Tsui-Chun Tsou

Genomic analysis of smooth muscle cells in 3-dimensional collagen matrix

The proliferation, differentiation, and protein synthesis of vascular smooth muscle cells (SMCs) play important roles in vascular remodeling. Here, we compared the genetic programming and signaling of SMCs in collagen matrix as a three‐dimensional (3‐D) environment and on a two‐dimensional (2‐D) surface. By using DNA microarrays with 9600 genes, we showed that 77 genes were expressed more than twofold and 22 genes were less than one‐half in 3‐D matrix, when compared with the 2‐D condition. The higher expression level of cyclin‐dependent kinase inhibitor 1 (p21) in 3‐D matrix suggests that p21 may be responsible for the lower proliferation rate in 3‐D matrix. The expression level of collagen I was higher in 3‐D matrix, suggesting that SMCs in 3‐D matrix have increased matrix synthesis. In addition, SMCs in 3‐D matrix had less stress fibers and focal adhesions, and a lower level of tyrosine phosphorylation of focal adhesion kinase (FAK). Overexpression of FAK attenuated the expression of p21 and collagen I in 3‐D matrix, suggesting that FAK functions as a molecular switch for cell cycle regulation and matrix synthesis. The information generated in this study helps to elucidate the molecular basis of the modulation of SMC phenotypes by the extracellular matrix.

Rate sensitivity of shear-induced changes in the lateral diffusion of endothelial cell membrane lipids: a role for membrane perturbation in shear-induced MAPK activation

Vascular endothelium transduces the temporal gradients in shear stress (τ) originating from unsteady blood flow into functional responses. We measured the effects of step‐τ and ramp‐τ (i.e., τ with different temporal shear gradients) on the lipid lateral diffusion coefficient (D) in the apical membranes of confluent cultured bovine aortic endothelial cells by using fluorescence recovery after photobleaching. A step‐τ of 10 dynes/cm2 elicited a rapid (5 s) increase of D in the portion of the cell upstream of the nucleus and a concomitant decrease in the downstream portion. A ramp‐τ with a rate of 20 dynes/cm2 per min elicited a rapid (5 s) decrease of D in both the upstream and the downstream portions. The mitogen‐activated protein kinases (MAPKs) ERK and JNK were activated by step‐τ but not by ramping to the same τ level. Benzyl alcohol, which increases D, enhanced the activities of both MAPKs; cholesterol, which reduces D, diminished these activities. We conclude that the lipid bilayer can sense the temporal features of the applied τ with spatial discrimination and that the τ‐induced membrane perturbations can be transduced into MAPK activation. These results have implications for understanding the role of τ in modulating vascular functions in health and disease.

Levels of Breast Milk PBDEs From Southern Taiwan and Their Potential Impact on Neurodevelopment

In vivo studies have demonstrated that prenatal or neonatal exposure to polybrominated diphenyl ethers (PBDEs) causes developmental neurotoxicity. However, there is a lack of human data. Our hypothesis was that PBDEs would result in lower infant neurodevelopment scores. This is a post hoc analysis of previous studies. Fourteen PBDEs in 70 breast milk were analyzed using a high-resolution gas chromatograph/high-resolution mass spectrometer. Infant neurodevelopment at the age of 8–12 mo was determined using the Bayley Scales of Infants and Toddlers Development, third edition (Bayley-III). The median of Σ14 PBDEs (the sum of 14 PBDE congeners) was 2.92 ng/g lipid. The Σ14 PBDE concentrations were not correlated with Bayley-III scores on cognitive, language, motor, social-emotional, or adaptive behavior scales. A significantly inverse association between brominated diphenyl ether (BDE)-209 and the cognitive scale was found after multivariate stepwise linear regression analyses (B = −0.007, adjusted R = −0.224, p = 0.032). In contrast, the language scale was positively correlated with BDE-196 (B = 0.096, adjusted R = 0.315, p = 0.002). Our results are consistent with most in vivo studies, suggesting that prenatal or postnatal exposure to BDE-209 potentially delays the neurological development.

Roles of Microtubule Dynamics and Small GTPase Rac in Endothelial Cell Migration and Lamellipodium Formation under Flow

Endothelial cell (EC) migration is required for vascular development and wound healing. We investigated the roles of microtubule (MT) dynamics and the small GTPase Rac in the fluid shear stress-induced protrusion of lamellipodia and enhancement of migration of bovine aortic ECs (BAECs). Shear stress increased lamellipodial protrusion and cell migration. Treating BAECs with paclitaxel (Taxol), an MT-stabilizing agent, inhibited lamellipodial protrusion and reduced migration speed in both the static and sheared groups. After Taxol washout, both lamellipodial protrusion and cell migration increased in the flow direction. Taxol treatment also decreased the shear-induced Rac activation. Transfection of BAECs with a dominant negative mutant of Rac1 inhibited lamellipodial protrusion and cell migration under static and shear conditions. Transfection with an activated mutant of Rac1 induced lamellipodia in all directions and attenuated the shear-induced migration, suggesting that an appropriate level of Rac activity and a polarized lamellipodial protrusion are important for cell migration under static and shear conditions. Our findings suggest that MT dynamics and optimum Rac activation are required for the polarized protrusion of lamellipodia that drives the directional EC migration under flow.

Stent Implantation Activates Akt in the Vessel Wall: Role of Mechanical Stretch in Vascular Smooth Muscle Cells

Objective—The long-term efficacy of stent implantation is affected by in-stent restenosis (ISR). Multiple factors can contribute to ISR, and the underlying mechanism remains elusive. We investigated the possible role of mechanical stretch and the associated molecular signaling in ISR. Methods and Results—Stent implantation in rat abdominal aortas induced neointima formation. Immunohistochemical studies revealed the activation of Akt in the media and neointima of the stented vessels. Western blotting showed increased phosphorylation of Akt at both Thr308 and Ser473 and phosphorylation of GSK-3&bgr; in the stented vessels. A stretch device applying static equibiaxial stretch on cultured vascular smooth muscle cells was used to delineate the molecular mechanism underlying the stretch activation of Akt. Static mechanical stretch induced the sustained activation of Akt and its upstream phosphoinositide 3-kinase (PI3K) and the phosphorylation of GSK-3&bgr;, its downstream effector in vascular smooth muscle cells. LY294002, a PI3K inhibitor, and N-acetylcysteine, a scavenger of reactive oxygen species, inhibited the stretch activation of Akt. Furthermore, N-acetylcysteine and wortmannin, another PI3K inhibitor, reduced the neointima formation after stent implantation. Conclusions—Mechanical stretch of the vascular wall during stent deployment may contribute to ISR by activating the Akt pathway.

Formation of reactive oxygen species and DNA strand breakage during interaction of chromium(III) and hydrogen peroxide in vitro: Evidence for a chromium(III)-mediated Fenton-like reaction

The role of reactive oxygen species in causing DNA damage through interaction of chromium (III) and hydrogen peroxide was examined using plasmid relaxation assay and EPR spectroscopy. Marked DNA strand breakage was induced by CrCl3 plus H2O2 in a phosphate buffer at pH 6-8.9; whereas, only slight DNA strand breakage was observed during similar treatment at pH less than 4. DNA breakage also increased as the reaction temperature and Cr(III)/H2O2 concentrations increased. Control experiments with Cr(III) or H2O2 alone did not cause DNA breakage. Sodium azide, D-mannitol, Tris-HCl, or catalase completely inhibited Cr(III)/H2O2-induced DNA breakage, but superoxide dismutase did not. The D2O enhancing effect on DNA breaks was not observed. Cr(III) pre-incubated with a 30-fold molar excess of EDTA did not cause any significant DNA breakage in the presence of H2O2. In a phosphate buffer containing Cr(III) and H2O2, singlet oxygen and hydroxyl radicals were detected using EPR spectrometry with the spin traps 2,2,6,6-tetramethyl-4-piperidone and 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), respectively. DMPO/.OH adducts and DNA breakage induced by Cr(III)/H2O2 were markedly higher than those induced by Cr(VI)/H2O2. Furthermore, ascorbate decreased Cr(III)/H2O2-induced DNA breakage. EPR studies revealed that ascorbate (mole ratio to Cr(III) = 0.5:1) attenuated the DMPO/.OH signal generated by Cr(III)/H2O2/DMPO, but a Cr(V) signal and ascorbate radicals were detected. NADPH, GSH, and GSSG also decreased DMPO/.OH generated by Cr(III)/H2O2/DMPO; however, they were less efficient than ascorbate and no Cr(V) signals were detected. This study shows that Cr(III)/H2O2 generates oxidative damage to DNA through a Fenton-like reaction: Cr(III) + H2O2-->Cr(IV) + .OH + OH.

Zinc oxide particles induce inflammatory responses in vascular endothelial cells via NF-κB signaling.

This study investigated inflammatory effects of zinc oxide (ZnO) particles on vascular endothelial cells. The effects of 50 and 100-nm ZnO particles on human umbilical vein endothelial cells (HUVECs) were characterized by assaying cytotoxicity, cell proliferation, and glutathione levels. A marked drop in survival rate was observed when ZnO concentration was increased to 45 μg/ml. ZnO concentrations of ≤3 μg/ml resulted in increased cell proliferation, while those of ≤45 μg/ml caused dose-dependent increases in oxidized glutathione levels. Treatments with ZnO concentrations ≤45 μg/ml were performed to determine the expression of intercellular adhesion molecule-1 (ICAM-1) protein, an indicator of vascular endothelium inflammation, revealing that ZnO particles induced a dose-dependent increase in ICAM-1 expression and marked increases in NF-κB reporter activity. Overexpression of IκBα completely inhibited ZnO-induced ICAM-1 expression, suggesting NF-κB plays a pivotal role in regulation of ZnO-induced inflammation in HUVECs. Additionally, TNF-α, a typical inflammatory cytokine, induced ICAM-1 expression in an NF-κB-dependent manner, and ZnO synergistically enhanced TNF-α-induced ICAM-1 expression. Both 50 and 100-nm ZnO particles agglomerated to similar size distributions. This study reveals an important role for ZnO in modulating inflammatory responses of vascular endothelial cells via NF-κB signaling, which could have important implications for treatments of vascular disease.

Six-month follow-up study of health markers of nanomaterials among workers handling engineered nanomaterials

Abstract The aim of this study was to identify the health hazards and possible exposure surveillance markers of workers exposed to nanoparticles during manufacturing and application in comparison to a group of unexposed workers. For this longitudinal study, we recruited 158 nanomaterial-handling workers and 104 non-exposed workers from 14 manufacturing plants in Taiwan (baseline). Among them, 124 nanomaterial-handling workers and 77 unexposed workers were monitored 6 months later. We investigated pulmonary and cardiovascular disease markers, inflammation and oxidative stress markers, antioxidant enzymes and genotoxicity markers. Antioxidant enzymes (superoxide dismutase, glutathione peroxidase) and cardiovascular markers (vascular cell adhesion molecule, paraoxonase) were significantly associated with nanomaterial-handling during the 6-month follow-up period. In addition, the small airway damage marker (Clara cell protein 16) and lung function test parameters were also significantly associated with handling nanomaterials. The study markers and lung function tests are possible markers that could be useful for surveillance of nanomaterial-handling workers.

Impact of non-occupational exposure to polybrominated diphenyl ethers on menstruation characteristics of reproductive-age females

Polybrominated diphenyl ethers (PBDEs) have documented effects on thyroid functions and rodent behavior in vivo. Epidemiological studies, however, have revealed only limited information about associations between PBDE exposure and menstruation characteristics. Our goal was to examine whether high breast milk PBDE levels in reproductive-age females lead to interference with menstruation characteristics. We analyzed 15 PBDE congeners in 46 breast milk samples. Fifteen PBDE congeners (BDE-15, 28, 47, 49, 99, 100, 153, 154, 183, 196, 197, 203, 207, 208, and 209) were analyzed using a gas chromatograph equipped with a high resolution mass spectrometer. The mean sum of PBDEs (SigmaPBDEs) in breast milk was 3.42 ng/g lipid. Womens age at menarche was not correlated with breast milk PBDE levels. Increased BDE-208 and 209 levels were significantly associated with the prolonged length of average and the longest menstrual cycle independent of age, pre-pregnant BMI, and parity. Higher concentrations of SigmaPBDEs and the higher brominated PBDEs from BDE-183 to 209, except 197, were significantly linked to women whose menstruation periods were still coming irregularly at the sampling time. Age-adjusted odds ratios (ORs) of BDE-153, 183, 207, 208, and SigmaPBDEs were significantly higher in women with length of average menstrual cycle >32 days, compared to the control. Women whose menstruation periods still came irregularly when they were 18 years old had higher age-adjusted ORs of BDE-207, 208, 209, and SigmaPBDEs than those whose periods came regularly at the same age. Although SigmaPBDEs and certain higher brominated PBDEs appear to have potential to prolong length of average menstrual cycle and delay the age when menstruation periods begin coming regularly, these findings are not conclusive because our sample size is small and more scientific evidence is needed.

Effects of arecoline on adipogenesis, lipolysis, and glucose uptake of adipocytes—A possible role of betel-quid chewing in metabolic syndrome

To investigate the possible involvement of betel-quid chewing in adipocyte dysfunction, we determined the effects of arecoline, a major alkaloid in areca nuts, on adipogenic differentiation (adipogenesis), lipolysis, and glucose uptake by fat cells. Using mouse 3T3-L1 preadipocytes, we showed that arecoline inhibited adipogenesis as determined by oil droplet formation and adipogenic marker gene expression. The effects of arecoline on lipolysis of differentiated 3T3-L1 adipocytes were determined by the glycerol release assay, indicating that arecoline induced lipolysis in an adenylyl cyclase-dependent manner. The diabetogenic effects of arecoline on differentiated 3T3-L1 adipocytes were evaluated by the glucose uptake assay, revealing that > or = 300 microM arecoline significantly attenuated insulin-induced glucose uptake; however, no marked effect on basal glucose uptake was detected. Moreover, using 94 subjects that were randomly selected from a health check-up, we determined the association of betel-quid chewing with hyperlipidemia and its related risk factors. Hyperlipidemia frequency and serum triglyceride levels of betel-quid chewers were significantly higher than those of non-betel-quid chewers. In this study, we demonstrated that arecoline inhibits adipogenic differentiation, induces adenylyl cyclase-dependent lipolysis, and interferes with insulin-induced glucose uptake. Arecoline-induced fat cell dysfunction may lead to hyperlipidemia and hyperglycemia/insulin-resistance. These findings provide the first in vitro evidence of betel-quid chewing modulation of adipose cell metabolism that could contribute to the explanation of the association of this habit with metabolic syndrome disorders.