data, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), MRI sacroiliitis score (MRISIS), and computer tomography sacroiliitis score (CTSIS) were recorded. Removed hip ligament tissue in the lesions after arthroplasty was collected and the lymphocytes and the peripheral blood mononuclear cells were prepared. Second, pathological section in hematoxylin–eosin stain were analyzed and flow cytometry and quantitative polymerase chain reaction analyses were carried out to detect the levels of Th17, Treg, IL-10, and nuclear factor (NF)-κB, and the relevance between them. The effect of Treg on Th17 was further analyzed by using Transwell coculturing. Results. Compared to AS patients without NBF, AS patients with NBF had significantly higher CTSIS and complications (P < 0.05 and 0.01, respectively), but significantly lower BASDAI (3.0 ± 0.4) and MRISIS (3.3 ± 0.8) (P < 0.01 and 0.05, respectively) and no acute inflammation in HE stain for hip joint. Compared to healthy donors, the ratio of Th17/Treg was significantly higher in AS patients without NBF and lower in AS patient with NBF (both P < 0.01) in flow cytometry analysis (FCA). Furthermore, Th17 significantly decreased after indirectly coculturing with Treg in FCA (P < 0.01). Finally, IL-10 had significantly higher mRNA expression in AS patients with NBF (P < 0.01), and NF-κB had significantly higher mRNA expression in AS patients without NBF (P < 0.05) than healthy donors. Only the mRNA expression of IL-10 was significantly correlated to the ratio of Th17/Treg (r = −0.93, P < 0.01). Conclusion. Treg-induced NBF of AS through suppressing Th17 by secreting IL10 and declining of the ratio of Th17/Treg indicated the development of NBF. This is important not only for screening development of NBF, but also for control of NBF of AS by immune therapy. Level of Evidence: N/A

Introduction: Spontaneous rupture of the urinary bladder(SRUB) is a relatively rare cause of acute abdomen disease, whcih was easily complicated with diffuse peritonitis, septic shock, acute renal failure. Athough a few cases about postpartum patient sufferred from SRUB have been reported recently,the management of postpartum SRUB was not well established. Case Report: The subject of this case was a 37-year-old postpartum woman who presented with abdominal pain for one day.Based on his clinical presentation, investigation results, it was highly suspected for urinary disease complicated with diffuse peritonitis, such as SRUB, ureteral rupture. Given that vital signs were unstable and peritonitis signs were aggravating gradually, emergency laparoscopic exploration were performed. There was a visible rupture in the left posterior of the bladder wall, with a diameter of 2cm.Laparoscopic repair was employed and patient recovered well. In this article, we also reviewed relevant literatures to initially establish main diagnostic basis for postpartum SRUB. Conlcusion: Postpartum women with a complaint of acute abdomen pain maybe caused by SRUB, especially companied with oliguria and dysuria. And laparoscopy have an obvious advantage to the diagnosis and treatment for highly suspected postpartum SRUB.

Objective To investigate the effect of microRNA-133b(miR-133b)on cardiac fibrosis and its mechanism.Methods Human cardiac fibroblasts(CFs)were harvested.The proliferation of CFs was detected by CCK8 during the overexpression and knock-down of miR-133b.The expressions of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),collagen Ⅰ,and collagen Ⅲ were detected with qRT-PCR and Western blot analysis after miR-133b overexpression or downexpression.Target genes of miR-133b were predicted by bioinformatics software.Dual-luciferase activity assay were used to verify a target gene of miR-133b.Results qRT-PCR showed that the expression level of miR-133b in the miR-133b mimic group was significantly higher than that in the negative control group(t=26.219,P=0.000).The expression level of miR-133b in the miR-133b inhibitor group was significantly lower than that in the negative control group(t=6.738,P=0.003).After 21,45,69,93,and 117 hours of transfection,the proliferation ability of CFs significantly decreased in the miR-133b mimic group but significantly increased in the miR-133b group(all P<0.05,compared with the negative control group).After overexpression of miR-133b,the mRNA and protein levels of CTGF(t=9.213,P=0.001;t=8.195,P=0.001),α-SMA(t=6.511,P =0.003;t=4.434,P=0.011),collagenⅠ(t=3.172,P=0.034;t=4.053,P=0.015)and collagen Ⅲ(t=6.404,P=0.003;t=5.319,P=0.006)were significantly down-regulated.After the expression of miR-133b was knocked down,the mRNA and protein levels of CTGF(t=9.439,P=0.001;t=14.100,P=0.000),α-SMA(t=4.519,P=0.011;t=4.377,P=0.012),collagen Ⅰ(t=5.966,P=0.004;t=5.514,P=0.005)and collagen Ⅲ(t=4.622,P=0.010;t=4.996,P=0.008)were significantly increased.The relative luciferase activity of the cells co-transfected with miR-133b mimic and WT 3'UTR expression vector was significantly lower than that of the cells co-transfected with mimic control and WT 3'UTR expression vectors(t=5.654,P=0.005);however,there was no significant difference in relative luciferase activity between cells co-transfected with miR-133b mimic and MUT 3'UTR expression vectors and cells co-transfected with mimic control and MUT 3'UTR expression vectors(t=0.380,P=0.724).Conclusion miR-133b may affect the activation and proliferation of CFs by targeting CTGF and thus improve cardiac fibrosis.

Esophageal adenocarcinoma (EAC) is one of the most common malignancies in the world which is associated the increased prevalence of obesity. In the context of obesity, leptin can directly contribute to progression of EAC. Adiponectin inhibits leptin-induced oncogenic signaling in EAC cells. However, the exact molecular mechanisms linking obesity, adipokines, and EAC remain far from completely understood. In the present study, we tested the role of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) in adiponectin-induced protective effects against leptin-induced EAC cell proliferation. We found that globular adiponectin (gAD) significantly inhibited leptin-induced increase of cell proliferation and decrease of apoptosis in OE 19 cells. Moreover, leptin-induced increase of UHRF1 expression was suppressed by gAD. Compared with normal controls, UHRF1 expression was markedly increased in EAC tissues and cell lines. Silence of UHRF1 increased the expression of cleaved caspase 3 and 9 and Bax, reduced the expression of Bcl-2, promoted apoptosis, and inhibited cell proliferation in OE 19 cells. Overexpression of UHRF1 significantly blocked gAD-induced decrease of cell proliferation and increase of apoptosis in leptin-treated cells. Silence of adiponectin receptor 1/2 (AdipoR1/2) could inhibit gAD-induced decrease of cell proliferation and increase of apoptosis in leptin-treated cells. Silence of AdipoR2, but not AdipoR1, suppressed gAD-induced decrease of UHRF1 expression in leptin-treated cells. The results indicated that gAD inhibited the prooncogenic effects of leptin via AdipoR2-mediated suppression of UHRF1. Our study provides novel insights into the role of UHRF1 in the development of EAC and the mechanism of antitumor effect of gAD.

Background Macrophage migration inhibitory factor ( MIF ) is a key regulator of inflammatory responses, including in the heart. Plasma MIF is elevated early in the course of acute myocardial infarction. In this study, we hypothesized that plasma MIF may also be increased in acute myocardial ischemia. Methods and Results Patients undergoing cardiac stress test (stress nuclear myocardial perfusion scan or stress echocardiography) were recruited. Twenty‐two patients had a stress test indicative of myocardial ischemia and were compared with 62 patients who had a negative stress test. Plasma MIF was measured by ELISA before and after the stress test. MIF was also measured in patients with peripheral arterial occlusive disease before and after exercise causing claudication. Gene and protein expression of MIF was measured in mouse cardiac and skeletal muscle tissue by real‐time polymerase chain reaction and western blot, respectively. Plasma MIF was elevated at 5 and 15 minutes after stress (relative to before stress) in patients with a positive test, compared with those with a negative test. In contrast, high‐sensitivity troponin T and C‐reactive protein were not altered after stress in either group. MIF was not altered after exercise in PAOD patients, despite the occurrence of claudication, suggesting that plasma MIF is not a marker for skeletal muscle ischemia. This may be explained by a lower gene and protein expression of MIF in skeletal muscle than the heart. Conclusions Our results suggest that plasma MIF is an early marker for acute myocardial ischemia.

Atherosclerosis and its complications rank as the leading cause of death with the hallmarks of lipid deposition and inflammatory response. MicroRNAs (miRNAs) have recently garnered increasing interests in cardiovascular disease. In this study, we investigated the function of miR-223 and the underlying mechanism in atherosclerosis. In the atherosclerotic ApoE−/− mice models, an obvious increase of miR-223 was observed in aortic atherosclerotic lesions. In lipopolysaccharide (LPS) activated macrophages, its expression was decreased. The miR-223 overexpression significantly attenuated macrophage foam cell formation, lipid accumulation and pro-inflammatory cytokine production, which were reversed by anti-miR-223 inhibitor transfection. Mechanism assay corroborated that miR-223 negatively regulated the activation of the toll-like receptor 4 (TLR4)-nuclear factor-κB (NF-κB) pathway. Pretreatment with a specific inhibitor of NF-κB (pyrrolidinedithiocarbamate, PDTC) strikingly abrogated miR-223 silence-induced lipid deposition and inflammatory cytokine production. Furthermore, PI3K/AKT was activated by miR-223 up-regulation. Pretreatment with PI3K/AKT inhibitor LY294002 strikingly ameliorated the inhibitory effects of miR-223 on the activation of TLR4 and p65, concomitant with the increase in lipid deposition and inflammatory cytokine production. Together, these data indicate that miR-223 up-regulation might abrogate the development of atherosclerosis by blocking TLR4 signaling through activation of the PI3K/AKT pathway, and provides a promising therapeutic avenue for the treatment of atherosclerosis.

Platypnea orthodeoxia syndrome is associated with dyspnea and arterial oxygen desaturation accentuated by an upright posture. It can be secondary to an intracardiac shunt. We report a case of platypnea-orthodeoxia syndrome (POS) in a 58-year old male patient who had a pre-existing patent foramen ovale (PFO) and substantial pulmonary pathologies. He was successfully treated by percutaneous transcatheter closure of the PFO. Our case highlights the importance of recognition of this rare syndrome in patients who present with unexplained hypoxia for whom transcatheter closure of the interatrial shunt can be safely carried out.

Nonanastomotic strictures (NAS) are common biliary complications after liver transplantation (LT). Delayed rearterialization induces biliary injury in several hours. However, whether this injury can be prolonged remains unknown. The correlation of this injury with NAS occurrence remains obscure. Different delayed rearterialization times were compared using a porcine LT model. Morphological and functional changes in bile canaliculus were evaluated by transmission electron microscopy and real‐time PCR. Immunohistochemistry and TUNEL were performed to validate intrahepatic bile duct injury. Three months after LT was performed, biliary duct stricture was determined by cholangiography; the tissue of common bile duct was detected by real‐time PCR. Bile canaliculi were impaired in early postoperative stage and then exacerbated as delayed rearterialization time was prolonged. Nevertheless, damaged bile canaliculi could fully recover in subsequent months. TNF‐α and TGF‐β expressions and apoptosis cell ratio increased in the intrahepatic bile duct only during early postoperative period in a time‐dependent manner. No abnormality was observed by cholangiography and common bile duct examination after 3 months. Delayed rearterialization caused temporary injury to bile canaliculi and intrahepatic bile duct in a time‐dependent manner. Injury could be fully treated in succeeding months. Solo delayed rearterialization cannot induce NAS after LT.

OBJECTIVE:To observe the effect of oversized occluder on endothelialization post percutaneous closure of experimental atrial septal defect (ASD) in dogs.METHODS:ASD was established with the help of transthoracic echocardiography in 18 dogs. ASD size was (6.0 ± 0.2) mm. Dogs were randomly divided into normal size group (implanted with 8 mm occlude, n = 9) and oversized group (implanted with 12 mm occluder, n = 9). Dogs were randomly killed at 3, 6 and 14 months after percutaneous closure. The endothelialization process on device surface was observed by scanning electron microscope.RESULTS:Four animals died around 1 month post procedure. Microscopic sections from normal group showed nearly complete endothelialization at 3 months after device implantation and complete endothelialization at 6 and 14 months after device implantation. While microscopic sections showed lack of endothelialization at 3 months post implantation, nearly endothelialization at 6 months, and complete endothelialization at 14 months after device implantation in oversized group.CONCLUSION:Incomplete endothelialization of occluder surface is observed at 6 months after implantation of an oversized ASD occluder device in this model.

SUMMARY We report the synthesis and characterization of an insulin sensitizer – Rosiglitazone (RSG) – loaded lipid-polymer hybrid, biomimetic nanoparticle (NP) by single step nanoprecipitation. These NPs were systemically administered for 1 month, twice a week, to LDLR -/mice on high-fat diet. The expression of genes regulating lipid metabolism and inflammation was analyzed in different organs, including the white adipose tissue (WAT), liver and heart. As compare to the freely administered RSG (oral RSG), the hybrid nanoparticle formulation resulted in reduced inflammation and less pronounced side effects. INTRODUCTION Drug delivery systems (DDS), including polymeric nanoparticles (NPs), metal/ceramic NPs, liposomes, and dendrimers have been exploited as carriers for drugs and other bioactive substances in recent decades. In this context, biodegradable and biocompatible polymer and lipid combination, the lipid-polymer hybrid NPs, offer many advantages over the other more traditional carriers. This includes the versatility in surface modification with specific targeting ligands or homing devices such as IgG or IgM. Also, the components used are nonimmunogenic, biodegradable, and biocompatible; they freely circulate throughout the body and offer ease of preparation. These NPs have the capacity to carry large amounts of drugs, and can behave as a slow-release, long-acting system [1]. Given the clinical success of polymeric and liposomal NPs, we aim to use lipid and polymer hybrid NPs to deliver rosiglitazone (RSG), which activates PPARγ target genes. RSG is a member of the thiazolidinedione class of drugs, which reduces glucose, fatty acid, and insulin blood concentrations. Unfortunately, RSG has been also found to increases fatalities from heart dysfunction and eventually failure. It has been reported that the risk of heart attack increased as much as 43% [2]. Due to the extreme cytotoxic effect of RSG, the US Food and Drug Administration (FDA) issued an alert of having higher risk of death from cardiovascular diseases and revised its prescribing information and medication guides for all RSG containing medicines. Considering the fact that the RSG is one of the important classes of drug that targets peroxisome proliferator-activated receptors (PPARγ), we proposed to reformulate the drug in a nanoparticle envelope in order to minimize its toxic effect. We hypothesize that encapsulating RSG into the nanoenvelope may reduce the side effects and enhance the therapeutic efficacy of RSG by conferring cell selective drug delivery, particularly to macrophages residing within the WAT and atherosclerotic plaques. EXPERIMENTAL METHODS L-α-phosphatidylcholine hydrogenated (EGG chicken, Avanti polar Lipid), 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[carboxy (polye thylene glycol) -2000] (ammonium salt, DSPE-PEGCOOH), and carboxy terminated poly(DL-lactideco-glycolide) (PLGA-COOH) was used to prepare lipid-polymer hybrid NPs by nanoprecipation technique [1]. Male LDLR -/mice were purchased from The Jackson Laboratory and maintained as reported earlier [3]. Mice were injected RSG-hybrid (10 ug of RSG twice a week) and bare (control, same concentration of polymer as in drug treated mice) lipid-polymer hybrid NPs systemically for a period of one months. After one month period, mice were euthanized by isoflurane overdose and the organs were collected for the gene expression studies. RESULTS AND DISCUSSION In this study, the NP consists of inner hydrophobic PLGA polymeric core surrounded by a lipid monolayer in which the small hydrophobic molecules of RSG were embedded. Further the NPs were PEGylated in order to prevent from aggregation in physiological environment by creating steric repulsion that evade protein opsonization in vivo leading to higher plasma residence time. NPs were characterized for their hydrodynamic diameter, surface property and drug release behavior. The dynamic light scattering (DLS) measurement confirmed the formation of 100±6 nm size NPs with very narrow PDI of 0.18±0.02 (Figure 1A). The NP spherical morphology was also confirmed by scanning electron microscopy (SEM), returning a uniform size Figure 1. Physicochemical characterization of RSGhybrid NPs. A) Size and morphology (B) Drug loading and release kinetics. of 70±3 nm (Figure 1B). SEM measured size is slightly smaller than DLS due to the different states of the measurements. The long term stability of RSG-loaded NPs was estimated over the time period of 11 days. As shown in Figure 1A, these NPs are highly stable in PBS at pH 7.4. Next we focused our attention on in vitro drug release kinetics of RSG-hybrid NPs (loading efficiency of 3.0 wt%, Figure 1B-inset) at pH 7.4 and 5.0. These two different ionic strengths were selected to mimic physiological and acidic endosomal environment, respectively. Only 20% of drug was released in first 5h at pH 7.4, whereas at pH 5.0, 50% of the drug was released within the first 5h as shown in Figure 1B. Drug release at pH 5.0 was relatively fast, which can be attributed to the Figure 2. In vitro gene expression studies using bone marrow derived monocytes (BMDM) at RSG concentration of 10 μM. erosion of outer stabilizing lipid layer and the degradation of PLGA in acidic medium. These results could highlight that the pharmokinetic profile in acidic pH is dramatically increased, so that this NP, after internalization via endocytotic pathway, can immediately release RSG thereby increasing the local drug concentration. As observed in in vitro gene expression studies with bone marrow derived monocytes (BMDM), low dose (10 μM) of RSGhybrid NPs up regulates the CD36, FABP4, ABCG1 and PPARγ to the same extent to that of Free RSG (Figure 2). Considering the slow releasing profile of the NP system, the treatment with NP would further facilitate in vivo transciptome profile. As expected, we observed reduced expression of inflammatory genes in liver, WAT, kidney and heart as compared to oral RSG, and no alteration in the expression of lipid metabolism genes. CONCLUSION In conclusion, the lipid-polymer hybrid NPs mediated the sustained delivery of RSG and modulated the overall inflammatory response. We expect this approach to have potential in the treatment of cardiovascular diseases by enhancing the therapeutic efficacy of thiazolidinedione-based drugs. REFERENCES 1. Zhang L.; Chan J. M.; Gu F. X.; Rhee j. W.; Wang A. Z.; Radovic-Moreno A. F.; Alexis F.; Langer R.; Farokhzad O. C. ACS Nano 2008, 2, 1696-1702. 2. Steven E. N.; Kathy W. New England Journal of Medicine 2007, 356, 2457-2471 3. Collins A. R.; Lyon C. J.; Xia X.; Liu J. Z.; Tangirala R. K.; Boyadjian R.; Bikineyeva A.; Pratico D.; Harrison D. G.; Hsueh W. A. Circulation Research 2009, 104: e42-e54. 0 2 4 6 8 10 12 0 50 100 150 200 250 300

PPARγ nuclear receptor agonists have been shown to attenuate macrophage inflammatory responses implicated in the metabolic complications of obesity and in atherosclerosis. However, PPARγ agonists currently in clinical use, including rosiglitazone (RSG), are often associated with severe side effects that limit their therapeutic use. Here, 200nm PLGA/PVA nanospheres were formulated for the systemic delivery of RSG specifically to macrophages. RSG was encapsulated with over 50% efficiency in the hydrophobic PLGA core and released specifically within the acidifying macrophage phagosomes. In bone marrow derived macrophages, RSG-loaded nanoparticles (RSG-NPs) induce a dose dependent upregulation (1.5 to 2.5-fold) of known PPARγ target genes, with maximal induction at 5μM; and downregulate the expression of genes related to the inflammatory process, with a maximum effect at 10μM. In Ldlr−/− mice fed high fat diet, treatment with RSG-NPs alleviated inflammation in white adipose tissue and liver but, unlike treatment with free RSG, did not alter genes associated with lipid metabolism or cardiac function, indicating a reduction in the RSG side effect profile. These biocompatible, biodegradable RSG-NPs represent a preliminary step towards the specific delivery of nuclear receptor agonists for the treatment of macrophage-mediated inflammatory conditions associated with obesity, atherosclerosis and other chronic disease states.