Endothelial cells (ECs) are constantly exposed to blood pressure-induced mechanical strain. We have previously demonstrated that cyclic strain can induce gene expression of monocyte chemotactic protein-1 (MCP-1). The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence indicates that intracellular reactive oxygen species (ROS) can act as a second messenger for signal transduction and thus affect gene expression. The potential role of ROS in strain-induced MCP-1 expression was investigated. ECs under cyclic strain induced a sustained elevated production of intracellular superoxide. ECs under strain or pretreated with either H2O2 or xanthine oxidase/hypoxanthine induced MCP-1 expression. Strain- or oxidant-induced MCP-1 mRNA levels could be inhibited by treating ECs with catalase or antioxidant N-acetyl-cysteine (NAC). Functional analysis of MCP-1 promoter and site-specific mutations indicates that the proximal tissue plasminogen activator-responsive element (TRE) in the -60-bp promoter region is sufficient for strain or H2O2 inducibility. Electrophoretic mobility shift assays demonstrated an increase of nuclear proteins binding to TRE sequences from ECs subsequent to strain or H2O2 treatment. NAC or catalase pretreatment of ECs inhibited the strain- or H2O2-induced AP-1 binding. These results clearly indicate that cyclic strain inducibility of MCP-1 in ECs uses the interaction of AP-1 proteins with TRE sites via the elevation of intracellular ROS levels in strained ECs. These findings emphasize the importance of intracellular ROS in the modulation of hemodynamic force-induced gene expression in vascular ECs.

Macrophage infiltration contributes to the instability of atherosclerotic plaques. In the present study, we investigated whether selective inhibition of PI3K/Akt/mTOR signaling pathway can enhance the stability of atherosclerotic plaques by activation of macrophage autophagy. In vitro study, selective inhibitors or siRNA of PI3K/Akt/mTOR pathways were used to treat the rabbit's peritoneal primary macrophage cells. Inflammation related cytokines secreted by macrophages were measured. Ultrastructure changes of macrophages were examined by transmission electron microscope. mRNA or protein expression levels of autophagy related gene Beclin 1, protein 1 light chain 3 II dots (LC3-II) or Atg5-Atg12 conjugation were assayed by quantitative RT-PCR or Western blot. In vivo study, vulnerable plaque models were established in 40 New Zealand White rabbits and then drugs or siRNA were given for 8 weeks to inhibit the PI3K/Akt/mTOR signaling pathway. Intravascular ultrasound (IVUS) was performed to observe the plaque imaging. The ultrastructure of the abdominal aortic atherosclerosis lesions were analyzed with histopathology. RT-PCR or Western blot methods were used to measure the expression levels of corresponding autophagy related molecules. We found that macrophage autophagy was induced in the presence of Akt inhibitor, mTOR inhibitor and mTOR-siRNA in vitro study, while PI3K inhibitor had the opposite role. In vivo study, we found that macrophage autophagy increased significantly and the rabbits had lower plaque rupture incidence, lower plaque burden and decreased vulnerability index in the inhibitors or siRNA treated groups. We made a conclusion that selective inhibition of the Akt/mTOR signal pathway can reduce macrophages and stabilize the vulnerable atherosclerotic plaques by promoting macrophage autophagy.

Endothelial cells (ECs) exposed to cyclic strain induce gene expression. To elucidate the signaling mechanisms involved, we studied the effects of cyclic strain on ECs by using early growth response-1 (Egr-1) as a target gene. Cyclic strain induced a transient increase of Egr-1 mRNA levels that resulted in an increase of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. ECs subjected to strain enhanced Egr-1 transcription as revealed by promoter activities. Catalase pretreatment inhibited this induction. ECs, transfected with a dominant positive mutant of Ras (RasL61), increased Egr-1 promoter activities. In contrast, transfection with a dominant negative mutant of Ras (RasN17) attenuated this strain inducibility. ECs transfected with a dominant negative mutant of Raf-1 (Raf301) or the catalytically inactive mutant of extracellular signal-regulated kinase (ERK)-2 (mERK2) diminished strain-induced promoter activities. However, little effect on strain inducibility was observed in ECs transfected with a dominant negative mutant of Rac (RacN17) or a catalytically inactive mutant of JNK (JNK[K-R]). Consistently, strain-induced Egr-1 expression was inhibited after ECs were treated with a specific inhibitor (PD98059) to mitogen-activated protein kinase kinase. Moreover, strain to ECs induced mitogen-activated protein kinase/ERK activity. The activation of the ERK pathway was further substantiated by an increase of strain-induced transcriptional activity of Elk1, an ERK substrate. This strain-induced ERK activity was attenuated after ECs were treated with N-acetylcysteine or catalase. Consequently, this Egr-1 gene induction was abolished after ECs were treated with N-acetylcysteine or catalase. Deletion analyses of the promoter region (-698 bp) indicated that cyclic strain and H2O2 shared a common serum response element. Our data clearly indicate that cyclic strain-induced Egr-1 expression is mediated mainly via the Ras/Raf-1/ERK pathway and that strain-induced reactive oxygen species can modulate Egr-1 expression at least partially via this signaling pathway.

Since endothelial cells are constantly subjected to pressure-induced strain, we examined how cyclic strain affects the expression of intercellular adhesion molecule-1 (ICAM-1). Endothelial cells grown on a flexible membrane base were deformed by different sinusoidal negative pressures (-10, -15, or -20 kPa) to produce an average strain of 9%, 11%, and 12%, respectively, for various times. The release of the soluble form of ICAM-1 from strained endothelial cells increased in a time- and strain-dependent manner. Using flow cytometric analysis, we showed the induction of ICAM-1 expression on the endothelial cell surface to depend on both time and the amount of strain. Northern blot analysis revealed a sustained, approximately 1.8-fold increase in ICAM-1 mRNA levels in 11% strained cells. Strain-induced expression of ICAM-1 correlated with a strain-dependent increase in adhesion of monocytic cells to strained cells. This increase in monocytic cell adhesion could be partially inhibited by pretreatment of strained cells with antibody to ICAM-1. These results indicate that mechanical strain can stimulate the expression of ICAM-1 by endothelial cells and thus contribute to the increased adhesion of monocytes to strained cells. Such strain-induced expression of ICAM-1 may contribute to the trapping of monocytes on local vascular walls where strain is high and to the initiation of atherogenesis, thus providing a possible link between hypertension and atherogenesis.

Abstract-Vascular endothelial cells (ECs) are constantly subjected to pressure-induced strain. We have previously demonstrated that strain can induce intercellular adhesion molecule-1 (ICAM-1) expression in ECs. The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence suggests that intracellular reactive oxygen species (ROS) may act as second messengers. The potential role of ROS in strain-induced ICAM-1 expression was examined. ECs grown on a flexible membrane base were deformed with various sinusoidal negative pressures to produce an average strain of 12%. Cyclic strain induced an increase in intracellular ROS measured by fluorescent intensity of dichlorofluorescein formed after peroxidation. Maximal levels of ROS were seen after 30 minutes. Levels subsequently decreased but remained elevated compared with unstrained groups. Concomitantly, a sustained increase of H 2 O 2 decomposition activity was observed in strained ECs. Both ROS and H 2 O 2 decomposition activity returned to basal levels after removal of the strain. ECs treated with an antioxidant (N-acetylcysteine or catalase) inhibited strain-induced ROS generation and ICAM-1 mRNA levels followed by decreased ICAM-1 expression on EC surfaces. This inhibition may account for the reduced monocytic cell adhesion in antioxidant-treated ECs but not in strained controls.

Hypoxia induces endothelial dysfunction that results in a series of cardiovascular injuries. Early growth response-1 (Egr-1) has been indicated as a common theme in vascular injury. Here we demonstrates that in bovine aortic endothelial cells (ECs) subjected to hypoxia (PO(2) approximately 23 mmHg), rapidly increased Egr-1 mRNA expression which peaked within 30 min and decreased afterwards. Treatment of ECs with PD98059, a specific inhibitor to mitogen-activated protein kinase (MAPK/ERK), inhibited this hypoxia-induced Egr-1 expression. The involvement of ERK pathway was further substantiated by the inhibition of Egr-1 promoter activities when ECs were co-transfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf 301), or a catalytically inactive mutant of ERK2 (mERK). In addition, the hypoxia-induced transcriptional activity of Elk-1, an ERK substrate, was abolished by administration of PD98059. Addition of calphostin C, a protein kinase C (PKC) inhibitor, completely blocked the hypoxia-augmented Egr-1 expression. The likewise occurred while exposing ECs to D609 to inhibit phospholipase C and BAPTA/AM to chelate intracellular calcium. Hypoxia to ECs increased ERK phosphorylation within 10 min and which was abolished by administration of PD98095, calphostin C, and BAPTA/AM. Hypoxia triggered a transient translocation of PKCalpha from cytosol to membrane fraction concurrent with the association of PKCalpha to Raf-1. Involvement of PKCalpha in mediating ERK activation was further confirmed by the inhibition of ERK and the subsequent Egr-1 gene induction with antisense oligonucleotides to PKCalpha. These results indicate that ECs under hypoxia induce Egr-1 expression and this induction requires calcium, phospholipase C activation, and PKCalpha-mediated Ras/Raf-1/ERK1/2 signaling pathway. Our finding support the importance of specific PKC isozyme linked to MAPK pathway in the regulation of endothelial responses to hypoxia.

Background: Enhanced IL1R2 expression has frequently been observed in tumors, but its function is unclear. Results: Intracellular IL1R2 transcriptionally activates IL-6 and VEGF-A by complexing with c-Fos and promotes the angiogenesis of colon cancer cells. Conclusion: IL1R2 functions as a transcriptional coactivator to enhance the expression of angiogenic factors. Significance: IL1R2 activates angiogenic factors and hence may serve as a clinical marker for prognosis or treatment.

Endothelial cells (ECs) are constantly subjected to hemodynamic forces including cyclic pressure-induced strain. The role of protein kinase C (PKC) in cyclic strain-treated ECs was studied. PKC activities were induced as cyclic strain was initiated. Cyclic strain to ECs caused activation of PKC-␣ and -⑀. The translocation of PKC-␣ and -⑀ but not PKC-␤ from the cytosolic to membrane fraction was observed. An early transient activation of PKC-␣ versus a late but sustained activation of PKC-⑀ was shown after the onset of cyclic strain. Consistently, a sequential association of PKC-␣ and -⑀ with the signaling molecule Raf-1 was shown. ECs treated with a PKC inhibitor (calphostin C) abolished the cyclic strain-induced Raf-1 activation. ECs under cyclic strain induced a sustained activation of extracellular signalregulated protein kinases (ERK1/2), which was inhibited by treating ECs with calphostin C. ECs treated with a specific Ca 2؉ -dependent PKC inhibitor (Go 6976) showed an inhibition in the early phase of ERK1/2 activation but not in the late and sustained phase. ECs transfected with the antisense to PKC-␣, the antisense to PKC-⑀, or the inhibition peptide to PKC-⑀ reduced strain-induced ERK1/2 phosphorylation in a temporal manner. PKC-␣ mediated mainly the early ERK1/2 activation, whereas PKC-⑀ was involved in the sustained ERK1/2 activation. Strained ECs increased transcriptional activity of Elk1 (an ERK1/2 substrate). ECs transfected with the antisense to each PKC isoform reduced Elk1 and monocyte chemotactic protein-1 promotor activity. Our findings conclude that a sequential activation of PKC isoform (␣ and ⑀) contribute to Raf/ERK1/2 activation, and PKC-⑀ appears to play a key role in endothelial adaptation to hemodynamic environment.