Plexin D1 negatively regulates macrophage-derived foam cell migration via the focal adhesion kinase/Paxillin pathway.

BACKGROUND: Macrophage-derived foam cell formation is a hallmark of atherosclerosis and is retained during plaque formation. Strategies to inhibit the accumulation of these cells hold promise as viable options for treating atherosclerosis. Plexin D1 (PLXND1), a member of the Plexin family, has elevated expression in atherosclerotic plaques and correlates with cell migration; however, its role in macrophages remains unclear. We hypothesize that the guidance receptor PLXND1 negatively regulating macrophage mobility to promote the progression of atherosclerosis. METHODS: We utilized a mouse model of atherosclerosis based on a high-fat diet and an ox-LDL- induced foam cell model to assess PLXND1 levels and their impact on cell migration. Through western blotting, Transwell assays, and immunofluorescence staining, we explored the potential mechanism by which PLXND1 mediates foam cell motility in atherosclerosis. RESULTS: Our study identifies a critical role for PLXND1 in atherosclerosis plaques and in a low-migration capacity foam cell model induced by ox-LDL. In the aortic sinus plaques of ApoE-/- mice, immunofluorescence staining revealed significant upregulation of PLXND1 and Sema3E, with colocalization in macrophages. In macrophages treated with ox-LDL, increased expression of PLXND1 led to reduced pseudopodia formation and decreased migratory capacity. PLXND1 is involved in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK. Additionally, FAK inhibitors counteract the ox-LDL-induced migration suppression by modulating the phosphorylation states of FAK, Paxillin and their downstream effectors CDC42 and PAK. CONCLUSION: Our findings indicate that PLXND1 plays a role in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK to promoting atherosclerosis.

Identification of antibacterial activity of liver-expressed antimicrobial peptide 2 (LEAP2) from primitive vertebrate lamprey.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a member of the antimicrobial peptides family and plays a key role in the innate immune system of organisms. LEAP2 orthologs have been identified from a variety of fish species, however, its function in primitive vertebrates has not been clarified. In this study, we cloned and identified Lc-LEAP2 from the primitive jawless vertebrate lamprey (Lethenteron camtschaticum) which includes a 25 amino acids signal peptide and a mature peptide of 47 amino acids. Although sequence similarity was low compared to other species, the mature Lc-LEAP2 possesses four conserved cysteine residues, forming a core structure with two disulfide bonds between the cysteine residues in the relative 1-3 (Cys 58 and Cys 69) and 2-4 (Cys 64 and Cys 74) positions. Lc-LEAP2 was most abundantly expressed in the muscle, supraneural body and buccal gland of lamprey, and was significantly upregulated during LPS and Poly I:C stimulations. The mature peptide was synthesized and characterized for its antibacterial activity against different bacteria. Lc-LEAP2 possessed inhibition of a wide range of bacteria with a dose-dependence, disrupting the integrity of bacterial cell membranes and binding to bacterial genomic DNA, although its inhibitory function is weak compared to that of higher vertebrates. These data suggest that Lc-LEAP2 plays an important role in the innate immunity of lamprey and is of great value in improving resistance to pathogens. In addition, the antimicrobial mechanism of LEAP2 has been highly conserved since its emergence in primitive vertebrates.

Based on 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474), design, synthesis and biological evaluation of novel PI3Kα selective inhibitors.

Based on 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474), three series of novel 1,3,5-triazine or pyrimidine derivatives containing semicarbazones have been designed and synthesized to obtain new potent and selective PI3Kα inhibitors. Their inhibitory activities in vitro were evaluated against PI3Kα and three tumor-derived cell lines (U87-MG, MCF-7, and PC-3). We also tested promising compounds (A4, A6, A10, and B1) for other PI3K class I subtype (PI3Kß, PI3Kδ, and PI3Kγ) activity. The representative compound A10 exhibited an IC50 value of 0.32 nM against PI3Kα, and demonstrated extraordinary subtype selectivity. Furthermore, compound A10 obviously inhibited proliferation of MCF-7 cell lines, induced a great decrease in mitochondrial membrane potential leading to apoptosis of cancer cells, and arrested G2 phase in a dose-dependent manner. Additionally, compound A10 induced significant tumor regressions in a xenograft mouse model of U87-MG cell line without an obvious sign of toxicity upon 20 mg/kg oral administration. Compound A10 may serve as a PI3Kα-selective inhibitor and provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family.

Lamprey immunity protein enables early detection and recurrence monitoring for bladder cancer through recognizing Neu5Gc-modified uromodulin glycoprotein in urine.

The clinical management of bladder cancer (BCa) is hindered by the lack of reliable biomarkers. We aimed to investigate the potential of lamprey immunity protein (LIP), a lectin that specifically binds to multi-antennary sialylated N-glycolylneuraminic acid (Neu5Gc) structures on UMOD glycoproteins in the urine of BCa patients. Primary BCa patients had higher levels of LIP-bound Neu5Gc in urine than healthy participants and patients receiving postoperative treatment did. In addition, lectin chip assay and mass spectrometry were used to analyze the glycan chain structure, which can recognize the UMOD glycoprotein decorated with multi-antennary sialylated Neu5Gc structures. Furthermore, compared with urine samples from healthy patients (N = 2821, T/C = 0.12 ± 0.09) or benign patients (N = 360, T/C = 0.11 ± 0.08), the range of the urine T/C ratio detected using LIP test paper was 1.97 ± 0.32 in patients with bladder cancer (N = 518) with significant difference (P < 0.0001). Our results indicate that LIP may be a tool for early BCa identification, diagnosis, and monitoring. Neu5Gc-modified UMOD glycoproteins in urine and Neu5Gc-modified N-glycochains and sialyltransferases may function as potential markers in clinical trials.

Lamprey immunity protein enables detection for bladder cancer through recognizing N-hydroxyacetylneuraminic acid (Neu5Gc)-modified as a diagnostic marker and exploration of its production mechanism.

Previous studies have demonstrated that Neu5Gc is highly expressed in breast, ovarian, prostate, colon and lung cancers, but not in normal human cells. The presence of Neu5Gc is important for prognosis and is associated with aggressiveness, metastasis, and tumor grade. However, increased Neu5Gc in bladder cancer remains unclear. LIP from lamprey binds the carbohydrate receptor of N-glycolylneuraminic acid (Neu5Gc). The combination of Neu5Gc and LIP suggested that it might be used as a diagnostic tool for the detection of Neu5Gc tumor antigen. Here, the classical animal model of bladder cancer was successfully induced by MNU bladder perfusion. The ELISA results showed that the expression level of Neu5Gc in the urine of normal rats was 94.96 ± 21.01ng/mg, and that of bladder cancer rats was 158.28 ± 34.86 ng/mg. In addition, the results of SNA and LIP immunohistochemistry demonstrated the high expression of Neu5Gc in bladder cancer. After the addition of Neu5Gc to BIU-87 and SV-HUC-1 cells, transcriptomic sequencing and real-time quantitative PCR analysis demonstrated that the gene expression of Neu5Gc synthesis pathway was significantly increased. These data suggest that LIP provides a new tool for the detection of biological samples, especially urine from patients with bladder cancer or suspected cancer, and that revealing the mechanism of abnormal glycosylation can provide theoretical basis for clinical studies.

Expression and clinical significance of HSP27 and its phosphorylation in lower extremity arteriosclerosis obliterans.

OBJECTIVES: This study set out to analyze the difference of heat shock protein 27 (HSP27) and its phosphorylation in patients with lower extremity arteriosclerosis obliterans (LEASO) at different stages. This research also examined their clinical significance in this disease. METHODS: Blood samples from 60 patients with LEASO were collected and divided into two groups according to ankle-brachial index (ABI): group A (ABI ≤ 0.43) and group B (ABI > 0.43). The expression of HSP27 in each stage of Fontaine was measured by ELISA, and the difference of HSP27 concentration and ABI between the two groups was analyzed. Meanwhile, three normal femoral artery specimens (normal group) and three atherosclerotic femoral artery specimens (lesion group) were collected, and HSP27 and its Phospho-HSP27 (Ser15), Phospho-HSP27 (Ser78) and Phospho-HSP27 (Ser82) were detected by western blotting. The data of the protein level between the normal group and the lesion group was made a statistical analysis. RESULTS: HSP27 concentration in group A was (40.73 ± 15.99) ng/ml, and ABI was 0.26 ± 0.20. HSP27 concentration in group B was (66.30 ± 24.70) ng/ml, and ABI was 0.64 ± 0.20. The protein expression of HSP27 and its phosphorylation in the normal group was 0.82 ± 0.13, 0.66 ± 0.12, 0.91 ± 0.24 and 0.90 ± 0.16, respectively; the protein expression of the lesion group was 0.45 ± 0.08, 0.42 ± 0.09, 0.39 ± 0.12 and 0.58 ± 0.11. CONCLUSION: Patients with higher LEASO Fontaine stage and lower ABI had a lower HSP27 concentration. Serum HSP27 concentration was negatively correlated with the severity of LEASO, while HSP27 concentration was positively correlated with ABI value. The content of HSP27 and its phosphorylation of lesion group is significantly lower than that of normal group, which may be closely related to the occurrence and development of atherosclerosis.

[Clinical application of transcatheter embolization for Klippel-Trenaunay syndrome].

OBJECTIVE: To explore the feasibility of interventional treatment of Klippel-Trenaunay syndrome(KTS). METHODS: The clinical data of 20 KTS patients admitted into our hospital from March 2005 to October 2010 were retrospectively analyzed. RESULTS: All 20 patients underwent angiography after the relevant examinations. Among them, 18 patients underwent interventional treatment (embolization with PVA particles and spring coils). Two patients received no interventional treatment due to non-cooperation, high risk or extremely thin vessels. And 18 patients achieved excellent results after interventional treatment. Neither complications nor peri-operative mortality occurred. The patients were followed-up for an average period of 12 months. Mild symptoms recurred in 2 patients. CONCLUSION: Interventional treatment is an effective, safe and mini-invasive procedure for KTS with satisfactory long-term outcomes.

Epithelial-mesenchymal transition induced by hepatitis C virus core protein in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CC) is associated with chronic hepatitis C virus (HCV) infection. We investigated the effect of hepatitis C virus core protein (HCVc) on epithelial-mesenchymal transition (EMT) in CC and tried to identify its target trigger. METHODS: First, we examined expression of HCVc and epithelial and mesenchymal markers in CC tissues. Then we transient-transfected HCVc gene into a CC cell line and examined expression of lysyl oxidase-like 2 (LOXL2) and epithelial and mesenchymal markers by quantitative real-time polymerase chain reaction (PCR) and Western blotting. Finally, LOXL2 gene silencing was shown in QBC939/HCVc cells by RNA interference (RNAi), and we further examined expression of epithelial and mesenchymal markers by quantitative real-time PCR and Western blotting. RESULTS: Through immunohistochemical staining, the present study showed that HCVc is significantly associated with CC invasion and metastasis. In vitro study showed that HCVc expression induces EMT in CC cell line QBC939, and a mechanism through LOXL2 pathway is suggested. Expression of HCVc was significantly correlated with greater migratory and invasive potential of CC cells. CONCLUSIONS: These observations indicate that HCVc plays a critical role in promoting invasion and metastasis of CC cells.

[Effect of hepatitis C virus core protein on cholangiocarcinoma tissues' epithelial-mesenchymal transition].

OBJECTIVE: To explore the role of hepatitis C virus core protein on the infiltration and metastasis of cholangiocarcinoma tissues. METHODS: From January 2001 to November 2006, 34 patients with cholangiocarcinoma who had intact follow-up data randomly were chosen. The expression of HCVc protein, epithelium markers and mesenchymal markers in cholangiocarcinoma tissues were examined by SP methods of immunohistochemistry, clinical-pathological data were recorded and analyzed. RESULTS: The positive expression rate was observed in 47.1% for HCVc protein, 50% for N-cadherin, 44.1% for Vimentin, 55.9% for Fibronectin and the decreased expression rate was E-cadherin for 55.9%, alpha-catenin for 70.6%, beta-catenin for 55.9%. The positive expression of HCVc protein was associated with the decreased expression of E-cadherin, alpha-catenin and the positive expression of N-cadherin, Vimentin, Fibronectin (chi(2) = 4.480, 4.163, 4.250, 7.438, 12.260, P < 0.05). A positive-correlation between the expression of HCVc protein and metastasis of lymph nodes and other organs were found (chi(2) = 5.708, 4.163, P < 0.05). CONCLUSION: HCVc protein might promote cholangiocarcinoma tissues' infiltration and metastasis by inducing it's epithelial-mesenchymal transition.

[Therapeutic effect of qingyi decoction and tetrandrine in treating severe acute pancreatitis in miniature pigs and serum drug level determination].

OBJECTIVE: To investigate the therapeutic effect of Qingyi Decoction (QYD) and tetrandrine (Tet), used singly or combind, in treating miniature pigs with severe acute pancreatitis (SAP) and its mechanism. METHODS: Thirty-two Guizhou miniature pigs were made into SAP model by pancreatic duct retrograde injection of 5% sodium taurocholate. They were randomly divided into 4 groups: the control group, the QYD group, the Tet group and the combined treated group. The serum amylase activity and interleukin-1 and 6 (IL-1, IL-6) contents in serum from vena cava and portal vein were tested by biochemistry and radioimmunoassay (RIA). Serum emodin and plasma Tet levels were measured by high performance liquid chromatography (HPLC) 24, 48 and 72 hrs after treatment. And the pathological changes of pancreas, lung and liver were observed under microscope. RESULTS: The mortality of SAP pigs was reduced significantly and the inflammatory injury of the organs was ameliorated obviously in all treated groups, and the increased amylase activity and IL-1, IL-6 levels was attenuated. The therapeutic effect was much more obvious, and the plasma Tet level at different time points were much higher in the combined treated group than those in the other two groups treated by single drug (P < 0.01). CONCLUSION: Both QYD and Tet could treat effectively SAP through multiple pathways, combination of them reveals an elevation of serum drug concentration and shows a synergistic effect.