Yi Shuan Li

Flow-Dependent Regulation of Krüppel-Like Factor 2 Is Mediated by MicroRNA-92a

BACKGROUNDnUpregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the posttranscriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow-regulated KLF2.nnnMETHODS AND RESULTSnDicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells, suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3 untranslated region of KLF2 mRNA. Overexpression of miR-92a decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase and thrombomodulin at mRNA and protein levels. A complementary finding is that miR-92a inhibitor increased the mRNA and protein expression of KLF2, endothelial nitric oxide synthase, and thrombomodulin. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a precursor to induce KLF2, and the level of this flow-induced KLF2 was reduced by miR-92a precursor. Furthermore, miR-92a level was lower in human umbilical vein endothelial cells exposed to the atheroprotective pulsatile shear flow than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunoprecipitation coupled with real-time polymerase chain reaction revealed that pulsatile shear flow decreased the functional targeting of miR-92a precursor/KLF2 mRNA in human umbilical vein endothelial cells. Consistent with these findings, mouse carotid arteries receiving miR-92a precursor exhibited impaired vasodilatory response to flow.nnnCONCLUSIONSnAtheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

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.

Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell-secreted MicroRNA-126: Role of Shear Stress

Rationale: Endothelial microRNA-126 (miR-126) modulates vascular development and angiogenesis. However, its role in the regulation of smooth muscle cell (SMC) function is unknown. Objective: To elucidate the role of miR-126 secreted by endothelial cells (ECs) in regulating SMC turnover in vitro and in vivo, as well as the effects of shear stress on the regulation. Methods and Results: Coculture of SMCs with ECs or treatment of SMCs with conditioned media from static EC monoculture (EC-CM) increased SMC miR-126 level and SMC turnover; these effects were abolished by inhibition of endothelial miR-126 and by the application of laminar shear stress to ECs. SMC miR-126 did not increase when treated with EC-CM from ECs subjected to inhibition of miR biogenesis, or with CM from sheared ECs. Depletion of extracellular/secreted vesicles in EC-CM did not affect the increase of SMC miR-126 by EC-CM. Biotinylated miR-126 or FLAG (DYKDDDDK epitope)-tagged Argonaute2 transfected into ECs was detected in the cocultured or EC-CM–treated SMCs, indicating a direct EC-to-SMC transmission of miR-126 and Argonaute2. Endothelial miR-126 represses forkhead box O3, B-cell lymphoma 2, and insulin receptor substrate 1 mRNAs in the cocultured SMCs, suggesting the functional roles of the transmitted miR-126. Systemic depletion of miR-126 in mice inhibited neointimal lesion formation of carotid arteries induced by cessation of blood flow. Administration of EC-CM or miR-126 mitigated the inhibitory effect. Conclusions: Endothelial miR-126 acts as a key intercellular mediator to increase SMC turnover, and its release is reduced by atheroprotective laminar shear stress.

Flow Activation of AMP-Activated Protein Kinase in Vascular Endothelium Leads to Krüppel-Like Factor 2 Expression

Objective—Vascular endothelial cells (ECs) confer atheroprotection at locations of the arterial tree where pulsatile laminar flow (PS) exists with a high shear stress and a large net forward direction. We investigated whether the PS-induced expression of the transcription factor Krüppel-Like Factor 2 (KLF2) in cultured ECs and its expression in the mouse aorta is regulated by AMP-activated protein kinase (AMPK). Methods and Results—AMPK inhibition by Compound C or siRNA had a significant blocking effect on the PS-induced KLF2 expression. The induction of KLF2 by PS led to the increase in eNOS and the suppression of ET-1, which could be reversed by KLF2 siRNA. In addition, PS induced the phosphorylation of ERK5 and MEF2 which are necessary for the KLF2 expression. These mechanotransduction events were abrogated by the blockade of AMPK. Furthermore, the phosphorylation levels of ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in the aorta of AMPKα2 knockout mice when compared with wild-type control mice. Conclusion—The flow-mediated AMPK activation is a newly defined KLF2 regulatory pathway in vascular endothelium that acts via ERK5/MEF2.

ERK activation and αvβ3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts

Osteoblast growth and differentiation are critical processes for bone development and maintenance, and are regulated by both humoral and mechanical factors. Humoral (hormonal) factors can affect gene transcription via MAPkinases, e.g., extracellular signal‐regulated kinase (ERK). We studied whether the ERK pathway is also involved in processing mechanical inputs in human bone cells. Exposing MG63 cells to physiologically relevant levels of fluid flow resulted in ERK phosphorylation. Genistein blocked this response, indicating that it is dependent on tyrosine phosphorylation. Furthermore, αvβ3 integrins were activated in response to fluid flow, as shown by recruitment of adaptor molecule Shc and clustering of αvβ3 in focal adhesion‐like structures. Antibodies blocking formation of β1 or β3 integrin‐matrix interactions or RGD peptides could not inhibit fluid flow‐induced ERK phosphorylation, suggesting that formation of new integrin‐matrix interactions is not essential for this response and that other upstream mechanosensors regulate induction of ERK phosphorylation in response to fluid flow in human bone cells. J. Cell. Biochem. 87: 85–92, 2002.

Directional shear flow and Rho activation prevent the endothelial cell apoptosis induced by micropatterned anisotropic geometry

To study the roles of anisotropic cell morphology and directionality of mechanical force in apoptosis, the spreading of human umbilical vein endothelial cells (HUVECs) was constrained by growing on micropatterned (MP) strips of fibronectin (FN, 20 μg/cm2) with widths of 15, 30, and 60 μm on silicone membrane. Cells on 30- and 60-μm strips, like cells on a nonpatterned (NP) surface coated with FN, showed clear actin stress fibers with anchoring spots of phosphorylated focal adhesion kinase (p-FAK) and no significant apoptosis. On 15-μm strips, cells had few stress fibers, no p-FAK, and significant apoptosis. After seeding for 12 h, the cells were subjected to pulsatile shear stress (12 ± 4 dyn/cm2) parallel or perpendicular to MP strips, or kept under static condition. Parallel flow caused cell elongation with enhanced stress fibers and p-FAK, and a reduction in apoptosis, but perpendicular flow did not. The Rho inhibitory C3 exoenzyme abolished the effects of parallel flow. RhoV14, the constitutively active Rho, enhanced stress fibers and p-FAK, and prevented apoptosis of HUVECs on 15-μm strips under static condition. RhoV14 also reduced cell apoptosis under both parallel and perpendicular flows. Our results indicate that cell apoptosis can be modulated by changes in ECM micropatterning, anisotropic cell morphology, and mechanical forces. These extracellular microenvironment factors affect cell survival through alterations in Rho GTPase activity, stress fiber organization, and FAK phosphorylation.

Molecular basis of mechanical modulation of endothelial cell migration.

Vascular endothelial cells (ECs) play important roles in the regulation of vascular functions. Loss of endothelial integrity can lead to vascular diseases such as stenosis resulting from atherosclerosis. The migration of ECs into wounded area in the vessel wall is required for the restoration of its integrity and functions. EC migration results from a balance of externally applied forces (e.g. shear stress), intracellular forces (e.g., those generated by contractile and cytoskeletal proteins), adhesion force between ECs and extracellular matrix (ECM) proteins, and the force of EC-EC coupling through junction proteins. Shear stress modulates EC migration through the regulation of multiple signaling pathways, gene expression, and the reorganization of cytoskeleton, focal adhesion sites, and cell junctions. Investigations of EC migration under shearing can provide valuable knowledge on vascular remodeling process under physiological and pathological conditions.

Roles of MAP kinases in the regulation of bone matrix gene expressions in human osteoblasts by oscillatory fluid flow

We investigated the effects of oscillatory flow in regulating the gene expressions of type I collagen (COL1, the main component of human bone tissues) and osteopontin (OPN, the key gene for calcium deposition) in human osteoblast‐like (MG‐63) cells, and the roles of mitogen‐activated protein kinases (MAPKs) in this regulation. The cells were subjected to oscillatory flow (0.5u2009±u20094 dyn/cm2) or kept under static condition for various time periods (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h). Oscillatory flow caused significant up‐regulations of both COL1 and OPN gene expressions over the 16 h of study, and a transient activation of MAPKs was starting at 15 min and declining to basal level in 2 h. The flow‐induction of COL1 was blocked by an ERK inhibitor (PD98059) and reduced by a JNK inhibitor (SP600125), whereas that of OPN was abolished by PD98059. Analysis of the cis‐elements in the COL1 and OPN promoters suggests the involvement of transacting factors Elk‐1 and AP‐1 in the transcription regulation. The ERK inhibitor (PD98059) blocked Elk‐1 phosphorylation, as well as COL1 and OPN gene expression. The JNK inhibitor (SP600125) abolished c‐jun phosphorylation and COL1 expression. These results suggest that the flow‐induction of OPN was mediated through the ERK‐Elk1‐OPN pathway, and that COL1 was regulated by both the ERK‐Elk1‐COL1 and JNK‐c‐JUN‐COL1 pathway. J. Cell. Biochem. 98: 632–641, 2006.

Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention

Little is known regarding the interplays between the mechanical and molecular bases for vein graft restenosis. We elucidated the stenosis initiation using a high-frequency ultrasonic (HFU) echogenicity platform and estimated the endothelium yield stress from von-Mises stress computation to predict the damage locations in living rats over time. The venous-arterial transition induced the molecular cascades for autophagy and apoptosis in venous endothelial cells (ECs) to cause neointimal hyperplasia, which correlated with the high echogenicity in HFU images and the large mechanical stress that exceeded the yield strength. The ex vivo perfusion of arterial laminar shear stress to isolated veins further confirmed the correlation. EC damage can be rescued by inhibiting autophagy formation using 3-methyladenine (3-MA). Pretreatment of veins with 3-MA prior to grafting reduced the pathological increases of echogenicity and neointima formation in rats. Therefore, this platform provides non-invasive temporal spatial measurement and prediction of restenosis after venous-arterial transition as well as monitoring the progression of the treatments.