Search

Background and Aims: Increased O-GlcNAc transferase (OGT)–induced O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification is linked with diabetic complications. MicroRNA-146a-5p (miR-146a-5p) is a negative inflammatory regulator and is downregulated in diabetes. Here, we investigated the interaction between miR-146a-5p and OGT.Methods: Human aortic endothelial cells (HAECs) were stimulated with high glucose (25 mM) and glucosamine (25 mM) for 24 h. Western blot, real time PCR, bioinformatics analysis, luciferase reporter assay, miR-146a-5p mimic/inhibitor transfection, siRNA OGT transfection, miR-200a/200b mimic transfection, and OGT pharmacological inhibition (ST045849) were performed. The aorta from miR-146a-5p mimic-treated db/db mice were examined by immunohistochemistry staining.Results: HG and glucosamine upregulated OGT mRNA and protein expression, protein O-GlcNAcylation, and IL-6 mRNA and protein expression. Real time PCR analysis found that miR-146a-5p was decreased in HG- and glucosamine-stimulated HAECs. This suggested that OGT-induced protein O-GlcNAcylation as a mechanism to downregulate miR-146a-5p. Bioinformatic miR target analysis excluded miR-146a-5p as a post-transcriptional regulator of OGT. However, a luciferase reporter assay confirmed that miR-146a-5p mimic bound to 3′-UTR of human OGT mRNA, indicating that OGT is a non-canonical target of miR-146a-5p. Transfection with miR-146a-5p mimic and inhibitor confirmed that miR-146a-5p regulated OGT/protein O-GlcNAcylation/IL-6 expression levels. Furthermore, OGT siRNA transfection, miR-200a/miR-200b mimic transfection, and ST045849 increased HG-induced miR-146a-5p expression levels, indicating that HG-induced miR-146a-5p downregulation is partially mediated through OGT-mediated protein O-GlcNAcylation. In vivo, intravenous injections of miR-146a mimic decreased endothelial OGT and IL6 expression in db/db mice.Conclusion: A non-canonical positive feedback interaction between miR-146a-5p and OGT is involved in a vicious cycle to aggravate HG-induced vascular complications.

Background and Aims: Increased O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins by O-GlcNAc transferase (OGT) is associated with diabetic complications. Furthermore, oxidative stress promotes endothelial inflammation during diabetes. A previous study reported that microRNA-200 (miR-200) family members are sensitive to oxidative stress. In this study, we examined whether miR-200a and miR-200b regulate high-glucose (HG)-induced OGT expression in human aortic endothelial cells (HAECs) and whether miRNA-200a/200b downregulate OGT expression to control HG-induced endothelial inflammation.Methods: HAECs were stimulated with high glucose (25 mM) for 12 and 24 h. Real-time polymerase chain reaction (PCR), western blotting, THP-1 adhesion assay, bioinformatics predication, transfection of miR-200a/200b mimic or inhibitor, luciferase reporter assay, and transfection of siRNA OGT were performed. The aortic endothelium of db/db diabetic mice was evaluated by immunohistochemistry staining.Results: HG upregulated OGT mRNA and protein expression and protein O-GlcNAcylation levels (RL2 antibody) in HAECs, and showed increased intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Bioinformatics analysis revealed homologous sequences between members of the miR-200 family and the 3′-untranslated region (3′-UTR) of OGT mRNA, and real-time PCR analysis confirmed that members of miR-200 family were significantly decreased in HG-stimulated HAECs. This suggests the presence of an impaired feedback restraint on HG-induced endothelial protein O-GlcNAcylation levels because of OGT upregulation. A luciferase reporter assay demonstrated that miR-200a/200b mimics bind to the 3′-UTR of OGT mRNA. Transfection with miR-200a/200b mimics significantly inhibited HG-induced OGT mRNA expression, OGT protein expression; protein O-GlcNAcylation levels; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Additionally, siRNA-mediated OGT depletion reduced HG-induced protein O-GlcNAcylation; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion, confirming that HG-induced endothelial inflammation is partially mediated via OGT-induced protein O-GlcNAcylation. These results were validated in vivo: tail-vein injection of miR-200a/200b mimics downregulated endothelial OGT and ICAM-1 expression in db/db mice.Conclusion: miR-200a/200b are involved in modulating HG-induced endothelial inflammation by regulating OGT-mediated protein O-GlcNAcylation, suggesting the therapeutic role of miR-200a/200b on vascular complications in diabetes.

Background and Aims: Endothelial dysfunction is a hallmark of cardiovascular diseases. The straight region of an artery is protected from atherosclerosis via its laminar blood flow and high shear stress. This study investigated the cytoprotective effects of a new laminar shear medium (LSM) derived from a modified cone-and-plate shear device and identified basic fibroblast growth factor (bFGF) secreted by human aortic endothelial cells (HAECs) as the dominant protective factor in the LSM.Methods: Based on a modified cone-and-plate shear device system, HAECs were exposed to laminar shear (15 dynes/cm2) and static control for 24 h to produce a new supernatant LSM and static medium (SM). Evaluation of the protective effects of LSM and SM on endothelial dysfunction induced by tumor necrosis factor (TNF)-α (10 ng/mL), which leads to production of reactive oxygen species (ROS), inflammatory monocyte adhesion, and tissue factor activity. ROS induction-, inflammation-, and thrombosis-related genes and protein expression were evaluated by quantitative-PCR and western blotting. To identify the cytokines that played a key role in the cytoprotective action of the LSM, we used cytokine antibody arrays, selected an abundant marker cytokine, bFGF, and validated the different cytoprotective effects of recombinant bFGF (rbFGF) and neutralization by monoclonal antibody (rbFGF+Ab) co-treatment. Aortic and lung tissues from different groups of C57BL/6J mice were examined by immunohistochemistry. SB203580 (specific inhibitor of p38) and BIX02189 (specific inhibitor of MEK5) were used to identify bFGF as the main cytoprotective factor acting via p38/MAPK and MEK5-KLF2 pathways.Results: Compared with traditional LSM, the new LSM not only significantly decreased TNF-α-induced intracellular adhesion molecule 1 and plasminogen activator inhibitor type 1 gene expression, but also significantly increased heme oxygenase 1 gene expression. The new LSM and bFGF attenuated TNF-α-induced ROS induction, inflammation, and tissue factor activity and inhibited the inflammatory- and thrombosis-related gene/protein overexpression both in vitro and in vivo. Mechanistically, the cytoprotective action of bFGF was mediated via the p38/MAPK and MEK5-KLF2 pathways.Conclusion: bFGF was identified as the critical factor mediating the cytoprotective effects of LSM derived from the modified laminar shear system.

Background and Aims: The angiotensin-(1-7)/angiotensin-converting enzyme 2/Mas receptor axis counter-regulates the detrimental effects of angiotensin II. Beneficial effects of angiotensin-(1-7), including anti-inflammation, oxidative stress reduction, and anti-thrombosis, have been reported. Previous studies documented that ramipril decreased thrombin generation in human hypertension and that the anti-thrombotic effects of captopril and losartan were angiotensin-(1-7)-dependent, suggesting an interaction between thrombin and angiotensin-(1-7). However, it is not clear whether angiotensin-(1-7) can alleviate the endothelial phenotypic changes induced by thrombin. We have previously documented cytoskeleton remodeling, cell adhesion, and cell migration as dominant altered phenotypes in thrombin-stimulated human aortic endothelial cells (HAECs). In this study, we investigated whether angiotensin-(1-7) can modulate thrombin-induced phenotypic changes. Furthermore, we investigated whether NAPDH oxidase 5 (Nox5)-produced reactive oxygen species (ROS) play a significant role in angiotensin-(1-7)-mediated phenotypic changes.Methods: HAECs were pretreated with 100 nM angiotensin-(1-7) for 1 h, followed by stimulation with 2 units/mL thrombin for different times. Immunofluorescent assay, monocyte adhesion assay, wound-healing assay, ROS assay, real-time PCR, Western blotting, and Nox5 siRNA transfection were conducted. HAECs were pretreated with the ROS scavenger N-acetylcysteine (NAC) to determine whether thrombin-induced phenotypic changes depended on ROS production.Results: Angiotensin-(1-7) prevented thrombin-induced actin cytoskeleton derangements, monocyte adhesion, and migratory impairment. Nox5 siRNA transfection confirmed that thrombin-induced Nox5 expression stimulated ROS production and increased HO-1/NQO-1/ICAM-1/VCAM-1 gene expression, all of which were decreased by angiotensin-(1-7). Phenotypic changes induced by thrombin were prevented by NAC pretreatment.Conclusion: Angiotensin-(1-7) prevents actin cytoskeleton derangement, monocyte adhesion, and migration impairment induced by thrombin via downregulation of ROS production. In addition, thrombin-induced Nox5 expression is involved in the production of ROS, and angiotensin-(1-7) decreases ROS through its inhibitory effect on Nox5 expression.

Background and Aims: Interleukin-1 receptor-associated kinase-1 (IRAK-1) is critical for mediating toll-like receptor and interleukin-1 receptor signaling. In this study, we have examined whether IRAK-1 expression is altered in high glucose (HG)-stimulated human aortic endothelial cells (HAECs), and whether microRNAs (miRs) target IRAK-1 to regulate HG-induced endothelial inflammation.Methods: HAECs were treated with HG for 24 and 48 h. Real-time PCR, Western blot, monocyte adhesion assay, bioinformatics analysis, TaqMan® arrays, microRNA mimic or inhibitor transfection, luciferase reporter assay and siRNA IRAK-1 transfection were performed. The aortic tissues from db/db type 2 diabetic mice were examined by immunohistochemistry staining.Results: HG time-dependently increased IRAK-1 mRNA and protein levels in HAECs, and was associated with increased VCAM-1/ICAM-1 gene expression and monocyte adhesion. Bioinformatic analysis, TaqMan® arrays, and real-time PCR were used to confirm that miR-146a-5p, miR-339-5p, and miR-874-3p were significantly downregulated in HG-stimulated HAECs, suggesting impaired feedback restraints on HG-induced endothelial inflammation via IRAK-1. However, only miR-146a-5p mimic transfection reduced the HG-induced upregulation of IRAK-1 expression, VCAM-1/ICAM-1 expression, and monocyte adhesion. Additionally, IRAK-1 depletion reduced HG-induced VCAM-1/ICAM-1 gene expression, and monocyte adhesion, indicating that HG-induced endothelial inflammation was mediated partially through IRAK-1. In vivo, intravenous injections of miR-146a-5p mimic prevented endothelial IRAK-1 and ICAM-1 expression in db/db mice.Conclusion: These results suggest that miR-146a-5p is involved in the regulation of HG-induced endothelial inflammation via modulation of IRAK-1; indicating that miR-146a-5p may be a novel target for the treatment of diabetic vascular complications.

Gastric adenocarcinoma originates from an abnormal epithelium. The aim of this study was to investigate the expression of sodium-potassium ATPase (NKA), a transmembrane protein located in the epithelium for Na+ and K+ transportation, and E-cadherin, which are both crucial for the epithelium and adherens junction, as potential gastric cancer biomarkers.45 patients diagnosed with gastric adenocarcinoma were recruited. Immunohistochemistry and immunofluorescence were conducted to for localization of NKA α1-, β1-isoform, and E-cadherin. NKA enzyme activity was determined by NADH-linked methods and immunoblotting of NKA α1-, β1-isoform, and E-cadherin were performed to evaluate protein expression.Immunostaining revealed that NKA was co-localized with E-cadherin in the glands of the gastric epithelium. Both NKA activity and α1-isoform protein expression were reduced in the study group (P < 0.05), indicating impaired NKA functions. In the adherens junctions, the NKA β1-isoform and E-cadherin were significantly reduced in the study groups (P < 0.05), indicating the adhesion force between cells may have been weakened.A significant decrease in NKA function (protein and activity) and E-cadherin in tumor lesions appear promising biomarker for gastric adenocarcinoma. Therefore, developing screening methods for detecting NKA function may be beneficial for the early diagnosis of gastric cancer. In our knowledge, this study was the first to investigate the NKA and E-cadherin expression in the relation of gastric adenocarcinoma in human patients.

Metastatic spinal cord compression (MSCC) treatment depends on life expectancies. Data regarding palliative decompression outcomes is scarce. We demonstrate that surgical timing has a significant impact on survival in MSCC patients treated with palliative decompression.Eighty-nine consecutive MSCC patients at a tertiary referral medical center were enrolled between January 2012 and February 2016. Wide laminectomy was performed for tumors invading the vertebral body. Debulking surgery was done for tumors damaging the posterior column of the spine. Patient records were retrospectively analyzed.Better survival was observed in patients with preoperative intact motor function (Group A, n = 37) than in those with motor deficit (Group B, n = 52, p = 0.0031). In Group B, survival was better in those who underwent surgery within 7 days of motor deficit onset than in those who underwent surgery 7 days after onset (p = 0.0444) and in postoperative ambulant patients than in nonambulant patients (p = 0.0120). In Group B, Frankel grade improved in patients who underwent surgery within 48 h than in those who underwent surgery after 48 h (p = 0.0992). Group A patients had a shorter hospital stay and higher revised Tokuhashi score than Group B patients. Overall survival was better in patients with a lower Tomita score (≤5, p = 0.0012), higher revised Tokuhashi score (≥9, p = 0.0009), better preoperative Frankel grade (p < 0.0001), and younger age (≤55 years, p = 0.0179). There were no significant differences in age, sex, tumor type, involved vertebrae level, Tomita score, intraoperative blood loss, operation time, incidence of infection, and postoperative complications between groups.We can improve the survival of MSCC patients with palliative decompression before motor deficits occur. After motor deficit onset, survival can still be improved with surgery within 7 days. Overall survival was better in patients aged ≤55 years.

A tropical species, the euryhaline milkfish (Chanos chanos), is a crucial economic species in Southeast Asia and is intolerant of water temperature below 12°C. Large numbers of milkfish die during cold periods in winter. Hypothermal environments usually increase oxidative stress in teleosts, and the liver is the major organ for anti-oxidative responses in the body. Peroxiredoxin-6 (Prdx6) in mammals is a multi-functional enzyme and acts as both glutathione peroxidase, phospholipase A2 and acyl-transferase for maintenance of redox status and prevention of cell membrane peroxidation. Prdx6 can protect cells from oxidant-induced membrane damage by translocating the Prdx6 protein from the cytosol to the membrane. Upon cold stress, Ccprdx6 transcript levels were up-regulated after 24 h and 96 h in livers of fresh water (FW)- and seawater (SW)-acclimated milkfish, respectively. In the hypothermal FW group, the Prdx6 protein was up-regulated in the cytosol of hepatocytes with a similar role as glutathione peroxidase to reduce oxidative stress upon hypothermal challenge. Conversely, in hypothermal SW milkfish, total Prdx6 protein was down-regulated. However, cytosolic Prdx6 protein was translocated to the membrane, using the ability of phospholipase A2 to stabilize the membrane redox state. Moreover, H2O2 content was increased in FW-acclimated milkfish livers upon hypothermal challenge. Ex vivo H2O2 treatment of milkfish livers also induced Ccprdx6 transcriptional expression, which provided more evidence of the antioxidant role of milkfish Prdx6. Taken together, upon hypothermal challenge, greater oxidative stress in livers of FW-acclimated milkfish rather than SW-acclimated individuals led to different profiles of hepatic CcPrdx6 expression between the FW and SW group. The results indicated that CcPrdx6 played the role of antioxidant with different mechanisms, i.e., binding to reactive oxygen species and stabilizing membrane fluidity, in livers of hypothermal FW and SW milkfish, respectively.

Milkfish is an important aquaculture species in Taiwan, and its high mortality during cold snaps in winter usually causes huge economic losses. To understand the effect of hypothermal stress and the corresponding compensatory stress response in milkfish, this study aimed to compare liver and gill protein levels between milkfish exposed to nonlethal (18°C), lethal (16°C), and control (28°C) temperatures. Using a proteomics approach based on two-dimensional electrophoresis and nano-LC-MS/MS analysis, this study identified thirty unique protein spots from milkfish livers and gills for which protein abundance was significantly different between nonlethal, lethal, and control temperature groups. Proteins identified in the liver were classified into three different categories according to their cellular function: (1) anti-oxidative stress, (2) apoptotic pathway, and (3) cytoskeleton. Similarly, proteins identified in the gill were sorted in five different functional categories: (1) cytoskeleton, (2) immune response, (3) protein quality control, (4) energy production, and (5) intracellular homeostasis. Based on functional information derived from the identified proteins, we assumed that different levels of hypothermal stress had a different effect and induced a different cellular response. Upon nonlethal hypothermal stress, the identified proteins were involved in anti-oxidative stress and anti-inflammation pathways, suggesting that milkfish had high levels of oxidative stress in the liver and exhibited inflammation response in the gill. Upon lethal hypothermal stress, however, identified proteins were associated with apoptosis in the liver and regulation of intracellular homeostasis in the gill. The present study provided evidence to illustrate different multi-physiological responses to nonlethal and lethal hypothermal stress in milkfish livers and gills.

Oral cancer is the fourth most common cause of death from cancer in Taiwanese men. Indirubin-3'-monoxime (I3M), a potent cyclin-dependent kinase inhibitor, has therapeutic effects in other cancer cells. In this study, we carried out in vitro assays to test cell viability, cell cycle progression, apoptosis, cell migration and invasion in this cancer type. In addition, using an oral tumorigenic animal model, we examined target gene and protein expression using real time qPCR, immunoblotting and immunohistochemical staining. Our results demonstrate that I3M has an anti-proliferative effect in both Cal-27 and HSC-3 oral cancer cell lines and that treatment of Cal-27 and HSC-3 cells with I3M results in apoptosis through the activation of cytochrome c. In addition, I3M interrupts the cell cycle in Cal-27 cells in a dose-dependent manner by arresting cells in the G2/M phase. We also found that I3M suppresses migration and invasion in Cal-27 cells by inhibiting the expression of focal adhesion kinase, urokinase-type plasminogen inhibitor, and matrix metalloproteinase 9. Moreover, we identified survivin as a target protein in I3M-treated oral cancer cells. Using an oral cancer mouse model, we demonstrate that topical application of an adhesive gel composed of I3M and poly(vinyl alcohol) (I3M/PVA) has dose-dependent anti-tumorigenic effects. Following treatment, the expression of survivin protein and mRNA was downregulated in cancerous tissues. Furthermore, plasma survivin levels were also reduced in the I3M-treated mice. These results suggest that topical application of I3M, a drug synthesized from indirubin, which is found in Qing-Dai - has therapeutic potential for treating oral cancer.

AbstractAim:Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) provides neuroprotection against apoptosis in a transient middle cerebral artery occlusion (MCAo) model. This study was to further investigate the anti-apoptotic effect of FA during reperfusion after cerebral ischemia.Methods:Rats were subjected to 90 min of cerebral ischemia followed by 3 or 24 h of reperfusion after which they were sacrificed.Results:Intravenous FA (100 mg/kg) administered immediately after middle cerebral artery occlusion (MCAo) or 2 h after reperfusion effectively abrogated the elevation of postsynaptic density-95 (PSD-95), neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine, and cleaved caspase-3 levels as well as apoptosis in the ischemic cortex at 24 h of reperfusion. FA further inhibited Bax translocation, cytochrome c release, and p38 mitogen-activated protein (MAP) kinase phosphorylation. Moreover, FA enhanced the expression of gamma-aminobutyric acid type B receptor subunit 1 (GABAB1) in the ischemic cortex at 3 and 24 h of reperfusion. In addition, nitrotyrosine-positive cells colocalized with cleaved caspase-3-positive cells, and phospho-p38 MAP kinase-positive cells colocalized with nitrotyrosine- and Bax-positive cells, indicating a positive relationship among the expression of nitrotyrosine, phospho-p38 MAP kinase, Bax, and cleaved caspase-3. The mutually exclusive expression of GABAB1 and nitrotyrosine revealed that there is a negative correlation between GABAB1 and nitrotyrosine expression profiles. Additionally, pretreatment with saclofen, a GABAB receptor antagonist, abolished the neuroprotection of FA against nitric oxide (NO)-induced apoptosis.Conclusion:FA significantly enhances GABAB1 receptor expression at early reperfusion and thereby provides neuroprotection against p38 MAP kinase-mediated NO-induced apoptosis at 24 h of reperfusion. [ABSTRACT FROM AUTHOR]

Our previous studies have shown that Uncaria rhynchophylla (UR) can reduce epileptic seizures. We hypothesized that UR and its major component rhynchophylline (RH), reduce epileptic seizures in rats treated with kainic acid (KA) by inhibiting nuclear factor-κB (NF-κB) and activator-protein-1 (AP-1) activity, and by eliminating superoxide anions. Therefore, the level of superoxide anions and the DNA binding activities of NF-κB and AP-1 were measured. Sprague-Dawley (SD) rats were pre-treated with UR (1.0 g/kg, i.p.), RH (0.25 mg/kg, i.p.), or valproic acid (VA, 250 mg/kg, i.p.) for 3 days and then KA was administered intra-peritoneal (i.p.). The results indicated that UR, RH, and VA can reduce epileptic seizures and the level of superoxide anions in the blood. Furthermore, KA was demonstrated to induce the DNA binding activities of NF-κB and AP-1. However, these inductions were inhibited by pre-treatment with UR, RH, or VA for 3 days. Moreover, UR and RH were shown to be involved in the suppression of c-Jun N-terminal kinase (JNK) phosphorylation. This study suggested that UR and RH have antiepileptic effects in KA-induced seizures and are associated with the regulation of the innate immune system via a reduction in the level of superoxide anions, JNK phosphorylation, and NF-κB activation. [ABSTRACT FROM AUTHOR]

An end-point quantitative nucleic acid sequence-based amplification (NASBA) reaction with two exogenous internal standards for the detection of the model analyte E. coli clpB mRNA was developed and statistically analyzed. Electrochemiluminescence was chosen as a highly sensitive detection means allowing careful evaluation of the internal standards used. The two internal standards examined had been designed previously using a novel and rapid NASBA-based method. Initially, each standard was used separately in a NASBA reaction; subsequently, two internal standards were added into one reaction at different concentrations. The accuracy and precision of the data obtained were analyzed using linear and multiple regression analysis. In the case of single-standard reactions, the accuracy was >95% and the precision >98.5%. In the case of double-standard reactions, the accuracy increased to >97%. With a single internal standard, 3 orders of magnitude of target sequence could be quantified; using three different concentrations of one internal standard, the dynamic range increased to 5 orders of magnitude. In both cases, a detection limit as low as 0.14 pg of target sequence was obtained. In the case of double-internal standard reactions, a dynamic range with 5 orders of magnitude and a detection limit of 1.76 pg was determined. The high-performance quality of the internal standards was assumed to be in part due to the unique synthesis process using two NASBA reactions rather than traditional cloning techniques. [ABSTRACT FROM AUTHOR]

Introduction The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. Materials and methods HAECs were stimulated with 2 units/mL thrombin for 5 h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore TM software. Results A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore TM pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Conclusions Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. [ABSTRACT FROM AUTHOR]