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/cm 2 ) to cultured ECs sustainably up-regulated class I and II HDACs and their nuclear accumulation, whereas PSS (12 ± 4 dynes/cm 2 ) 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 downregulation of NQO1 and KLF2, respectively. OSS up-regulated cyclin A and down-regulated p21 CIP1 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.epigenetics | mechanotransduction | transcriptional regulation

Abstract, "En face examination of rat aortas reveals high levels of phospho-Smad1/5 in ECs of the straight segment of thoracic aorta and the inner, but not the outer, curvature of aortic arch" should instead appear as "En face examination of rat aortas reveals high levels of phospho-Smad1/5 in ECs of the inner, but not the outer, curvature of aortic arch, nor the straight segment of thoracic aorta."www.pnas.org/cgi

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.

Summary. Background: 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. Objectives: Here we elucidated the molecular mechanisms by which OSS activates EC Smad1/5 and its in vivo functional role. Methods: Lentiviral Smad5-specific short hairpin RNA (Lenti-shSmad5) was constructed and intra-arterially injected into the lumen of stenosed abdominal aorta in bromodeoxyuridine-infused rats. Co-immunoprecipitation and in situ proximity ligation assays were performed on ECs exposed to OSS (0.5 AE 4 dynes/cm 2 ) in a parallel-plate flow chamber to investigate BMPR-integrin interactions and their regulatory role in OSS-activation of EC Smad1/5. Results: Intra-arterial administration of Lenti-shSmad5 inhibited bromodeoxyuridine uptake of ECs at post-stenotic sites, where disturbed flow with OSS occurs. OSS induced sustained BMPRIB-a v b 3 integrin association in ECs, which was mediated by the intracytoplasmic kinase domain of BMPRII and subsequently activated the Shc/ focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) cascade, leading to Smad1/5 activation. This OSS-activation of Smad1/5 induced its association with and activation of runt-related transcription factor-2 (Runx2), leading to activations of mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K), a pathway critical for EC proliferation in response to OSS. Conclusions: Oscillatory shear stress induces synergistic interactions between specific BMPRs and integrin to activate Smad1/5 through the Shc/FAK/ERK pathway, which leads to the activation of the Runx2/mTOR/p70S6K pathway to promote EC proliferation.

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 Co-culturing ECs with sSMCs under static condition causes initial increases of four 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 h and those for miR-451/98 lasted for only 6-12 h. Shear stress (12 dynes/cm2) to co-cultured ECs for 24 h augments these four miR expressions. In vivo, these four miRs are highly expressed in neointimal ECs in injured arteries under physiological levels of flow, but not expressed under flow stagnation. MiR-146a, -708, -451, and -98 target interleukin (IL)-1 receptor-associated kinase, inhibitor of nuclear factor-κB (NF-κB) kinase subunit-γ, IL-6 receptor, and conserved helix-loop-helix ubiquitous kinase, respectively, to inhibit NF-κB signaling, which exerts negative feedback control on the biogenesis of these miRs. NF-E2-related factor-2 (Nrf-2) is critical for shear-induction of miR-146a in co-cultured 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.

T-lymphocyte migration under flow is critical for immune responses, but the mechanisms by which flow modulates the migratory behaviors of T-lymphocytes remain unclear. Human peripheral blood T-lymphocytes (PBTLs), when stimulated with phorbol 12-myristate 13-acetate (PMA), stretched their cell bodies dramatically and moved along the flow direction. In contrast, stromal cell-derived factor-1α-stimulated PBTLs deformed and migrated in a random manner. Here we elucidated the molecular mechanisms underlying flow-induced directionality and deformation of PMA-stimulated PBTLs. PMA primed PBTLs for polarization under flow, with protein kinase C (PKC)-δ enriched in the leading edge, PKC-βI in the microtubule organizing center, and PKC-βII in the uropod and peripheral region. PKC-δ regulated cell protrusions in the leading edge through Tiam1/Rac1/calmodulin, whereas PKC-β regulated RhoA/Rho-associated kinase activity and microtubule stability to modulate uropod contractility and detachment. Our findings indicate that PKC-δ and -β coordinate in the cell leading edge and uropod, respectively, to modulate the directionality and deformability of migratory T-lymphocytes under flow.