Chih-I Lee

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.

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.

BMP receptor-integrin interaction mediates responses of vascular endothelial Smad1/5 and proliferation to disturbed flow.

Vascular endothelial cells (ECs) are constantly exposed to blood flow‐induced shear stress. Our previous study demonstrated that disturbed flow with low and oscillatory shear stress (OSS) induces bone morphogenetic protein receptor (BMPR)‐specific Smad1/5 activation in ECs, but the underlying mechanisms and the in vivo functional role of Smad1/5 remain unclear.

Estrogen Augments Shear Stress–Induced Signaling and Gene Expression in Osteoblast-like Cells via Estrogen Receptor–Mediated Expression of β1-Integrin

Estrogen and mechanical forces are positive regulators for osteoblast proliferation and bone formation. We investigated the synergistic effect of estrogen and flow‐induced shear stress on signal transduction and gene expression in human osetoblast‐like MG63 cells and primary osteoblasts (HOBs) using activations of extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK) and expressions of c‐fos and cyclooxygenase‐2 (I) as readouts. Estrogen (17β‐estradiol, 10 nM) and shear stress (12 dyn/cm2) alone induced transient phosphorylations of ERK and p38 MAPK in MG63 cells. Pretreating MG63 cells with 17β‐estradiol for 6 hours before shearing augmented these shear‐induced MAPK phosphorylations. Western blot and flow cytometric analyses showed that treating MG63 cells with 17β‐estradiol for 6 hrs induced their β1‐integrin expression. This estrogen‐induction of β1‐integrin was inhibited by pretreating the cells with a specific antagonist of estrogen receptor ICI 182,780. Both 17β‐estradiol and shear stress alone induced c‐fos and Cox‐2 gene expressions in MG63 cells. Pretreating MG63 cells with 17β‐estradiol for 6 hrs augmented the shear‐induced c‐fos and Cox‐2 expressions. The augmented effects of 17β‐estradiol on shear‐induced MAPK phosphorylations and c‐fos and Cox‐2 expressions were inhibited by pretreating the cells with ICI 182,780 or transfecting the cells with β1‐specific small interfering RNA. Similar results on the augmented effect of estrogen on shear‐induced signaling and gene expression were obtained with HOBs. Our findings provide insights into the mechanism by which estrogen augments shear stress responsiveness of signal transduction and gene expression in bone cells via estrogen receptor–mediated increases in β1‐integrin expression.

MicroRNA Mediation of Endothelial Inflammatory Response to Smooth Muscle Cells and Its Inhibition by Atheroprotective Shear Stress

RATIONALE In atherosclerotic lesions, synthetic smooth muscle cells (sSMCs) induce aberrant microRNA (miR) profiles in endothelial cells (ECs) under flow stagnation. Increase in shear stress induces favorable miR modulation to mitigate sSMC-induced inflammation. OBJECTIVE To address the role of miRs in sSMC-induced EC inflammation and its inhibition by shear stress. METHODS AND RESULTS Coculturing ECs with sSMCs under static condition causes initial increases of 4 anti-inflammatory miRs (146a/708/451/98) in ECs followed by decreases below basal levels at 7 days; the increases for miR-146a/708 peaked at 24 hours and those for miR-451/98 lasted for only 6 to 12 hours. Shear stress (12 dynes/cm(2)) to cocultured ECs for 24 hours augments these 4 miR expressions. In vivo, these 4 miRs are highly expressed in neointimal ECs in injured arteries under physiological levels of flow, but not expressed under flow stagnation. MiR-146a, miR-708, miR-451, and miR-98 target interleukin-1 receptor-associated kinase, inhibitor of nuclear factor-κB kinase subunit-γ, interleukin-6 receptor, and conserved helix-loop-helix ubiquitous kinase, respectively, to inhibit nuclear factor-κB signaling, which exerts negative feedback control on the biogenesis of these miRs. Nuclear factor-E2-related factor (Nrf)-2 is critical for shear-induction of miR-146a in cocultured ECs. Silencing either Nrf-2 or miR-146a led to increased neointima formation of injured rat carotid artery under physiological levels of flow. Overexpressing miR-146a inhibits neointima formation of rat or mouse carotid artery induced by injury or flow cessation. CONCLUSIONS Nrf-2-mediated miR-146a expression is augmented by atheroprotective shear stress in ECs adjacent to sSMCs to inhibit neointima formation of injured arteries.

MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

Significance This study demonstrates that hormone receptor RARα plays a vital role in the selective activation of proinflammatory and anti-inflammatory signaling to modulate the miR-10a/GATA6/VCAM-1 cascade in endothelial cells in response to proatherogenic oscillatory shear stress (OS) vs. atheroprotective pulsatile shear stress (PS). HDAC-3/5/7 and RXRα are induced by OS and PS to serve as mechanosensitive “repressors” and “enhancers,” respectively, to associate with RARα to modulate its binding to RA-responsive element (RARE) to switch miR-10a expression. Our findings provide insight into the relationship between two different epigenetic factors (HDACs and miRs) and hormone receptors (RARα and RXRα) in the regulation of endothelial functions and elucidate new mechanisms of hemodynamic-based pathophysiology of the atherosclerotic vascular wall. Histone deacetylases (HDACs) and microRNAs (miRs) have emerged as two important epigenetic factors in the regulation of vascular physiology. This study aimed to elucidate the relationship between HDACs and miRs in the hemodynamic modulation of endothelial cell (EC) dysfunction. We found that miR-10a has the lowest expression among all examined shear-responsive miRs in ECs under oscillatory shear stress (OS), and a relatively high expression under pulsatile shear stress (PS). PS and OS alter EC miR-10a expression to regulate the expression of its direct target GATA6 and downstream vascular cell adhesion molecule (VCAM)-1. PS induces the expression, nuclear accumulation, and association of retinoid acid receptor-α (RARα) and retinoid X receptor-α (RXRα). RARα and RXRα serve as a “director” and an “enhancer,” respectively, to enhance RARα binding to RA-responsive element (RARE) and hence miR-10a expression, thus down-regulating GATA6/VCAM-1 signaling in ECs. In contrast, OS induces associations of “repressors” HDAC-3/5/7 with RARα to inhibit the RARα-directed miR-10a signaling. The flow-mediated miR-10a expression is regulated by Krüppel-like factor 2 through modulation in RARα–RARE binding, with the consequent regulation in GATA6/VCAM-1 in ECs. These results are confirmed in vivo by en face staining on the aortic arch vs. the straight thoracic aorta of rats. Our findings identify a mechanism by which HDACs and RXRα modulate the hormone receptor RARα to switch miR-10a expression and hence the proinflammatory vs. anti-inflammatory responses of vascular endothelium under different hemodynamic forces.

Convergence of physical and chemical signaling in the modulation of vascular smooth muscle cell cycle and proliferation by fibrillar collagen-regulated P66Shc

Arterial smooth muscle cell (SMC) phenotype and proliferation is regulated by their surrounding collagens, which transform from fibrillar to monomeric type in atherogenesis, and platelet-derived growth factor (PDGF)-BB/interleukin (IL)-1β. This study aims at elucidating the mechanisms by which physical (monomeric vs. fibrillar collagens) and chemical (PDGF-BB/IL-1βvs. vehicle controls) stimuli modulate SMC cycle and proliferation. SMCs were cultured on monomeric vs. fibrillar type I collagens. In parallel experiments, SMCs on fibrillar collagen were co-stimulated with PDGF-BB/IL-1β. These physical and chemical factors induced common SMC cycle signaling events, including up-regulations of cyclin-dependent kinase-4/6 and cyclins A/D1, phosphorylation of retinoblastoma (Rb) and its dissociations with E2F2/3. The physical and chemical inductions of SMC cycle signaling and progression were oppositely regulated by phosphatidylinositol 3-kinase (PI3K)-mediated Akt and p38 mitogen-activated protein kinase (MAPK). Fibrillar collagen degraded p66Shc, whose Ser36-phosphorylation plays important roles in the modulation of SMC cycle. Monomeric collagen and PDGF-BB/IL-1β co-stimulation induced p66Shc expression and Ser36-phosphorylation through β(1) integrin and PDGF receptor-β, respectively. In conclusion, our results demonstrate that fibrillar collagen-regulated p66Shc converges the physical and chemical stimuli to modulate SMC cycle and proliferation through PI3K-mediated Akt and p38 MAPK and their opposite regulation in downstream common cell cycle signaling cascades.

Differential regulations of fibronectin and laminin in Smad2 activation in vascular endothelial cells in response to disturbed flow

BackgroundAtherosclerosis occurs in arterial curvatures and branches, where the flow is disturbed with low and oscillatory shear stress (OSS). The remodeling and alterations of extracellular matrices (ECMs) and their composition is the critical step in atherogenesis. In this study, we investigated the effects of different ECM proteins on the regulation of mechanotransduction in vascular endothelial cells (ECs) in response to OSS.MethodsThrough the experiments ranging from in vitro cell culture studies on effects of OSS on molecular signaling to in vivo examinations on clinical specimens from patients with coronary artery disease (CAD), we elucidated the roles of integrins and different ECMs, i.e., fibronectin (FN) and laminin (LM), in transforming growth factor (TGF)-β receptor (TβR)-mediated Smad2 activation and nuclear factor-κB (NF-κB) signaling in ECs in response to OSS and hence atherogenesis.ResultsOSS at 0.5±12 dynes/cm2 induces sustained increases in the association of types I and II TβRs with β1 and β3 integrins in ECs grown on FN, but it only transient increases in ECs grown on LM. OSS induces a sustained activation of Smad2 in ECs on FN, but only a transient activation of Smad2 in ECs on LM. OSS-activation of Smad2 in ECs on FN regulates downstream NF-κB signaling and pro-inflammatory gene expression through the activation of β1 integrin and its association with TβRs. In contrast, OSS induces transient activations of β1 and β3 integrins in ECs on LM, which associate with type I TβR to regulate Smad2 phosphorylation, resulting in transient induction of NF-κB and pro-inflammatory gene expression. In vivo investigations on diseased human coronary arteries from CAD patients revealed that Smad2 is highly activated in ECs of atherosclerotic lesions, which is accompanied by the concomitant increase of FN rather than LM in the EC layer and neointimal region of atherosclerotic lesions.ConclusionsOur findings provide new insights into the mechanisms of how OSS regulates Smad2 signaling and pro-inflammatory genes through the complex signaling networks of integrins, TβRs, and ECMs, thus illustrating the molecular basis of regional pro-inflammatory activation within disturbed flow regions in the arterial tree.