Hsyue-Jen Hsieh

Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods.

Freeze-fixation and freeze-gelation methods are presented in this paper which can be used to prepare highly porous scaffolds without using the time and energy consuming freeze-drying process. The porous structure was generated during the freeze of a polymer solution, following which either the solvent was extracted by a non-solvent or the polymer was gelled under the freezing condition; thus, the porous structure would not be destructed during the subsequent drying stage. Compared with the freeze-drying method, the presented methods are time and energy-saving, with less residual solvent, and easier to be scaled up. Besides, the problem of formation of surface skin can be resolved and the limitation of using solvent with low boiling point can be lifted by the presented methods. With the freeze-extraction and freeze-gelation methods, porous PLLA, PLGA, chitosan and alginate scaffolds were successfully fabricated. In addition to the presentation of the morphologies of the fabricated scaffolds, preliminary data of cell culture on them are as well included in the present work.

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.

Fabrication of a novel porous PGA-chitosan hybrid matrix for tissue engineering

Polyglycolide (PGA) and chitosan mixture solution was prepared using solvents of low toxicity to create novel, porous, biocompatible, degradable, and modifiable hybrid matrices for biomedical applications. The porosity of these PGA-chitosan hybrid matrices (P/C matrices) was created by a thermally induced phase separation method. Two types of the P/C hybrid matrices containing 70 wt% PGA (P/C-1 matrix) and 30 wt% PGA (P/C-2 matrix) were fabricated. Chitosan matrix was also prepared for comparison. A 35-day in vitro degradation revealed that the weight losses for the P/C-1 and P/C-2 matrices were similar ( approximately 61%), but the releases of glycolic acid from the P/C-1 and P/C-2 matrices were 95% and 60%, respectively. The P/C-1 matrix had higher porosity and higher mechanical strength than the P/C-2 and chitosan matrices. Fibroblast cells cultivated in these matrices proliferated well and the cell density was the highest in the P/C-1 matrix, followed by the chitosan and P/C-2 matrices, suggesting good biocompatibility for the P/C-1 matrix. We thereby concluded that the P/C-1 matrix, due to its high strength, porosity, biocompatibility and degradability, is a promising biomaterial. The presence of chitosan in the P/C matrices provides many amino groups for further modifications such as biomolecule conjugation and thus enhances the application potential of the P/C hybrid matrices in tissue engineering.

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.

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.

Nitric Oxide Regulates Shear Stress-Induced Early Growth Response-1 Expression via the Extracellular Signal-Regulated Kinase Pathway in Endothelial Cells

Endothelial cells (ECs) subjected to shear stress constantly release nitric oxide (NO). The effect of NO on shear stress-induced endothelial responses was examined. ECs subjected to shear stress induced a transient and shear force-dependent increase in early growth response-1 (Egr-1) mRNA levels. Treatment of ECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1), inhibited this shear stress-induced Egr-1 expression. Conversely, an NO synthase inhibitor to ECs, N(G)-monomethyl-L-arginine, augmented this Egr-1 expression. NO modulation of Egr-1 expression was demonstrated by functional analysis of Egr-1 promoter activity using a chimera containing the Egr-1 promoter region (-698 bp) and reporter gene luciferase. In contrast to the enhanced promoter activity after N(G)-monomethyl-L-arginine treatment, shear stress-induced Egr-1 promoter activity was attenuated after ECs were treated with an NO donor. ECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal-regulated kinase (ERK)-2 (mERK) inhibited shear stress-induced Egr-1 promoter activity. NO modulation of the signaling pathway was shown by its inhibitory effect on shear stress-induced ERK1/ERK2 phosphorylation and activity. This inhibitory effect was further substantiated by the inhibition of NO on both the shear stress-induced transcriptional activity of Elk-1 (an ERK substrate) and the promoter activity of a reporter construct containing serum response element. NO-treated ECs resulted in a reduction of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. These results indicate that shear stress-induced Egr-1 expression is modulated by NO via the ERK signaling pathway in ECs. Our findings support the importance of NO as a negative regulator in endothelial responses to hemodynamic forces.

Regulation of shear-induced nuclear translocation of the Nrf2 transcription factor in endothelial cells

BackgroundVascular endothelial cells (ECs) constantly experience fluid shear stresses generated by blood flow. Laminar flow is known to produce atheroprotective effects on ECs. Nrf2 is a transcription factor that is essential for the antioxidant response element (ARE)-mediated induction of genes such as heme-oxygenase 1 (HO-1). We previously showed that fluid shear stress increases intracellular reactive oxygen species (ROS) in ECs. Moreover, oxidants are known to stimulate Nrf2. We thus examined the regulation of Nrf2 in cultured human ECs by shear stress.ResultsExposure of human umbilical vein endothelial cells (HUVECs) to laminar shear stress (12 dyne/cm2) induced Nrf2 nuclear translocation, which was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, a protein kinase C (PKC) inhibitor, and an antioxidant agent N-acetyl cysteine (NAC), but not by other protein kinase inhibitors. Therefore, PI3K, PKC, and ROS are involved in the signaling pathway that leads to the shear-induced nuclear translocation of Nrf2. We also found that shear stress increased the ARE-binding activity of Nrf2 and the downstream expression of HO-1.ConclusionOur data suggest that the atheroprotective effect of laminar flow is partially attributed to Nrf2 activation which results in ARE-mediated gene transcriptions, such as HO-1 expression, that are beneficial to the cardiovascular system.

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.

Use of Dicarboxylic Acids To Improve and Diversify the Material Properties of Porous Chitosan Membranes

Several nontoxic dicarboxylic acid solutions (oxalic acid, succinic acid, malic acid, and adipic acid solutions) instead of an acetic acid solution were used as solvents for chitosan dissolution. The amount of free amino groups of the chitosan in the solution decreased due to the ionic cross-linking of the dicarboxylic acids with chitosan. These solutions were used to fabricate porous chitosan membranes. Replacing acetic acid with these dicarboxylic acids for membrane preparation improved the water uptake (by 35% at most), tensile strength (by 110% at most), and elongation capability (by 50% at most) of the membranes. These dicarboxylic acid solutions not only act as solvents but also improve the material properties of the chitosan membranes due to the ionic cross-linking and hydrogen bond formation. In brief, a nontoxic and straightforward cross-linking method has been developed for chitosan material; this method does not result in a brittle product, thus making it better than the use of toxic cross-linking reagents.

Chitosan/pectin/gum Arabic polyelectrolyte complex: process-dependent appearance, microstructure analysis and its application.

Novel chitosan/pectin/gum Arabic polyelectrolyte complex (PEC) solutions and membranes with various compositions were prepared for biomedical applications. The appearance of the PEC solutions, either clear or turbid, was process-dependent and depended on how the three components were dissolved and mixed. The addition of gum Arabic to the chitosan and pectin significantly decreased the viscosities of the resultant PEC solutions due to the formation of globe-like microstructures that was accompanied by network-like microstructures and other molecular entanglements. The mechanical strength and hydrophilicity of the PEC membranes manufactured from the PEC solutions, especially for a weight ratio of 84/8/8 (chitosan/pectin/gum Arabic), were enhanced compared to pure chitosan membranes. Moreover, the use of the 84/8/8 PEC membranes as a drug carrier exhibited steady and fairly complete release of a drug (insulin) for 6h. Based on these promising results, the chitosan/pectin/gum Arabic PEC membranes have great potential in controlled drug release applications.