Quantitative proteomic study of arsenic treated mouse liver sinusoidal endothelial cells using a reverse super-SILAC method.

Liver sinusoidal endothelial cells are the border patrol in the liver. Their open transcellular fenestrations allow the transfer of small and dissolved substances from the blood into the liver parenchymal cells. Fenestrations are dynamic structures, and many drugs and diseases alter their size and number, thus making them an important target for modulation. There is an urgent need to understand how various diseases, toxic substances, and physiological conditions influence liver endothelial cell fenestrations, and how these changes affects liver function. This work represents a straightforward quantitative proteomics study of the in vivo arsenic-stressed liver sinusoidal endothelial cells using a reverse super-SILAC based method. The aim of this study was to identify proteins, which are up- or down-regulated in response to arsenic. This knowledge will aid in identification of potential targets and mechanisms of arsenic toxicity and novel ways to reverse these changes.

Targeting Endothelial Erk1/2-Akt Axis as a Regeneration Strategy to Bypass Fibrosis during Chronic Liver Injury in Mice.

Liver sinusoidal endothelial cells (LSECs) have great capacity for liver regeneration, and this capacity can easily switch to profibrotic phenotype, which is still poorly understood. In this study, we elucidated a potential target in LSECs for regenerative treatment that can bypass fibrosis during chronic liver injury. Proregenerative LSECs can be transformed to profibrotic phenotype after 4 weeks of carbon tetrachloride administration or 10 days of bile duct ligation. This phenotypic alternation of LSECs was mediated by extracellular regulated protein kinases 1 and 2 (Erk1/2)-Akt axis switch in LSECs during chronic liver injury; Erk1/2 was normally associated with maintenance of the LSEC proregenerative phenotype, inhibiting hepatic stellate cell (HSC) activation and promoting tissue repair by enhancing nitric oxide (NO)/reactive oxygen species (ROS) ratio and increasing expression of hepatic growth factor (HGF) and Wingless-type MMTV integration site family member 2 (Wnt2). Alternatively, Akt induced LSEC profibrotic phenotype, which mainly stimulated HSC activation and concomitant senescence by reducing NO/ROS ratio and decreasing HGF/Wnt2 expression. LSEC-targeted adenovirus or drug particle to promote Erk1/2 activity can alleviate liver fibrosis, accelerate fibrosis resolution, and enhance liver regeneration. This study demonstrated that the Erk1/2-Akt axis acted as a switch to regulate the proregenerative and profibrotic phenotypes of LSECs, and targeted therapy promoted liver regeneration while bypassing fibrosis, providing clues for a more effective treatment of liver diseases.

Changes in renal tissue proteome induced by mesenteric lymph drainage in rats after hemorrhagic shock with resuscitation.

Kidney injury commonly occurs after hemorrhagic shock. Previous studies have shown that post-hemorrhagic shock mesenteric lymph (PHSML) return negatively affects the kidneys and may induce injury. This study investigates the effect of PHSML drainage on the proteome in renal tissue. A controlled hemorrhagic shock model was established in the shock and shock+drainage groups. After 1 h of hypotension, fluid resuscitation was implemented within 30 min. Meanwhile, PHSML was drained in the shock+drainage group. After 3 h of resuscitation, renal tissue was extracted for proteome analysis using two-dimensional fluorescence difference gel electrophoresis. Differential proteins with intensities that either increased or decreased by 1.5-fold or greater were selected for trypsin digestion and analyzed by matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry and tandem TOF/TOF mass spectrometry. Enzyme-linked immunosorbent assay was used to validate the identified partial proteins. Compared with the sham group, hnRNPC and Starp decreased in the shock group, whereas Hadha, Slc25a13, Atp5b, hnRNPC, Starp, Rps3, and actin were downregulated in the shock+drainage group. Meanwhile, Atp5b and actin decreased in the shock+drainage group relative to the shock group. The identified proteins can be classified into different categories, such as cell proliferation (hnRNPC, Strap, and Rps3), energy metabolism (Hadha, Atp5b, and Slc25a13), cell motility, and cytoskeleton (actin). Moreover, enzyme-linked immunosorbent assay measurement validated the changed levels of Atp5b and Actg2. Our findings provide a starting point for investigating the functions of differentially expressed proteins in acute kidney injury induced by hemorrhagic shock. These findings hold great potential for the development of therapeutic interventions.

A new insight into the impact of different proteases on SILAC quantitative proteome of the mouse liver.

In this study, we examined the use of multiple proteases (trypsin, LysC, tandem LysC/trypsin) on both protein identification and quantification in the Lys-labeled SILAC mouse liver. Our results show that trypsin and tandem LysC/trypsin digestion are superior to LysC in peptides and protein identification while LysC shows advantages in quantification of Lys-labeled proteins. Combination of experimental results from different proteases (LysC and trypsin) enabled a significant increase in the number of identified protein and protein can be quantified. Thus, taking advantage of the complementation of different protease should be a good strategy to improve both qualitative and quantitative proteomics research.

Identification of α1-antitrypsin as a potential prognostic biomarker for advanced nonsmall cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors by proteomic analysis.

OBJECTIVE: This retrospective study attempted to identify serum biomarkers that could help to indicate treatment response in advanced nonsmall-cell lung cancer (NSCLC) patients receiving epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment. METHODS: Two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to identify proteins expressed in serum samples from NSCLC patients with long (>6-month) progression-free survival (PFS) periods, following EGFR-TKI treatment. RESULTS: Serum amyloid P component (APCS), α1-antitrypsin (AAT), fibrinogen-α (FGA), keratin type I cytoskeletal 10 (KRT10) and serotransferrin (TF) expression differed between samples taken from 18 patients before treatment (baseline) and when progressive disease (PD) was observed, during treatment. Changes in AAT, KRT10 and APCS levels were validated by Western blot analysis in the sample pool; findings were further validated by Western blot analysis in a random sample of four patients. These proteins were also present in serum samples obtained from the same patients at the partial response (PR) timepoint during EGFR-TKI treatment. AAT was upregulated at PD compared with baseline, but downregulated during the PR phase. CONCLUSION: These observations suggest that AAT could be used as a serological biomarker for predicting the utility of EGFR-TKI treatment for advanced NSCLC.

Identification of isocitrate dehydrogenase 1 as a potential diagnostic and prognostic biomarker for non-small cell lung cancer by proteomic analysis.

Lung cancer is the leading cause of cancer-related death in the world. To explore tumor biomarkers for clinical application, two-dimensional fluorescence difference gel electrophoresis and subsequent MALDI-TOF/TOF mass spectrometry were performed to identify proteins differentially expressed in 12 pairs of lung squamous cell tumors and their corresponding normal tissues. A total of 28 nonredundant proteins were identified with significant alteration in lung tumors. The up-regulation of isocitrate dehydrogenase 1 (IDH1), superoxide dismutase 2, 14-3-3ε, and receptor of activated protein kinase C1 and the down-regulation of peroxiredoxin 2 in tumors were validated by RT-PCR and Western blot analysis in independent 15 pairs of samples. Increased IDH1 expression was further verified by the immunohistochemical study in extended 73 squamous cell carcinoma and 64 adenocarcinoma clinical samples. A correlation between IDH1 expression and poor overall survival of non-small cell lung cancer (NSCLC) patients was observed. Furthermore, ELISA analysis showed that the plasma level of IDH1 was significantly elevated in NSCLC patients compared with benign lung disease patients and healthy individuals. In addition, knockdown of IDH1 by RNA interference suppressed the proliferation of NSCLC cell line and decreased the growth of xenograft tumors in vivo. These observations suggested that IDH1, as a protein promoting tumor growth, could be used as a plasma biomarker for diagnosis and a histochemical biomarker for prognosis prediction of NSCLC.