Jeng-Jiann Chiu

MicroRNA-21 targets peroxisome proliferators-activated receptor-α in an autoregulatory loop to modulate flow-induced endothelial inflammation

Adhesion of circulating monocytes to vascular endothelial cells (ECs) is a critical event leading to vascular inflammation and, hence, development of atherosclerosis. MicroRNAs (miRs) are a class of endogenous, highly conserved, noncoding small RNAs that play important roles in regulating gene expression and cellular function, as well as pathogenesis of atherosclerosis. Here, we showed that oscillatory shear stress (OSS) induces the expression of miR-21 at the transcriptional level in cultured human umbilical vein ECs via an increased binding of c-Jun, which is a component of transcription factor activator protein-1 (AP-1), to the promoter region of miR-21. OSS induction of miR-21 inhibited the translation, but not transcription, of peroxisome proliferators-activated receptor-α (PPARα) by 3′-UTR targeting. Overexpression of miR-21 up-regulated AP-1 activation, which was attenuated by exogenous expression of PPARα. OSS and overexpression of miR-21 enhanced the expression of adhesion molecules vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 and the consequential adhesion of monocytes to ECs. Overexpression of PPARα significantly attenuated the AP-1–mediated miR-21 expression. These results demonstrate a unique mechanism by which OSS induces AP-1–dependent miR-21 expression, which directly targets PPARα to inhibit its expression, thereby allowing activation of AP-1 and the promotion of monocyte adhesion. Our findings suggest the presence of a positive feedback loop that enables the sustained induction of miR-21, thus contributing to the proinflammatory responses of vascular endothelium under OSS.

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.

Vascular endothelial responses to altered shear stress: Pathologic implications for atherosclerosis

Atherosclerosis preferentially develops at branches and curvatures of the arterial tree, where blood flow is disturbed from a laminar pattern, and wall shear stress is non-uniform and has an irregular distribution. Vascular endothelial cells (ECs), which form an interface between the flowing blood and the vessel wall, are exposed to blood flow-induced shear stress. There is increasing evidence suggesting that laminar blood flow and sustained high shear stress modulate the expression of EC genes and proteins that function to protect against atherosclerosis; in contrast, disturbed blood flow and the associated low and reciprocating shear stress upregulate proatherosclerotic genes and proteins that promote development of atherosclerosis. Understanding of the effects of shear stress on ECs will provide mechanistic insights into its role in the pathogenesis of atherosclerosis. The aim of this review article is to summarize current findings on the effects of shear stress on ECs, in terms of their signal transduction, gene expression, structure, and function. These endothelial cellular responses have important relevance to understanding the pathophysiological effects of altered shear stress associated with atherosclerosis and thrombosis and their complications.

Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad

Interstitial flow in and around tumor tissue affects the mechanical microenvironment to modulate tumor cell growth and metastasis. We investigated the roles of flow-induced shear stress in modulating cell cycle distribution in four tumor cell lines and the underlying mechanisms. In all four cell lines, incubation under static conditions for 24 or 48 h led to G0/G1 arrest; in contrast, shear stress (12 dynes/cm2) induced G2/M arrest. The molecular basis of the shear effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 osteosarcoma cells. Shear stress induced increased expressions of cyclin B1 and p21CIP1 and decreased expressions of cyclins A, D1, and E, cyclin-dependent protein kinases (Cdk)-1, -2, -4, and -6, and p27KIP1 as well as a decrease in Cdk1 activity. Using specific antibodies and small interfering RNA, we found that the shear-induced G2/M arrest and corresponding changes in G2/M regulatory protein expression and activity were mediated by αvβ3 and β1 integrins through bone morphogenetic protein receptor type IA-specific Smad1 and Smad5. Shear stress also down-regulated runt-related transcription factor 2 (Runx2) binding activity and osteocalcin and alkaline phosphatase expressions in MG63 cells; these responses were mediated by αvβ3 and β1 integrins through Smad5. Our findings provide insights into the mechanism by which shear stress induces G2/M arrest in tumor cells and inhibits cell differentiation and demonstrate the importance of mechanical microenvironment in modulating molecular signaling, gene expression, cell cycle, and functions in tumor cells.

Role of histone deacetylases in transcription factor regulation and cell cycle modulation in endothelial cells in response to disturbed flow

Vascular endothelial cells (ECs) are exposed to different flow patterns (i.e., disturbed vs. laminar), and the associated oscillatory shear stress (OSS) or pulsatile shear stress (PSS) lead to differential responses. We investigated the roles of class I and II histone deacetylases (HDAC-1/2/3 and HDAC-5/7, respectively) in regulating NF-E2–related factor-2 (Nrf2) and Krüppel-like factor-2 (KLF2), two transcription factors governing many shear-responsive genes, and the cell cycle in ECs in response to OSS. Application of OSS (0.5 ± 4 dynes/cm2) to cultured ECs sustainably up-regulated class I and II HDACs and their nuclear accumulation, whereas PSS (12 ± 4 dynes/cm2) induced phosphorylation-dependent nuclear export of class II HDACs. En face immunohistochemical examination of rat aortic arch and experimentally stenosed abdominal aorta revealed high HDAC-2/3/5 levels in ECs in areas exposed to disturbed flow. OSS induced the association of HDAC-1/2/3 with Nrf2 and HDAC-3/5/7 with myocyte enhancer factor-2; deacetylation of these factors led to down-regulation of antioxidant gene NAD(P)H quinone oxidoreductase-1 (NQO1) and KLF2. HDAC-1/2/3– and HDAC-3/5/7–specific small interfering RNAs eliminated the OSS-induced down-regulation of NQO1 and KLF2, respectively. OSS up-regulated cyclin A and down-regulated p21CIP1 in ECs and induced their proliferation; these effects were mediated by HDAC-1/2/3. Intraperitoneal administration of the class I-specific HDAC inhibitor valproic acid into bromodeoxyuridine (BrdU)-infused rats inhibited the increased EC uptake of BrdU at poststenotic sites. The OSS-induced HDAC signaling and EC responses are mediated by phosphatidylinositol 3-kinase/Akt. Our findings demonstrate the important roles of different groups of HDACs in regulating the oxidative, inflammatory, and proliferative responses of ECs to disturbed flow with OSS.

Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells

The preferential adhesion of monocytes to vascular endothelial cells (ECs) at regions near branches and curvatures of the arterial tree, where flow is disturbed, suggests that hemodynamic conditions play significant roles in monocyte adhesion. The present study aims to elucidate the effects of disturbed flow on monocyte adhesion to ECs and the adhesive properties of ECs. We applied, for the first time, the micron-resolution particle image velocimetry (microPIV) technique to analyze the characteristics of the disturbed flow produced in our vertical-step flow (VSF) chamber. The results demonstrated the existence of a higher near-wall concentration and a longer residence time of the monocytic analog THP-1 cells near the step and the reattachment point. THP-1 cells showed prominent adhesion to ECs pretreated with TNFalpha in the regions near the step and the reattachment point, but they showed virtually no adhesion to un-stimulated ECs. Pre-incubation of the TNFalpha-treated ECs with antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin inhibited the THP-1 adhesion; the maximal inhibition was observed with a combination of these antibodies. Pre-exposure of ECs to disturbed flow in VSF for 24 h led to significant increases in their surface expressions of ICAM-1 and E-selectin, but not VCAM-1, and in the adhesion of THP-1 cells. Our findings demonstrate the importance of complex flow environment in modulating the adhesive properties of vascular endothelium and consequently monocyte adhesion in regions of prevalence of atherosclerotic lesions.

Integrin-mediated expression of bone formation-related genes in osteoblast-like cells in response to fluid shear stress: roles of extracellular matrix, Shc, and mitogen-activated protein kinase.

Integrins play significant roles in mechanical responses of cells on extracellular matrix (ECM). We studied the roles of integrins and ECM proteins (fibronectin [FN], type I collagen [COL1], and laminin [LM]) in shear‐mediated signaling and the expression of bone formation‐related genes (early growth response‐1 [Egr‐1], c‐fos, cyclooxygenase‐2 [Cox‐2], and osteopontin [OPN]) in human osteosarcoma MG63 cells. MG63 cells on FN, COL1, and LM were kept as controls or subjected to shear stress (12 dynes/cm2), and the association of αvβ3 and β1 integrins with Shc, phosphorylation of mitogen‐activated protein kinases (MAPKs, i.e., extracellular signal‐regulated kinase [ERK], c‐jun‐NH2‐terminal kinase [JNK], and p38), and expressions of Egr‐1, c‐fos, Cox‐2, and OPN were determined. In MG63 cells, shear stress induces sustained associations of αvβ3 and β1 with Shc when seeded on FN, but sustained associations of only β1 with Shc when seeded on COL1/LM. Shear inductions of MAPKs and bone formation‐related genes were sustained (24 h) in cells on FN, but some of these responses were transient in cells on COL1/LM. The shear activations of ERK, JNK, and p38 were mediated by integrins and Shc, and these pathways differentially modulated the downstream bone formation‐related gene expression. Our findings showed that β1 integrin plays predominant roles for shear‐induced signaling and gene expression in osteoblast‐like MG63 cells on FN, COL1, and LM and that αvβ3 also plays significant roles for such responses in cells on FN. The β1/Shc association leads to the activation of ERK, which is critical for shear induction of bone formation‐related genes in osteoblast‐like cells.

Shear Stress Induces Synthetic-to-Contractile Phenotypic Modulation in Smooth Muscle Cells via Peroxisome Proliferator-Activated Receptor α/δ Activations by Prostacyclin Released by Sheared Endothelial Cells

Rationale: Phenotypic modulation of smooth muscle cells (SMCs), which are located in close proximity to endothelial cells (ECs), is critical in regulating vascular function. The role of flow-induced shear stress in the modulation of SMC phenotype has not been well defined. Objective: The objective was to elucidate the role of shear stress on ECs in modulating SMC phenotype and its underlying mechanism. Methods and Results: Application of shear stress (12 dyn/cm2) to ECs cocultured with SMCs modulated SMC phenotype from synthetic to contractile state, with upregulation of contractile markers, downregulation of proinflammatory genes, and decreased percentage of cells in the synthetic phase. Treating SMCs with media from sheared ECs induced peroxisome proliferator-activated receptor (PPAR)-&agr;, -&dgr;, and -&ggr; ligand binding activities; transfecting SMCs with specific small interfering (si)RNAs of PPAR-&agr; and -&dgr;, but not -&ggr;, inhibited shear induction of contractile markers. ECs exposed to shear stress released prostacyclin (PGI2). Transfecting ECs with PGI2 synthase-specific siRNA inhibited shear-induced activation of PPAR-&agr;/&dgr;, upregulation of contractile markers, downregulation of proinflammatory genes, and decrease in percentage of SMCs in synthetic phase. Mice with PPAR-&agr; deficiency (compared with control littermates) showed altered SMC phenotype toward a synthetic state, with increased arterial contractility in response to angiotensin II. Conclusions: These results indicate that laminar shear stress induces synthetic-to-contractile phenotypic modulation in SMCs through the activation of PPAR-&agr;/&dgr; by the EC-released PGI2. Our findings provide insights into the mechanisms underlying the EC-SMC interplays and the protective homeostatic function of laminar shear stress in modulating SMC phenotype.

Oscillatory Flow-induced Proliferation of Osteoblast-like Cells Is Mediated by αvβ3 and β1 Integrins through Synergistic Interactions of Focal Adhesion Kinase and Shc with Phosphatidylinositol 3-Kinase and the Akt/mTOR/p70S6K Pathway

Interstitial flow in and around bone tissue is oscillatory in nature and affects the mechanical microenvironment for bone cell growth and formation. We investigated the role of oscillatory shear stress (OSS) in modulating the proliferation of human osteoblast-like MG63 cells and its underlying mechanisms. Application of OSS (0.5 ± 4 dynes/cm2) to MG63 cells induced sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, which was accompanied by increased expression of cyclins A and D1, cyclin-dependent protein kinases-2, -4, and -6, and bone formation-related genes (c-fos, Egr-1, and Cox-2) and decreased expression of p21CIP1 and p27KIP1. OSS-induced activation of PI3K/Akt/mTOR/p70S6K and cell proliferation were inhibited by specific antibodies or small interference RNAs of αvβ3 and β1 integrins and by dominant-negative mutants of Shc (Shc-SH2) and focal adhesion kinase (FAK) (FAK(F397Y)). Co-immunoprecipitation assay showed that OSS induces sustained increases in association of Shc and FAK with αvβ3 and β1 integrins and PI3K subunit p85, which were abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. OSS also induced sustained activation of ERK, which was inhibited by the specific PI3K inhibitor LY294002 and was required for OSS-induced activation of mTOR/p70S6K and proliferation in MG63 cells. Our findings provide insights into the mechanisms by which OSS induces osteoblast-like cell proliferation through activation of αvβ3 and β1 integrins and synergistic interactions of FAK and Shc with PI3K, leading to the modulation of downstream ERK and Akt/mTOR/p70S6K pathways.

Shear Stress Inhibits Smooth Muscle Cell–Induced Inflammatory Gene Expression in Endothelial Cells Role of NF-κB

Objectives—Vascular endothelial cells (ECs) are influenced by shear stress and neighboring smooth muscle cells (SMCs). We investigated the inflammation-relevant gene expression in EC/SMC cocultures under static condition and in response to shear stress. Materials and Methods—Under static condition, DNA microarrays and reverse-transcription polymerase chain reaction identified 23 inflammation-relevant genes in ECs whose expression was significantly affected by coculture with SMCs, with 18 upregulated and 5 downregulated. Application of shear stress (12 dynes/cm2) to the EC side of the coculture for 6 hours inhibited most of the proinflammatory gene expressions in ECs induced by coculture with SMCs. Inhibition of nuclear factor-&kgr;B (NF-&kgr;B) activation by the p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced EC expression of proinflammatory genes, indicating that the NF-&kgr;B binding sites in the promoters of these genes play a significant role in their expression as a result of coculture with SMCs. Chromatin immunoprecipitation assays demonstrated the in vivo regulation of NF-&kgr;B recruitment to selected target promoters. Shear stress inhibited the SMC coculture-induced NF-&kgr;B activation in ECs and monocytic THP-1 cell adhesion to ECs. Conclusions—Our findings suggest that shear stress plays an inhibitory role in the proinflammatory gene expression in ECs located in close proximity to SMCs.