Matrix metalloproteinases modulated by protein kinase Cε mediate resistin-induced migration of human coronary artery smooth muscle cells.

BACKGROUND: Emerging evidence showed that resistin induces vascular smooth muscle cell (VSMC) migration, a critical step in initiating vascular restenosis. Adhesion molecule expression and cytoskeletal rearrangement have been observed in this progress. Given that matrix metalloproteinases (MMPs) also regulate cell migration, we hypothesized that MMPs may mediate resistin-induced VSMC migration. METHODS: Human VSMCs were treated with recombinant human resistin at physiologic (10 ng/mL) and pathologic (40 ng/mL) concentrations for 24 hours. Cell migration was determined by the Boyden chamber assay. MMP and tissue inhibitor metalloproteinase (TIMP) mRNA and protein levels were measured with real-time PCR and ELISA. MMP enzymatic activity was measured by zymography. In another experiment, neutralizing antibodies against MMP-2 and MMP-9 were coincubated with resistin in cultured VSMCs. The regulation of MMP by protein kinase C (PKC) was determined by εV1-2, a selective PKCε inhibitor. RESULTS: Resistin-induced smooth muscle cell (SMC) migration was confirmed by the Boyden chamber assay. Forty nanograms/milliliter resistin increased SMC migration by 3.7 fold. Additionally, resistin stimulated MMP-2 and -MMP9 mRNA and protein expressions. In contrast, the TIMP-1 and TIMP-2 mRNA levels were inhibited by resistin. Neutralizing antibodies against MMP-2 and MMP-9 effectively reversed VSMC migration. Furthermore, resistin activated PKCε, but selective PKCε inhibitor suppressed resistin-induced MMP expression, activity, and cell migration. CONCLUSIONS: Our study confirmed that resistin increased vascular smooth muscle cell migration in vitro. In terms of mechanism, resistin-stimulated cell migration was associated with increased MMP expression, which was dependent on PKCε activation.

Resistin and cardiovascular disease.

Resistin has been implicated in coronary atherosclerotic disease and congestive heart failure. Recent studies have extended its involvement in peripheral artery disease. Despite some controversial data, the mainstream clinical literature supports that resistin is associated with both coronary and peripheral artery diseases including ischemic stroke. In this review, the multiple roles of resistin as screening, diagnostic, and prognostic marker for cardiovascular disease are discussed. The independence of resistin in disease prediction and diagnosis appears complicated by its confounders, such as C-reactive protein. A clear-cut biomarker function of resistin in cardiovascular disease needs be clarified by additional large-scale, well-designed prospective studies.

Proteomic profiling of proteins in rat spinal cord induced by contusion injury.

It is widely accepted that mechanical injury to spinal cord can cause nervous system dysfunction, which leads to the loss of movement and sensation. However, the exact molecular mechanism is currently unclear. In this study, contused rat spinal cords were collected at 8h, 1 day, 3, and 5 days after injury and the expression patterns of the proteins were monitored and quantified with two-dimensional gel electrophoresis-based proteomics. Fifty-one protein spots showed significant regulation at least at one time point. Of the 39 proteins, identified by mass spectrometry analysis and clustered into three down-regulation profiles and two up-regulation profiles, eight contusion-related proteins have been reported in previous proteomic studies of spinal cord whereas 31 proteins were described for the first time. For example, apoptosis-related protein of heat shock 70 kDa protein 1B increased after contusion, reaching the peak at 1 day; septin 7, a protein involved in cytoskeleton organization, maintained a steady increase for the first 5 days after injury; metabolism-related protein of 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 was constantly down-regulated during the whole time course observed; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide, associated with cell cycle progression, showed a gradual increase after contusion. To our knowledge, this is the first case of detailed and dynamic proteomic snapshots of contusion-induced spinal cord injury. Most of the identified proteins were found for the first time to be differentially expressed after spinal cord contusion, which may help explore the complex molecular cascades underlying the progressive pathologic changes in the contused spinal cord.

Proteomic profiling of the insoluble pellets of the transected rat spinal cord.

This study investigated the proteomic changes at different time points in the precipitated pellets of rat spinal cords after applying complete spinal cord transection. By two-dimensional electrophoresis, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, MALDI-TOF/TOF and peptide mass fingerprinting analysis, 44 proteins were identified, most of which are membrane and/or organellar proteins. They are mainly involved in metabolic processes (75%), developmental processes (30%), or responses to stimuli (30%), playing negative or positive roles. In particular, decreases of pyruvate dehydrogenase beta, aconitase 2, fumarate hydratase 1, and ATP synthase subunit 6 can lead to ATP depletion by crippling tricarboxylic acid cycle and oxidative phosphorylation. Decreases of several antioxidant proteins such as catalase, peroxiredoxin 1, Parkinson disease 7, and stress-induced phosphoprotein 1 can contribute to the secondary injury of spinal cord. Decreases of development-related 3-phosphoglycerate dehydrogenase and stathmin 1 may be not propitious for spinal cord regeneration. On the other hand, increases of isocitrate dehydrogenase 3 alpha/gamma and glutamate dehydrogenase 1 can help compensate the impaired energy metabolism. Increases of sirtuin 2, crystallin alpha B (CRYAB), and heat shock 27-kDa protein 1 can help resist stresses induced by injury. Increases of adenylate cyclase-associated protein 1 and galactose binding lectin 3 can help regeneration by replaying their roles in neural development. To our knowledge, this is the first case of characterization of the proteomic changes seen in the precipitated fraction of injured spinal cord. Most of the identified proteins were found for the first time to be differentially expressed after spinal cord injury, which may provide new clues about the molecular mechanisms of spinal cord injury and repair.

Exercise affects energy metabolism and neural plasticity-related proteins in the hippocampus as revealed by proteomic analysis.

Studies were conducted to evaluate the effect of a brief voluntary exercise period on the expression pattern and post-translational modification of multiple protein classes in the rat hippocampus using proteomics. An analysis of 80 protein spots of relative high abundance on two-dimensional gels revealed that approximately 90% of the proteins identified were associated with energy metabolism and synaptic plasticity. Exercise up-regulated proteins involved in four aspects of energy metabolism, i.e. glycolysis, ATP synthesis, ATP transduction and glutamate turnover. Specifically, we found increases in fructose-bisphosphate aldolase C, phosphoglycerate kinase 1, mitochondrial ATP synthase, ubiquitous mitochondrial creatine kinase and glutamate dehydrogenase 1. Exercise also up-regulated specific synaptic-plasticity-related proteins, the cytoskeletal protein alpha-internexin and molecular chaperones (chaperonin-containing TCP-1, neuronal protein 22, heat shock 60-kDa protein 1 and heat shock protein 8). Western blot was used to confirm the direction and magnitude of change in ubiquitous mitochondrial creatine kinase, an enzyme essential for transducing mitochondrial-derived ATP to sites of high-energy demand such as the synapse. Protein phosphorylation visualized by Pro-Q Diamond fluorescent staining showed that neurofilament light polypeptide, glial fibrillary acidic protein, heat shock protein 8 and transcriptional activator protein pur-alpha were more intensely phosphorylated with exercise as compared with sedentary control levels. Our results, together with the fact that most of the proteins that we found to be up-regulated have been implicated in cognitive function, support a mechanism by which exercise uses processes of energy metabolism and synaptic plasticity to promote brain health.

Bacterial endotoxin lipopolysaccharide induces up-regulation of glyceraldehyde-3-phosphate dehydrogenase in rat liver and lungs.

Bacterial endotoxin or lipopolysaccharide (LPS) can trigger inflammatory responses and cause damage in organs such as liver and lungs when it is introduced into mammals, but the exact molecular events that mediate these responses have remained obscure. In this study, by using 2D gel electrophoresis and cDNA microarray analysis, we found that both protein and mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were significantly increased in rat liver and lungs after treatment with LPS. The results were further confirmed by Western blot and Northern blot. Given the known role of GAPDH in inducing apoptosis, our results suggest that LPS-induced GAPDH up-regulation may be an important mechanism responsible for the damage induced by Gram negative bacteria in mammalian tissue and GAPDH may be involved in the signaling pathway of LPS induced apoptosis. Our results also demonstrate that GAPDH is not a suitable internal control in gene expression studies, especially when bacterial infection is involved.

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury.

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.

[Influence of hyperoxic fluid on the down-regulated proteins of intestinal mucosa in scalded rats].

OBJECTIVE: To investigate the influence of hyperoxic fluid on the down-regulated proteins of intestinal mucosa in scalded rats, so as to provide a theoretical basis for the clinical use of hyperoxic fluid. METHODS: Sprague-Dawley (SD) rats with 35% TBSA full-thickness scald were randomly divided into scald control (S, n = 6,with intraperitoneal fluid infusion after scalding), hyperoxic (H, n = 6, with hyperoxic fluid infusion after scalding) groups. Six rats without scald injury served as normal group. The proteins in the intestinal mucosa were separated with the two-dimensional electrophoresis (2-DE), and were analyzed with ImageMaster 2D Elite. The influence of hyperoxic fluid on the down-regulated proteins of intestinal mucosa in scalded rats was studied with bio-spectrum, protein bank and document analysis. RESULTS: (1) Among the 34 down-regulated protein spots in S group, 9 of them definitely exhibited up-regulation compared with those in H group. (2) The expression of mitochondrial aconitase, alpha-propionyl-CoA carboxylase, short chain of hydroxyacyl-Coenzyme A dehydrogenase, transcription factor EB (estradiol benzoate), triosephosphate isomerase 1, T cell receptor V delta 6, and dynein-like protein-5 in H group were significantly up-regulated. CONCLUSION: The hyperoxic fluid could up-regulate the down-regulated proteins in rat intestinal mucosa at early postburn stage, so that the barrier function of intestinal mucosa of rats with severe burns could be partially recovered.

[Study on serum proteome of rat endotoxemia treated by figwort root].

OBJECTIVE: To study the serum proteome of rat endotoxemia treated by figwort root (FR). METHOD: The differences of serum proteome among rats treated with lipopolysaccharide (LPS), FR, LPS + FR and saline respectively were analyzed by two-dimensional electrophoresis (2DE) assay. RESULT: The volumes of sixteen serum proteins (xPr) in LPS induced-endotoxemia group were greatly changed compared with those of the control group. Among them, the volumes of xPr 16, 19 were significantly decreased, and the volumes of xPr 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 18, 23 were significantly increased. When treated with FR, the volumes of xPr 1, 6, 7, 8, 9, 11, 12, 14, 18, 23 were significantly decreased, and the volumes of xPr 8, 9, 11, 12, 23, 14 were back to normal level. Two factors statistic analysis showed that FR had interaction with LPS for xPr 1, 5, 8, 10, 11, 12, 18, 19, 20, 21, 22, and FR might be the functional antagonist of LPS. We also observed that the volumes of xPr 10, 13, 15, 20, 21, 22 were found to change significantly only in FR treated group but not in LPS treated group or control group. Interestingly, the volume of xPr 13, 20, 21, 22 were increased and the volume of xPr 10, 15 were decreased. CONCLUSION: The molecular basis of therapeutic effect of FR on endotoxemia might be through the regulation of xPr 1, 6, 7, 8, 9, 11, 12, 14, 18, 23. We can use proteomic techniques to study the molecular mechanisms of diseases treated by functional Chinese herbs and the combination of different herbs is necessary for the treatment of endotoxemia, as FR can not regulated all the changed proteins induced by LPS.

[Separation and identification of down-regulated proteomics of intestinal mucosa in scalded rats].

OBJECTIVE: To explore the pathogenesis of postburn intestinal mucosal injury in scalded rats. METHODS: The rats inflicted with full thickness burn were employed as the model. The two-dimensional electrophoresis (2-DE) was employed to identify the down-regulating proteins from the differential proteins in scalded rats. Spot detection and matching were performed with Image Master 2D Elite. Mass spectrometry was performed on Bruker BIFLEX III TOF. RESULTS: There are 34 points of proteins in intestinal mucosa in scalded rats which were down-regulated at 6 and 12 postburn hours. Among them 22 proteins were employed for the identification and analysis. Mitochondrial aconitase, alpha-propionyl -CoA carboxylase heavy chain A of F1-ATPase in rat liver, Troponin short-chain of hydroxyacyl-coenzyme A dehydrogenase, alpha-subunit of P-electronic transferring flavoprotein (ETF) were down-regulating proteins correlated with mitochondria in intestinal mucosa in severely scalded rats. Triosephosphate isomerase 1 and cytosolic epoxide hydrolase were down-regulating proteins participated in metabolism of scalded rats. Fibromodulin, dynein-like protein, Troponin-2 and myosin light chain 3 alkali (MLC) were down-regulating proteins correlated with cellular skeletal protein. Glucocorticoid-inducible protein, nuclear factor1-B2, BRCA1, transcriptive factor EB (estradiol benzoate), beta2 subunit of G-protein, N-methyl-D-aspartate receptor1 (NMDAR) were down-regulating proteins participated in postburn regulation of intestinal mucosa in scalded rats. T-cell receptor-V-delta 6 and Ig heavy chain V region protein 1 were down-regulating proteins correlated with the immunomodulation of intestinal mucosa in scalded rats. CONCLUSION: The down-regulating proteins of intestinal mucosa in scalded rats exhibited close relationship with mitochondria.

Unmatched masses in peptide mass fingerprints caused by cross-contamination: an updated statistical result.

Unmatched masses are often observed in the experimental peptide mass spectra when database searching is performed with the ProFound program. Comparison between theoretical and experimental mass spectra of standard proteins shows that contamination accounts for most of the unmatched masses. In this retrospective analysis, the top 100 most probable contaminating masses, as listed in order of their probability, are statistically filtered out from 118 different experimental peptide mass fingerprinting (PMF) maps. Most of the interfering masses originate from trypsin autolysis and human keratins. Subtraction of known contaminants from raw data and using cleaner masses for searching can enhance protein identification by PMF.

A new method to improve sensitivity and resolution in matrix-assisted laser desorption/ionization time of flight mass spectrometry.

High detection sensitivity and resolution are two critical parameters for recording good peptide mass fingerprints (PMF) of low abundance proteins. This paper reports a mass spectrometry (MS) sample preparation technique that could improve sensitivity and resolution. By coating the MS steel target with a thin layer of pentadecafluorooctamido propyltrimethoxysilane, which was both polar and nonpolar solvent repellent, the transferred sample droplets on its surface were significantly smaller. As a result, the analyte of the peptide mixture became more concentrated and homogeneous, which helped to improve the sensitivity. The advantages of a modified MS target were documented by mass spectra improvement of attomole level standard peptides and silver-stained proteins from polyacrylamide gels. The mass signal of angiotensin II at 100 attomole was difficult to record on the conventional support, whereas it was easily detected on the modified one. The PMF of cytochrome C was also better recorded on the modified support, in terms of both signal-to-noise ratio and the number of detected peptides. When silver-stained proteins from two-dimensional electrophoresis gels were analyzed, in most cases more satisfactory peptide mass spectra were obtained from the modified support. Searching protein databases with more mass data from the improved PMFs, several unknown proteins were successfully identified.