Danny Ling Wang

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression.

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm2 in a parallel plate flow chamber to demonstrate the potential role of ROS in shear‐induced cellular response. The use of 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5‐ to 1.5‐fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear‐induced elevation of ROS was blocked by either antioxidant N‐acetyl‐cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (·OH) scavenger, 1,3‐dimethyl‐2‐thiourea (DMTU), suggesting that hydrogen peroxide (H2O2), ·OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3‐fold increase of decomposition of exogenously added H2O2 was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3‐amino‐1,2,4‐triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c‐fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c‐fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear‐induced c‐fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow. J. Cell. Physiol. 175:156–162, 1998.

Increased ferritin gene expression in atherosclerotic lesions

To identify genes potentially implicated in atherogenesis, a cDNA library was constructed from human atherosclerotic aorta and differentially screened with 32P-labeled-cDNAs prepared from human normal and atherosclerotic aortas. Two cDNA clones exhibiting higher hybridization to the 32P-labeled cDNAs from atherosclerotic vessels were isolated and identified to be genes encoding L-ferritin and H-ferritin, respectively. Northern blot analysis confirmed that the expression of both ferritin genes was notably higher in human and rabbit atherosclerotic aortas than in their normal counterparts. A time-course study illustrated that both L- and H-ferritin mRNAs were markedly increased in aortas of rabbits after feeding with a high cholesterol diet for 6 wk, which was also the time period after which the formation of lesions became evident. In situ hybridization revealed that both L- and H-ferritin mRNAs were induced in endothelial cells and macrophages of human early lesions. The signals were also detected in the smooth muscle cells of advanced lesions. Immunostaining further identified the presence of ferritin protein in atherosclerotic lesions. On the other hand, Prussian blue stain revealed the presence of iron deposits in advanced lesions but not in early human or rabbit lesions. Further experiments with cultured human monocytic THP-1 cells and aortic smooth muscle cells demonstrated that ferritin mRNAs were subjected to up-regulation by treatment with IL-1 or TNF, while TGF, PDGF, and oxidized LDL did not affect the expression of either ferritin gene in both cell lines. Collectively, these results clearly demonstrate that ferritin genes are susceptible to induction in the course of plaque formation.

Mechanical Strain Induces Monocyte Chemotactic Protein-1 Gene Expression in Endothelial Cells Effects of Mechanical Strain on Monocyte Adhesion to Endothelial Cells

Monocyte chemotactic protein-1 (MCP-1), a potent monocyte chemoattractant secreted by endothelial cells (ECs), is believed to play a key role in the early events of atherogenesis. Since vascular ECs are constantly subjected to mechanical stresses, we examined how cyclic strain affects the expression of the MCP-1 gene in human ECs grown on a flexible membrane base deformed by sinusoidal negative pressure (peak level, -16 kPa at 60 cycles per minute). Northern blot analysis demonstrated that the MCP-1 mRNA levels in ECs subjected to strain for 1, 5, or 24 hours were double those in control ECs (P < .05). This strain-induced increase was mainly serum independent, and MCP-1 mRNA level returned to its control basal level 3 hours after release of strain. Culture media from strained ECs contained approximately twice the MCP-1 concentration and more than twice the monocyte chemotactic activity of media from control ECs (P < .05). Pretreatment of collected media with anti-MCP-1 antibody suppressed such activity. Monocyte adhesion to ECs subjected to strain for 12 hours was 1.8-fold greater than adhesion to unstrained control ECs (P < .05). A protein kinase C inhibitor, calphostin C, abolished the strain-induced MCP-1 gene expression. In addition, cAMP- or cGMP-dependent protein kinase inhibitors (KT5720 and KT5823, respectively) partially inhibited such expression. Pretreatment with EGTA or the intracellular Ca2+ chelator BAPTA/AM strongly suppressed the strain-induced MCP-1 mRNA. Verapamil, a Ca2+ channel blocker, greatly reduced MCP-1 mRNA levels in both strained and unstrained ECs.(ABSTRACT TRUNCATED AT 250 WORDS)

SIRT3 deacetylates FOXO3 to protect mitochondria against oxidative damage.

Progressive accumulation of defective mitochondria is a common feature of aged cells. SIRT3 is a NAD(+)-dependent protein deacetylase that regulates mitochondrial function and metabolism in response to caloric restriction and stress. FOXO3 is a direct target of SIRT3 and functions as a forkhead transcription factor to govern diverse cellular responses to stress. Here we show that hydrogen peroxide induces SIRT3 to deacetylate FOXO3 at K271 and K290, followed by the upregulation of a set of genes that are essential for mitochondrial homeostasis (mitochondrial biogenesis, fission/fusion, and mitophagy). Consequently, SIRT3-mediated deacetylation of FOXO3 modulates mitochondrial mass, ATP production, and clearance of defective mitochondria. Thus, mitochondrial quantity and quality are ensured to maintain mitochondrial reserve capacity in response to oxidative damage. Maladaptation to oxidative stress is a major risk factor underlying aging and many aging-related diseases. Hence, our finding that SIRT3 deacetylates FOXO3 to protect mitochondria against oxidative stress provides a possible direction for aging-delaying therapies and disease intervention.

Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications.

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects.

Endothelial exposure to hypoxia induces Egr-1 expression involving PKCα-mediated Ras/Raf-1/ERK1/2 pathway

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 (PO2 ≈ 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 PKCα from cytosol to membrane fraction concurrent with the association of PKCα to Raf‐1. Involvement of PKCα in mediating ERK activation was further confirmed by the inhibition of ERK and the subsequent Egr‐1 gene induction with antisense oligonucleotides to PKCα. These results indicate that ECs under hypoxia induce Egr‐1 expression and this induction requires calcium, phospholipase C activation, and PKCα‐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.

Angiotensin II Increases Expression of α1C Subunit of L-Type Calcium Channel Through a Reactive Oxygen Species and cAMP Response Element–Binding Protein–Dependent Pathway in HL-1 Myocytes

Angiotensin II (Ang II) is involved in the pathogenesis of atrial fibrillation (AF). L-type calcium channel (LCC) expression is altered in AF remodeling. We investigated whether Ang II modulates LCC current through transcriptional regulation, by using murine atrial HL-1 cells, which have a spontaneous calcium transient, and an in vivo rat model. Ang II increased LCC α1C subunit mRNA and protein levels and LCC current density, which resulted in an augmented calcium transient in atrial myocytes. An ≈2-kb promoter region of LCC α1C subunit gene was cloned to the pGL3 luciferase vector. Ang II significantly increased promoter activity in a concentration- and time-dependent manner. Truncation and mutational analysis of the LCC α1C subunit gene promoter showed that cAMP response element (CRE) (−1853 to −1845) was an important cis element in Ang II-induced LCC α1C subunit gene expression. Transfection of dominant-negative CRE binding protein (CREB) (pCMV-CREBS133A) abolished the Ang II effect. Ang II (1 μmol/L, 2 hours) induced serine 133 phosphorylation of CREB and binding of CREB to CRE and increased LCC α1C subunit gene promoter activity through a protein kinase C/NADPH oxidase/reactive oxygen species pathway, which was blocked by the Ang II type 1 receptor blocker losartan and the antioxidant simvastatin. In the rat model, Ang II infusion increased LCC α1C subunit expression and serine 133 phosphorylation of CREB, which were attenuated by oral losartan and simvastatin. In summary, Ang II induced LCC α1C subunit expression via a protein kinase C–, reactive oxygen species–, and CREB-dependent pathway and was blocked by losartan and simvastatin.

Shear flow increases S-nitrosylation of proteins in endothelial cells

AIMS Endothelial cells (ECs) constantly exposed to shear flow increase nitric oxide production via the activation of endothelial nitric oxide synthase. Nitric oxide-mediated S-nitrosylation has recently been identified as an important post-translational modification that may alter signalling and/or protein function. S-nitrosylation of endothelial proteins after shear flow treatment has not been fully explored. In this study, the CyDye switch method was utilized to examine S-nitrosylated proteins in ECs after exposure to shear flow. METHODS AND RESULTS Human umbilical vein ECs were subjected to shear flow for 30 min, and S-nitrosylated proteins were detected by the CyDye switch method. In principle, free thiols in proteins become blocked by alkylation, the S-nitrosylated bond is reduced by ascorbate, and then CyDye labels proteins. Proteins that separately labelled with Cy3 or Cy5 were mixed and subjected to two-dimensional gel electrophoresis for further analysis. More than 100 S-nitrosoproteins were detected in static and shear-treated ECs. Among these, 12 major proteins of heterogeneous function showed a significant increase in S-nitrosylation following shear flow. The S-nitrosylated residues in tropomyosin and vimentin, which were localized in the hydrophobic motif of each protein, were identified as Cys170 and Cys328, respectively. CONCLUSION Post-translational S-nitrosylation of proteins in ECs can be detected by a reliable CyDye switch method. This flow-induced S-nitrosylation of endothelial proteins may be essential for the adaptation and remodelling of ECs under flow conditions.

Cyclic strain activates redox-sensitive proline-rich tyrosine kinase 2 (PYK2) in endothelial cells.

Proline-rich tyrosine kinase 2 (PYK2), structurally related to focal adhesion kinase, has been shown to play a role in signaling cascades. Endothelial cells (ECs) under hemodynamic forces increase reactive oxygen species (ROS) that modulate signaling pathways and gene expression. In the present study, we found that bovine ECs subjected to cyclic strain rapidly induced phosphorylation of PYK2 and Src kinase. This strain-induced PYK2 and Src phosphorylation was inhibited by pretreating ECs with an antioxidant N-acetylcysteine. Similarly, ECs exposed to H2O2 increased both PYK2 and Src phosphorylation. An increased association of Src to PYK2 was observed in ECs after cyclic strain or H2O2 exposure. ECs treated with an inhibitor to Src (PPI) greatly reduced Src and PYK2 phosphorylation, indicating that Src mediated PYK2 activation. Whereas the protein kinase C (PKC) inhibitor (calphostin C) pretreatment was shown to inhibit strain-induced NADPH oxidase activity, ECs treated with either calphostin C or the inhibitor to NADPH oxidase (DPI) reduced strain-induced ROS levels and then greatly inhibited the Src and PYK2 activation. In contrast to the activation of PYK2 and Src with calcium ionophore (ionomycin), ECs treated with a Ca2+chelator inhibited both phosphorylation, indicating that PYK2 and Src activation requires Ca2+. ECs transfected with antisense to PKCα, but not antisense to PKCε, reduced cyclic strain-induced PYK2 activation. These data suggest that cyclic strain-induced PYK2 activity is mediated via Ca2+-dependent PKCα that increases NADPH oxidase activity to produce ROS crucial for Src and PYK2 activation. ECs under cyclic strain thus activate redox-sensitive PYK2 via Src and PKC, and this PYK2 activation may play a key role in the signaling responses in ECs under hemodynamic influence.

Activation of PKC‐ε and ERK1/2 participates in shear‐induced endothelial MCP‐1 expression that is repressed by nitric oxide

Vascular endothelial cells (ECs) continuously experience hemodynamic shear stress generated from blood flow. Previous studies have demonstrated that shear stress modulates monocyte chemotactic protein‐1 (MCP‐1) expression in ECs. This study explored the roles of protein kinase C (PKC), extracellular signal‐regulated protein kinase (ERK1/2), and nitric oxide (NO) in sheared‐induced MCP‐1 expression in ECs. The activation of PKC‐α and PKC‐ε isoforms was observed in ECs exposed to shear stress. The use of an inhibitor (calphostin C) to PKC‐α and PKC‐ε decreased ERK1/2 activation and MCP‐1 induction by shear, whereas an inhibitor (Go6976) to PKC‐α did not affect ERK1/2 activation or MCP‐1 induction. Inhibition of ERK1/2 activation by PD98059 blocked MCP‐1 induction. Transfection of ECs with an antisense to PKC‐ε abolished the shear inducibility of MCP‐1 promoter. These results demonstrate that PKC‐ε and ERK1/2 participate in shear‐induced MCP‐1 expression. We also examined the regulatory role of NO in MCP‐1 expression. An NO donor (NOC18) suppressed shear‐induced activation of PKC‐ε and ERK1/2, and also repressed MCP‐1 induction. Consistently, overexpression of endothelial nitric oxide synthase (eNOS) to enhance the endogenous generation of NO in ECs decreased the activation of PKC‐ε and ERK1/2, and also inhibited MCP‐1 expression. Taken together, these findings suggest that PKC‐ε and ERK1/2 are critical in the signaling pathway(s) leading to the MCP‐1 expression induced by shear stress. Additionally, this study indicates that NO, by repressing PKC‐ε activity and ERK pathway activation, attenuates shear‐induced MCP‐1 expression.