Biomedical subjects

D L Wang

Publications and source records attributed to D L Wang.

At least 37 records · Page 2Linked to original sources

PubMed1999

Modulation of Ras/Raf/extracellular signal-regulated kinase pathway by reactive oxygen species is involved in cyclic strain-induced early growth response-1 gene expression in endothelial cells.

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.

Animals↗

PubMed1999

Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes.

OBJECTIVES: Recent evidence indicates that reactive oxygen species (ROS) may act as second messengers in receptor-mediated signaling pathways. The possible role of ROS during Et-1 stimulation in cardiomyocytes was therefore investigated. METHODS: Intracellular ROS levels were measured with fluorescence probe 2',7'-dichlorofluorescin diacetate by confocal microscopy in cultured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was analyzed by Northern blotting and promoter activity. RESULTS: Et-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The increase of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomyocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) reduced Et-1-induced ROS levels. Et-1 or H2O2 treatment of cardiomyocytes rapidly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (-2.25 kb) and reporter gene chloramphenicol acetyltransferase. The induction of mRNA levels and the promoter activities of c-fos gene by Et-1 or H2O2 were abolished by pretreating cardiomyocytes with catalase or NAC. Cells transiently transfected with the dominant positive mutant of p21ras (RasL61) led to a significant increase in intracellular ROS. Concomitantly, the mRNA levels and the promoter activities of c-fos were also induced. In contrast, cells transfected with the dominant negative mutant of Ras (RasN17) inhibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels and promoter activities by Et-1 were also inhibited. CONCLUSIONS: These findings clearly indicate that Et-1 treatment to cardiomyocytes can induce ROS via Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second messengers in Et-1-induced responses on cardiomyocytes.

Animals↗

PubMed1999

Segmentation of medical images using LEGION.

Advances in visualization technology and specialized graphic workstations allow clinicians to virtually interact with anatomical structures contained within sampled medical-image datasets. A hindrance to the effective use of this technology is the difficult problem of image segmentation. In this paper, we utilize a recently proposed oscillator network called the locally excitatory globally inhibitory oscillator network (LEGION) whose ability to achieve fast synchrony with local excitation and desynchrony with global inhibition makes it an effective computational framework for grouping similar features and segregating dissimilar ones in an image. We extract an algorithm from LEGION dynamics and propose an adaptive scheme for grouping. We show results of the algorithm to two-dimensional (2-D) and three-dimensional (3-D) (volume) computerized topography (CT) and magnetic resonance imaging (MRI) medical-image datasets. In addition, we compare our algorithm with other algorithms for medical-image segmentation, as well as with manual segmentation. LEGION's computational and architectural properties make it a promising approach for real-time medical-image segmentation.

Algorithms↗

PubMed1999

Therapeutic efficacy and safety of one-week intermittent therapy with itraconazole for onychomycosis in a Chinese patient population.

OBJECTIVE: To evaluate the efficacy and safety of a 1-week intermittent itraconazole dosing schedule for onychomycosis. METHODS: In this multicenter, open-label study, 646 patients received itraconazole 200 mg twice daily for 1 week/month, followed by 3 weeks without therapy. Patients with fingernail infections received 2 treatment cycles, patients with toenail or combined toenail and fingernail infections received 3 cycles. Efficacy was evaluated at week 9 (2-month regimen), week 13 (3-month regimen) and 3, 6 or 9 (toenails only) months after completion of therapy. RESULTS: Clinical and mycologic cure rates for fingernails were greater than 90% 6 months after completion of 2 treatment cycles. Clinical and mycologic cure rates for toenails were 84 and 98%, respectively, 9 months after completion of 3 cycles. Treatment was well tolerated; adverse events (mostly mild) occurred in 4.6% of patients. CONCLUSION: A 1-week intermittent itraconazole dosing regimen is a safe and effective treatment for onychomycosis.

Adolescent↗

PubMed1998

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 (H202), .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.

Acetylcysteine↗

PubMed1998

Effects of disturbed flow on endothelial cells.

Atherosclerotic lesions tend to localize at curvatures and branches of the arterial system, where the local flow is often disturbed and irregular (e.g., flow separation, recirculation, complex flow patterns, and nonuniform shear stress distributions). The effects of such flow conditions on cultured human umbilical vein endothelial cells (HUVECs) were studied in vitro by using a vertical-step flow channel (VSF). Detailed shear stress distributions and flow structures have been computed by using the finite volume method in a general curvilinear coordinate system. HUVECs in the reattachment areas with low shear stresses were generally rounded in shape. In contrast, the cells under higher shear stresses were significantly elongated and aligned with the flow direction, even for those in the area with reversed flow. When HUVECs were subjected to shearing in VSF, their actin stress fibers reorganized in association with the morphological changes. The rate of DNA synthesis in the vicinity of the flow reattachment area was higher than that in the laminar flow area. These in vitro experiments have provided data for the understanding of the in vivo responses of endothelial cells under complex flow environments found in regions of prevalence of atherosclerotic lesions.

Arteriosclerosis↗

PubMed1998

Cyclic strain-induced reactive oxygen species involved in ICAM-1 gene induction in endothelial cells.

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 H2O2 decomposition activity was observed in strained ECs. Both ROS and H2O2 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. Our findings indicate that cyclic strain-induced monocyte adhesion to ECs is mediated, at least in part, by an increase of ICAM-1 gene expression via the elevation of ROS levels in strained ECs. Our results support the importance of intracellular ROS in the modulation of hemodynamic force-induced endothelial responses.

Antioxidants↗

PubMed1997

Reactive oxygen species are involved in shear stress-induced intercellular adhesion molecule-1 expression in endothelial cells.

Vascular endothelial cells (ECs) are constantly subjected to flow-induced shear stress. Although the effects of shear stress on ECs are well known, the intracellular signal mechanisms remain largely unclear. Reactive oxygen species (ROS) have recently been suggested to act as intracellular second messengers. The potential role of ROS in shear-induced gene expression was examined in the present study by subjecting ECs to a shear force using a parallel-plate flow chamber system. ECs under shear flow increased their intracellular ROS as indicated by superoxide production. This superoxide production was maintained at an elevated level as shear flow remained. Sheared ECs, similar to TNF(alpha)-, PMA-, or H2O2-treated cells, increased their intercellular adhesion molecule-1 (ICAM-1) mRNA levels in a time-dependent manner. Pretreatment of ECs with an antioxidant, N-acetyl-cysteine (NAC) or catalase, inhibited this shear-induced or oxidant-induced ICAM-1 expression. ROS that were involved in the shear-induced ICAM-1 gene expression were further substantiated by functional analysis using a chimera containing the ICAM-1 promoter region (-850 bp) and the reporter gene luciferase. Shear-induced promoter activities were attenuated by pretreating sheared ECs with NAC and catalase. Flow cytometric analysis and monocytic adhesion assay confirmed the inhibitory effect of NAC and catalase on the shear-induced ICAM-1 expression on ECs. These results clearly demonstrate that shear flow to ECs can induce intracellular ROS generation that may result in an increase of ICAM-1 mRNA levels via transcriptional events. Our findings thus support the importance of intracellular ROS in modulating hemodynamically induced endothelial responses.

Acetylcysteine↗

PubMed1997

Cyclic strain-induced monocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein 1.

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.

Acetylcysteine↗

PubMed1996

Cyclic strain-induced plasminogen activator inhibitor-1 (PAI-1) release from endothelial cells involves reactive oxygen species.

The molecular mechanisms of the endothelial fibrinolytic activities modulated by mechanical strain are not clear. Endothelial cells (ECs) grown on a flexible membrane base were deformed with sinusoidal negative pressures to produce an average strain of 12%. Cyclic strain induced PAI-1 release in a time-dependent manner. Strain cells resulted in a 5-fold increase in PAI-1 release. Strain induced a sustained elevated level in intracellular reactive oxygen species (ROS). Concomitantly, a sustained increase of catalase activity was observed. Both ROS and catalase activity returned to basal levels with the removal of strain. ECs pretreated with antioxidant, N-acetyl-cysteine, abolished the strain-induced ROS generation as well as strained-induced PAI-1 release. Our results indicate that cyclic strain-induced PAI-1 secretion may be mediated by an increase in ROS generation and thus emphasizes the importance of intracellular ROS in the modulation of hemodynamic force-induced cellular response of ECs.

Acetylcysteine↗

PubMed1996

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.

Animals↗

PubMed1996

Cyclical strain increases monocyte chemotactic protein-1 secretion in human endothelial cells.

The effects of mechanical strain on monocyte chemotactic protein-1 (MCP-1) secretion were examined on human endothelial cells (ECs) grown on a flexible membrane base. MCP-1 release into culture medium from strained ECs was demonstrated to be time and strain dose dependent. Northern blot analysis demonstrated a mainly serum-independent 1.8-fold induction of MCP-1 mRNA levels in ECs strained at 15 kPa compared with unstrained controls. ECs treated with actinomycin D abolished this strain-induced expression. Strained ECs at the periphery of wells showed higher MCP-1 gene expression than ECs at the center. Pretreatment of ECs with either cytochalasin D or phalloidin did not abolish strain-induced gene expression. ECs pretreated with stretch-activated ion channel blocker gadolinium or with ryanodine to deplete intracellular stored Ca2+ strongly inhibited the strain-induced MCP-1 levels. We conclude that 1) cyclical strain can modulate the secretion of MCP-1 in a dose-dependent manner, 2) strain-induced MCP-1 production is mediated by increasing MCP-1 mRNA levels via transcription, 3) cytoskeletal rearrangement is not essential for this strain-induced MCP-1 expression, and 4) both Ca2+ influx via stretch-activated ion channels and intracellular Ca2+ release contribute to the strain-induced effect. Such strain-induced MCP-1 secretion might contribute to the trapping of monocytes in the subendothelial space to initiate atherogenesis.

Blotting, Northern↗

PubMed1996

Cyclic strain enhances adhesion of monocytes to endothelial cells by increasing intercellular adhesion molecule-1 expression.

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.

Cell Adhesion↗

PubMed1995

Angiotensinogen gene expression is induced by cyclical mechanical stretch in cultured rat cardiomyocytes.

The effect of cyclical mechanical stretch on angiotensinogen gene expression was examined using a neonatal rat cardiocyte culture system. Cultured cardiocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. The angiotensinogen gene was activated 2 to 5 fold after stretch for 3 to 24 hr, as shown by quantitative reverse transcription polymerase chain reaction. The 5'-flanking region of the angiotensinogen promoter was activated after stretch for 24 hr. This gene expression could be completely suppressed by losartan, a specific antagonist of angiotensin II receptor. These results indicate that (1) cyclical mechanical stretching of cardiocytes is a good model for the study of cardiac hypertrophy-related gene expression; (2) cyclical stretch up-regulates expression of the angiotensinogen gene and (3) the increase in promoter activity may contribute to the induction of angiotensinogen mRNA by cyclical stretch.

Angiotensinogen↗

PubMed1995

Regulation of human cardiac myosin heavy chain genes by cyclical mechanical stretch in cultured cardiocytes.

We examined the effect of cyclical mechanical stretch on the regulation of cardiac myosin heavy chain genes using an isolated neonatal rat cardiocyte culture system. Cultured cardiocytes grown on a flexible membrane were deformed by vacuum to 20% of maximum elongation, at 60 cycles/min in a serum-free medium. Cyclical stretch did not cause myocyte damage as assessed by supernatant LDH measurement and trypan blue exclusion test. The levels of myosin heavy chain (MyHC) mRNA increased as early as 1 h after stretch, reaching 12-fold over the control in 24 h, as shown by Northern blot analysis. However, the proximal 5'-flanking regions of the alpha- and beta-MyHC gene which were linked to chloramphenicol acetyltransferase (CAT) reporter gene did not exhibit enhanced CAT activity following cyclical stretch. Deletion of the chimeric constructs to shorten the 5'-flanking regions of the MyHC genes generated by polymerase chain reaction amplification did not enhance the CAT activity under cyclical stretch. This finding suggests that the stretch-response element of the alpha- and beta-MyHC gene promoter is probably not present in the proximal region of either the alpha- or beta-MyHC genes.

Animals↗

PubMed1995

Recombinant Desulfovibrio vulgaris rubrerythrin. Isolation and characterization of the diiron domain.

The gene encoding Desulfovibrio (D.) vulgaris rubrerythrin (Prickril, B. C., Kurtz, D. M., Jr., LeGall, J., & Voordouw, G. (1991) Biochemistry 30, 1118), a protein of unknown function containing both FeS4 and (mu-oxo)diiron sites, was cloned and overexpressed in Escherichia coli. Upon cell lysis, the overexpressed protein was found in an insoluble form deficient in iron. Iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically and diluting the denaturant. This recombinant rubrerythrin was found to have properties very similar to those of rubrerythrin isolated from D. vulgaris, except that the recombinant rubrerythrin contained six rather than four (or five) iron atoms per 44 kDa homodimer. Analyses of UV-vis, Mössbauer, and EPR spectra showed that the six iron atoms in recombinant rubrerythrin are organized as two FeS4 and two (mu-oxo/hydroxo)diiron sites. In order to allow examination of the diiron sites in the absence of the FeS4 sites, a truncated gene encoding the N-terminal 152 residues of D. vulgaris rubrerythrin was also cloned and overexpressed as an insoluble protein in E. coli, and iron was incorporated by a procedure analogous to that for recombinant rubrerythrin. This so-called "chopped" rubrerythrin (CRr) was found to consist of an approximately 35 kDa homodimer containing four iron atoms. Spectroscopic characterization indicated that the four iron atoms in CRr are organized as two diiron sites, the majority of which closely resemble the (mu-oxo)diiron(III) sites in E. coli ribonucleotide reductase R2 protein, and a minor fraction of which resemble the mixed-valent diiron(II,III) site in methane monooxygenase hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1995

Mechanical strain increases endothelin-1 gene expression via protein kinase C pathway in human endothelial cells.

Vascular endothelial cells (ECs) are constantly subjected to mechanical strain due to relaxation and contraction of vessel walls. The effects of cyclical strain on endothelin-1 (Et-1) secretion and Et-1 mRNA levels in human umbilical vein ECs were examined. Cultured ECs grown on a flexible membrane base were deformed by negative pressure (16 kPa at 60 cycles/min). Cells subjected to strain showed increased Et-1 secretion (0.54 ng/hr/10(6) cells) compared with unstrained control cells (0.22 ng/hr/10(6) cells). Northern blot analysis of cells strained for 2 hours or longer demonstrated a sustained elevated Et-1 mRNA level at more than double the level in unstrained controls. This strain-induced ET-1 mRNA level returned to its basal level 2 hours after the release of strain. Cells treated with actinomycin D before or during strain treatment showed no strain-induced gene expression. Pretreatment of ECs with a protein kinase C (PKC) inhibitor, Calphostin C, strongly inhibited the strain-induced Et-1 gene expression. Pretreatment of ECs with cAMP- or cGMP-dependent protein kinase inhibitors (KT5720 or KT5823) only partially inhibited the increased Et-1 mRNA levels in strain-treated cells. EGTA strongly inhibited the Et-1 gene expression. The intracellular calcium chelator BAPTA/AM also showed an inhibitory effect on Et-1 mRNA levels. We conclude that mechanical strain can stimulate the secretion of Et-1 from ECs by increasing Et-1 mRNA levels via transcription, and that this gene induction is mediated predominantly via the PKC pathway and requires extracellular Ca2+. This strain-induced Et-1 gene expression in ECs may contribute to the regulation of vascular tone and structure in normal and pathological states of the cardiovascular system.

Cells, Cultured↗

PubMed1995

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)

Analysis of Variance↗