Unidirectional Laminar Shear Stress Research Articles

Abstract 14971: Loss of ZBTB46 Decreases BCL2A Expression and Increases Endothelial Cell Apoptosis

Introduction: Endothelial cell (EC) activation, injury, and apoptosis in areas of the vasculature experiencing low and oscillatory shear stress (or disturbed flow) play a key role in the development and pathogenesis of atherosclerosis. In in vivo and in vitro studies, we have demonstrated that ZBTB46, a member of the BTB-ZF protein family of transcription factors, is down-regulated in ECs exposed to disturbed flow when compared to cells experiencing unidirectional laminar shear stress. Additionally, we have observed that germline deletion of ZBTB46 leads to decreased BCL2A expression in mouse carotid arteries. Given that ZBTB46 down-regulation occurs in areas of the vasculature prone to increased EC apoptosis and loss of ZBTB46 in vivo is associated with down regulation of anti-apoptotic protein BCL2A, we explored the role of ZBTB46 in EC apoptosis. Hypothesis: Loss of ZBTB46 negatively regulates transcription of the anti-apoptotic mediator BCL2A and sensitizes EC to apoptosis. Methods: Human aortic endothelial cells (HAEC) were transfected with either ZBTB46-siRNA or control siRNA, grown to 2 days post-confluence to induce ZBTB46 expression and then harvested for RNA. Gene expression was assessed by quantitative real-time PCR. In assays to measure apoptosis, siZBTB46 transfected cells, and corresponding controls were treated with various concentrations (0-400 micro molar) of hydrogen peroxide (H 2 O 2 ) for 18 hours, stained for Annexin V and imaged by confocal microscopy Results: Down regulation of ZBTB46 using siRNA (siZBTB46) in HAEC led to a 78% ± 9% (SEM) reduction in BCL2A1 mRNA expression (n=4, p=0.004) when compared to controls (siCtrl). BCL2A1 downregulation also coincided with increased apoptosis in ZBTB46 deficient HAEC (siZBTB46 39% ± 2.9% v siCtrl 27% ± 3.5% n=6, p=0.026) as measured by Annexin V staining. Additionally, H 2 O 2 -induced apoptosis was exacerbated by loss of ZBTB46 in HAEC at 100 μM (siZBTB46 68% ± 2.1% v siCtrl 30% ± 3.1% n=5-6, p=0.001), 200 μM (siZBTB46 82% ± 1.2% v siCtrl 44% ± 3.2% n=4-5, p=0.001), and 400 μM H 2 O 2 (siZBTB46 88% ± 1.1% v siCtrl 58% ± 5.6% n=5-6, p=0.001). Conclusions: Loss of ZBTB46 in endothelial cells reduces BCL2A expression and increases the susceptibility of endothelial cells to apoptosis.

Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells.

Atherosclerosis is a chronic inflammatory disease and occurs preferentially in arterial regions exposed to disturbed blood flow (d-flow) while the stable flow (s-flow) regions are spared. D-flow induces endothelial inflammation and atherosclerosis by regulating endothelial gene expression partly through the flow-sensitive transcription factors (FSTFs). Most FSTFs, including the well-known Kruppel-like factors KLF2 and KLF4, have been identified from in vitro studies using cultured endothelial cells (ECs). Since many flow-sensitive genes and pathways are lost or dysregulated in ECs during culture, we hypothesized that many important FSTFs in ECs in vivo have not been identified. We tested the hypothesis by analyzing our recent gene array and single-cell RNA sequencing (scRNAseq) and chromatin accessibility sequencing (scATACseq) datasets generated using the mouse partial carotid ligation model. From the analyses, we identified 30 FSTFs, including the expected KLF2/4 and novel FSTFs. They were further validated in mouse arteries in vivo and cultured human aortic ECs (HAECs). These results revealed 8 FSTFs, SOX4, SOX13, SIX2, ZBTB46, CEBPβ, NFIL3, KLF2, and KLF4, that are conserved in mice and humans in vivo and in vitro. We selected SOX13 for further studies because of its robust flow-sensitive regulation, preferential expression in ECs, and unknown flow-dependent function. We found that siRNA-mediated knockdown of SOX13 increased endothelial inflammatory responses even under the unidirectional laminar shear stress (ULS, mimicking s-flow) condition. To understand the underlying mechanisms, we conducted an RNAseq study in HAECs treated with SOX13 siRNA under shear conditions (ULS vs. oscillatory shear mimicking d-flow). We found 94 downregulated and 40 upregulated genes that changed in a shear- and SOX13-dependent manner. Several cytokines, including CXCL10 and CCL5, were the most strongly upregulated genes in HAECs treated with SOX13 siRNA. The robust induction of CXCL10 and CCL5 was further validated by qPCR and ELISA in HAECs. Moreover, the treatment of HAECs with Met-CCL5, a specific CCL5 receptor antagonist, prevented the endothelial inflammation responses induced by siSOX13. In addition, SOX13 overexpression prevented the endothelial inflammation responses. In summary, SOX13 is a novel conserved FSTF, which represses the expression of pro-inflammatory chemokines in ECs under s-flow. Reduction of endothelial SOX13 triggers chemokine expression and inflammatory responses, a major proatherogenic pathway.

Laminar Shear Stress Enhances Cytosolic PINK1 Expression: Increased Mitophagic Sensitivity Toward Mitochondrial Dysfunction

PURPOSEPhosphatase and tensin homolog (PTEN)‐induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process and known to act as a cytoprotective protein in mammalian cells. Mutation or deficiency of PINK1 has been closely related to several disease conditions. The purpose of this study was to determine PINK1 expression levels and subcellular localization under exercise‐mimic laminar shear stress (LSS) condition in human aortic endothelial cells (HAECs) or in exercising mice, and its implication on endothelial homeostasis and cardiovascular disease (CVD) prevention.METHODS & RESULTSFirst, we measured full‐length PINK1 (FL‐PINK1) mRNA and protein expression levels in HAECs under unidirectional laminar shear stress (LSS) at 15 dyne/cm2 for 48h. LSS significantly elevated both FL‐PINK1 mRNA and protein expressions in HAECs compared to static control. Mitochondrial fractionation assays showed a decrease in FL‐PINK1 accumulation in the mitochondria with a compensatory increase in the cytosolic FL‐PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Also, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential (JC‐1) showed mitochondrial elongation and increased mitochondrial membrane potential, respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. Based on this observation, we further hypothesized that the elevation of the cytosolic pool of FL‐PINK1 would increase the sensitivity to mitophagic signals in pathological conditions. Preconditioned HAECs with LSS showed lower mtDNA lesions under angiotensin II stimulation (100 nM, 6h). Moreover, LSS‐preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e. CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS‐enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise‐preconditioned mice were more protective to angiotensin II‐induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1‐dependent manner.CONCLUSIONLSS increases a cytosolic pool of FL‐PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in endothelial cells. Our data suggest that exercise may support mitochondrial homeostasis in vascular endothelial cells by enhancing PINK1‐dependent cell protection mechanisms.

Coxsackievirus and adenovirus receptor mediates the responses of endothelial cells to fluid shear stress

Endothelial mechanotransduction by fluid shear stress (FSS) modulates endothelial function and vascular pathophysiology through mechanosensors on the cell membrane. The coxsackievirus and adenovirus receptor (CAR) is not only a viral receptor but also a component of tight junctions and plays an important role in tissue homeostasis. Here, we demonstrate the expression, regulatory mechanism, and role of CAR in vascular endothelial cells (ECs) under FSS conditions. Disturbed flow increased, whereas unidirectional laminar shear stress (LSS) decreased, CAR expression in ECs through the Krüppel-like factor 2 (KLF2)/activator protein 1 (AP-1) axis. Deletion of CAR reduced the expression of proinflammatory genes and endothelial inflammation induced by disturbed flow via the suppression of NF-κB activation. Consistently, disturbed flow-induced atherosclerosis was reduced in EC-specific CAR KO mice. CAR was found to be involved in endothelial mechanotransduction through the regulation of platelet endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation. Our results demonstrate that endothelial CAR is regulated by FSS and that this regulated CAR acts as an important modulator of endothelial mechanotransduction by FSS.

Shear-Induced CCN1 Promotes Atheroprone Endothelial Phenotypes and Atherosclerosis.

Atherosclerosis occurs preferentially at the blood vessels encountering blood flow turbulence. The matricellular protein CCN1 is induced in endothelial cells by disturbed flow, and is expressed in advanced atherosclerotic lesions in patients and in the Apoe-/- mouse model. The role of CCN1 in atherosclerosis remains undefined. To assess the function of CCN1 in vivo, knock-in mice carrying the integrin α6β1-binding-defective mutant allele Ccn1-dm on the Apoe-/- background were tested in an atherosclerosis model generated by carotid artery ligation. Additionally, CCN1-regulated functional phenotypes of human umbilical vein endothelial cells, or primary mouse aortic endothelial cells isolated from wild-type and Ccn1 dm/dm mice, were investigated in the in vitro shear stress experiments under unidirectional laminar shear stress (12 dyn/cm2) versus oscillatory shear stress (±5 dyn/cm2) conditions. We found that Ccn1 expression was upregulated in the arterial endothelium 3 days after ligation before any detectable structural changes, and intensified with the progression of atherosclerotic lesions. Compared with Apoe-/- controls, Ccn1 dm/dm/ Apoe-/- mice were remarkably resistant to ligation-induced plaque formation (n=6). These mice exhibited lower oxidative stress, expression of endothelin-1 and monocyte chemoattractant protein-1, and monocyte homing. CCN1/α6β1 critically mediated flow-induced activation of the pleiotropic transcription factor nuclear factor-κB and therefore the induction of atheroprone gene expression in the mouse arterial endothelium after ligation (n=6), or in cultured human umbilical vein endothelial cells or primary mouse aortic endothelial cells exposed to oscillatory shear stress (n=3 in triplicate). Interestingly, the activation of nuclear factor-κB by CCN1/α6β1 signaling prompted more production of CCN1 and α6β1. Blocking CCN1-α6β1 binding by the Ccn1-dm mutation or by T1 peptide (derived from an α6β1-binding sequence of CCN1) disrupted the positive-feedback regulation between CCN1/α6β1 and nuclear factor-κB, and prevented flow-induced atheroprone phenotypic alterations in endothelial cells or atherosclerosis in mice. These data demonstrate a causative role of CCN1 in atherosclerosis via modulating endothelial phenotypes. CCN1 binds to its receptor integrin α6β1 to activate nuclear factor-κB, thereby instigating a vicious circle to persistently promote atherogenesis. T1, a peptide antagonist selectively targeting CCN1-α6β1, can be further optimized for developing T1-mimetics to treat atherosclerosis.

ZBTB46 is a shear-sensitive transcription factor inhibiting endothelial cell proliferation via gene expression regulation of cell cycle proteins

ZBTB46 is a transcription factor identified in classical dendritic cells and keeps dendritic cells in a quiescent state. Chromatin immunoprecipitation sequencing in dendritic cells has identified over 1300 potential gene targets of ZBTB46, affecting many processes including cell cycle. Endothelial cells (ECs) also express ZBTB46 and are mostly in a quiescent non-proliferative state. While EC proliferation is a critical process in development, dysregulation of EC proliferation as seen in areas of disturbed flow play an important role in many disease processes such as atherosclerosis, pulmonary hypertension, transplant vasculopathy, neointimal hyperplasia, and in-stent restenosis. We studied the role of ZBTB46 in ECs, hypothesizing that it inhibits EC proliferation. Using a model of disturbed flow in mice, we found that ZBTB46 is expressed in murine arterial ECs in vivo, and is downregulated by disturbed flow. In vitro results using HAECs showed that cell confluence and laminar shear stress, both known physiological conditions promoting EC quiescence, led to upregulation of ZBTB46 expression. Adenoviral-mediated overexpression of ZBTB46 in vitro caused reduced EC proliferation, and increased number of cells in the G0/G1 phase of cell cycle, without affecting apoptosis or senescence, while siRNA knockdown of ZBTB46 negated the known inhibitory role of unidirectional laminar shear stress on EC proliferation. ZBTB46 overexpression also led to a broad suppression of genes involved in cell cycle progression including multiple cyclins and cyclin-dependent kinases, but an increase in the CDK inhibitor CDKN1A. Phosphorylation of the retinoblastoma protein was also decreased as assessed by Western blot. Tube formation on Matrigel was reduced, suggesting an inhibitory role for ZBTB46 in angiogenesis. Further research is required to investigate the potential role of ZBTB46 in specific pathologic conditions and whether it can be targeted in a therapeutic manner.

Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress

The endothelium is exposed to various flow patterns such as vasoprotective unidirectional laminar shear stress (LSS) and atherogenic oscillatory shear stress (OSS). A software-controlled, valve-operated OsciFlow device with parallel chambers was used to apply LSS and OSS to endothelial cells. Although LSS inhibited superoxide over time, OSS time-dependently increased superoxide production from endothelial cells. Immunocytochemical staining revealed that, at resting state, p47phox colocalizes with NOX2, whereas NOXO1 colocalizes with NOX1. RNAi of p47phox had no effects on superoxide or NO production in response to OSS but significantly reduced NO production in LSS, implicating a p47phox-bound NADPH oxidase (NOX) in mediating basal NO production. Indeed, RNAi of p47phox inhibited endothelial nitric oxide synthase (eNOS) serine 1179 phosphorylation, whereas PEG-catalase scavenging of intracellular hydrogen peroxide or RNAi of NOX2 produced similar results, indicating a role of NOX2/p47phox-derived hydrogen peroxide in mediating the basal activity of NO production from eNOS. In contrast, RNAi of NOXO1 resulted in no significant changes in NO and superoxide levels in response to LSS but significantly reduced superoxide while increasing NO in response to OSS. Furthermore, we identified, for the first time, that OSS uncouples eNOS, which was corrected by RNAi of NOXO1. In summary, LSS activates the NOX2-p47phox complex to activate eNOS phosphorylation and NO production. OSS instead activates the NOX1-NOXO1 complex to uncouple eNOS. These results demonstrate differential roles of NOXs in modulating the redox state in response to different shear stresses, which may promote the development of novel therapeutic agents to mimic the protective effects of LSS while inhibiting the injurious effects of OSS.

Flow-dependent expression of ectonucleotide tri(di)phosphohydrolase-1 and suppression of atherosclerosis.

The ability of cells to detect and respond to nucleotide signals in the local microenvironment is essential for vascular homeostasis. The enzyme ectonucleotide tri(di)phosphohydrolase-1 (ENTPD1, also known as CD39) on the surface of leukocytes and endothelial cells metabolizes locally released, intravascular ATP and ADP, thereby eliminating these prothrombotic and proinflammatory stimuli. Here, we evaluated the contribution of CD39 to atherogenesis in the apolipoprotein E-deficient (ApoE-deficient) mouse model of atherosclerosis. Compared with control ApoE-deficient animals, plaque burden was markedly increased along with circulating markers of platelet activation in Cd39+/-Apoe-/- mice fed a high-fat diet. Plaque analysis revealed stark regionalization of endothelial CD39 expression and function in Apoe-/- mice, with CD39 prominently expressed in atheroprotective, stable flow regions and diminished in atheroprone areas subject to disturbed flow. In mice, disturbed flow as the result of partial carotid artery ligation rapidly suppressed endothelial CD39 expression. Moreover, unidirectional laminar shear stress induced atheroprotective CD39 expression in human endothelial cells. CD39 induction was dependent upon the vascular transcription factor Krüppel-like factor 2 (KLF2) binding near the transcriptional start site of CD39. Together, these data establish CD39 as a regionalized regulator of atherogenesis that is driven by shear stress.

Disturbed Shear Stress Promotes Caveolae‐Mediated oxLDL Uptake Leading to Increased Endothelial Stiffness

IntroductionOxidized low density lipoprotein (oxLDL) is a major risk factor in the development of atherosclerotic plaques, which preferentially form at vascular bifurcations subject to bidirectional recirculating disturbed shear stress (DSS).MethodsA microfluidic channel with a step barrier was designed, tested and fabricated to generate atheroprotective unidirectional laminar shear stress (LSS) and atheroprone DSS onto an EC monolayer for 48 hours. EC stiffness was assessed using atomic force microscopy.ResultsOur results show a statistically significant increase in oxLDL uptake in DSS vs. LSS regions in three EC types: human aortic (55% increase), human microvascular (40% increase), and mouse microvascular (50% increase). OxLDL uptake was reduced in cells lacking Cav1, the main protein in caveolae (n=4, p<0.05). The mechanism of differential oxLDL uptake was further investigated using siRNA treated ECs for CD36 and Lox1, two oxLDL receptors. Our experiments revealed that CD36 (n=4, p<0.05), but not Lox1 (n=4, p<0.05), is required for increased uptake under DSS. Furthermore, the increased uptake under DSS results in a 60% increase in EC stiffness (n=3, p<0.05). This data is further supported by ex vivo stiffness measurements of vessels from WT mice which show a 50% increase in EC stiffness in the aortic arch (DSS) than the LSS region of the descending aorta (DA) (n=5, p<0.05). In Cav1 KO mice, EC stiffness was markedly reduced (n=5, p<0.05) and no differences observed between the DA and arch.ConclusionTaken together, this study suggests that DSS‐induced increase in oxLDL uptake is mediated by caveolae and plays a significant role in increasing localized EC stiffness.

Abrupt reflow enhances cytokine-induced proinflammatory activation of endothelial cells during simulated shock and resuscitation.

Circulatory shock and resuscitation are associated with systemic hemodynamic changes, which may contribute to the development of MODS (multiple organ dysfunction syndrome). In this study, we used an in vitro flow system to simulate the consecutive changes in blood flow as occurring during hemorrhagic shock and resuscitation in vivo. We examined the kinetic responses of different endothelial genes in human umbilical vein endothelial cells preconditioned to 20 dyne/cm unidirectional laminar shear stress for 48 h to flow cessation and abrupt reflow, respectively, as well as the effect of flow cessation and reflow on tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory activation. Endothelial CD31 and VE-cadherin were not affected by the changes in flow in the absence or presence of TNF-α. The messenger RNA levels of proinflammatory molecules E-selectin, VCAM-1 (vascular cell adhesion molecule 1), and IL-8 (interleukin 8) were significantly induced by flow cessation respectively acute reflow, whereas ICAM-1 (intercellular adhesion molecule 1) was downregulated on flow cessation and induced by subsequent acute reflow. Flow cessation also affected the Ang/Tie2 (Angiopoietin/Tie2 receptor tyrosine kinase) system by downregulating Tie2 and inducing its endothelial ligand Ang2, an effect that was further extended on acute reflow. Furthermore, the induction of proinflammatory adhesion molecules by TNF-α under flow cessation was significantly enhanced on subsequent acute reflow. This study demonstrated that flow alterations per se during shock and resuscitation contribute to endothelial activation and that these alterations interact with proinflammatory factors coexisting in vivo such as TNF-α. The abrupt reflow-related enhancement of cytokine-induced endothelial proinflammatory activation supports the concept that sudden regain of flow during resuscitation has an aggravating effect on endothelial activation, which may play a significant role in vascular dysfunction and consequent organ injury. This study implies that the improvement of resuscitation strategies and the pharmacological interference with proinflammatory signaling cascades at the right time of resuscitation of shock patients may be beneficial to regain and/or maintain organ function in patients after circulatory shock.

Distinctive Subcellular Akt‐1 Responses to Shear Stress in Endothelial Cells

Endothelial cells undergo a rapid cell-cell junction inclination following exposure to atheroprotective unidirectional flow. In contrast, atherosclerotic lesions correlate with a heterogeneous distribution of the junctional wall inclination in cells exposed to time-varying, reversing, and oscillatory flow as well as to low mean shear stress. However, the underlying biochemical events by which endothelial cells distinctively respond to unidirectional versus flow reversal remain unclear. Here, we show that the subcellular distribution of flow-induced Akt-1 phosphorylation in endothelial cells lining the mouse aorta varies depending on local hemodynamics. Activated Akt-1 accumulated in perinuclear areas of cells in regions predisposed to disturbed flow but were localized at the cell-cell junction in regions of high unidirectional laminar shear stress. In flow-adapted human endothelial cells, reversal in flow direction was associated within minutes with a subcellular concentration of phosphorylated Akt-1 at the upstream edge of cells. Interestingly, oscillatory flow (with a zero mean shear stress) failed to activate Akt-1, whereas a unidirectional pulsatile flow of similar amplitude induced an increase in Akt-1 phosphorylation. Finally, silencing of the G protein αq/11 subunit abrogated both flow-induced Akt-1 and GSK-3β activation. Together, these results characterize the existence of a Gαq/11-mediated Akt-1 signaling pathway that is dynamically responsive to flow direction, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to flow reversal.

Anti-Atherogenic Effect of Laminar Shear StressviaNrf2 Activation

Fluid shear stress plays a critical role in the regulation of vascular biology and its pathology, such as atherosclerosis, via modulation of redox balance. Both pro-atherogenic (either oscillatory or turbulent, nonunidirectional) shear stress and anti-atherogenic (either steady or pulsatile, unidirectional laminar) shear stress stimulate production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are involved in signal transduction of gene expression. Nonunidirectional shear stress induces pro-atherogenic genes encoding adhesion molecules and chemokines in a manner dependent on production of both superoxide and nitric oxide. Steady or pulsatile laminar shear stress induces expression of genes encoding cytoprotective enzymes for glutathione biosynthesis and detoxification, which are regulated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). We show that pulsatile laminar shear stress (PLSS)-induced expression of adhesion molecules and chemokines was enhanced in human umbilical vein endothelial cells (HUVEC) treated with Nrf2 siRNA and arterial endothelial cells isolated from Nrf2 knockout mice. Hence, we propose the hypothesis that PLSS maintains the endothelium in an anti-atherogenic state via intracellular antioxidant levels increased as a result of Nrf2 activation, thereby preventing excess ROS/RNS production required for pro-atherogenic gene expression.

Laminar Shear Stress Modulates Phosphorylation and Localization of RNA Polymerase II on the Endothelial Nitric Oxide Synthase Gene

In endothelial cells exposed to unidirectional laminar shear stress, endothelial nitric oxide synthase transcription (eNOS), mRNA stability, and protein levels are enhanced. We have previously demonstrated that these changes are associated with increased 3' polyadenylation of eNOS mRNA. Here, we investigated the effect of laminar shear stress on the phosphorylation and localization of RNA polymerase (Pol) II, the enzyme primarily responsible for coordinating transcription and posttranscriptional processing. Using Western and chromatin immunoprecipitation analyses, Pol II phosphorylation and localization on the eNOS gene were assessed in bovine aortic endothelial cells exposed to laminar shear stress. Total Pol II (phosphorylated and unphosphorylated) levels were increased 65% in response to laminar shear stress. This was associated with an increase in Pol II phosphoserine 2, but no change in levels of the unphosphorylated or phosphoserine 5 isoforms. Quantitative chromatin immunoprecipitation analysis showed that laminar shear stress enhanced binding of Pol II phosphoserine 2 to the 3' end of the eNOS gene, particularly exon 26, which encodes the 3'UTR. Treatment of cells with DRB attenuated laminar shear stress-induced Pol II phosphorylation, eNOS 3' polyadenylation, and eNOS expression. These data suggest that laminar shear stress enhances eNOS mRNA 3' polyadenylation by modulating phosphorylation and localization of Pol II.

Induction of the Cytoprotective Enzyme Heme Oxygenase-1 by Statins Is Enhanced in Vascular Endothelium Exposed to Laminar Shear Stress and Impaired by Disturbed Flow

In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H(2)O(2)-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2.

KLF2-dependent, Shear Stress-induced Expression of CD59

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.

Microvascular function and ageing: L‐arginine, tetrahydrobiopterin and the search for the fountain of vascular youth

Cardiovascular disease remains the most salient killer in the United States and ageing is a primary risk factor. With advancing age, there is pervasive macrovascular and microvascular dysfunction that manifests as stiffening of central elastic arteries, thickening of intimal–medial layers of the vascular wall, and diminished peripheral conduit artery/resistance artery vasodilatory capacity. Reduced endothelium-dependent vasodilatation has been shown in several prospective studies to be an independent predictor of adverse cardiovascular events. In the healthy vasculature, nitric oxide (NO) is released from endothelial cells in response to laminar shear stress and causes vasodilatation. In addition to its vasodilatory properties, NO is antiatherogenic: it inhibits platelet aggregation/adhesion, smooth muscle cell proliferation and lipid oxidation. Depletion of NO has been linked to several pathologies including atherosclerosis and hypertension. Understanding the mechanistic aspects of NO bioavailability, and loss thereof with ageing, has significant clinical relevance. Synthesizing NO requires a precise admixture of substrate and cofactors. Unidirectional laminar shear stress activates endothelial nitric oxide synthase (eNOS) via phosphorylation. l-Arginine is hydroxylated to N-hydroxy-l-arginine and then further oxidized to NO and l-citrulline. NO diffuses into smooth muscle cells, activates guanylate cyclase and induces cyclic GMP-mediated smooth muscle relaxation. Other substrate and cofactors required for this reaction include oxygen, NADPH, flavin, heme and tetrahydrobiopterin (BH4). Altering any one of these variables with ageing may set the reaction awry and attenuate vasodilatation. In an article recently published in The Journal of Physiology, Delp and associates examined several potential mechanisms that may contribute to reduced microvascular vasodilatory capacity with ageing (Delp et al. 2008). Delp et al. harvested arterioles from the soleus muscles of young and old rats and exposed them to graded increases in intraluminal flow in the absence of changes in intraluminal pressure. Results revealed that arterioles from old rats exhibited a 52% reduction in flow-mediated dilatation (FMD) compared to arterioles from young rats. Vessel dilatation was also assessed following administration of the NO donor sodium nitropruside (SNP). There were no group differences in SNP-mediated dilatation suggesting similar endothelial-independent vasodilatation in arterioles from young and old rats. Confirming that the age-associated difference in microvascular vasodilatation was endothelial dependent, Delp and associates proceeded to systematically and eloquently rule out several potential limiting factors regulating the NO signalling pathway.

Peroxiredoxin 1 is Upregulated by Laminar Shear Stress via Nrf2 Transcription Factor

Endothelial cells (EC) exposed to oscillatory shear stress (OS) experience oxidative stress as a signature of atherosclerosis. Conversely, unidirectional laminar shear stress (LS) reduces reactive oxygen species (ROS) production and subsequent inflammatory responses. LS induces many protective genes via a shear‐sensitive antioxidant response element (ARE). We showed that the antioxidant enzyme peroxiredoxin 1 (PRX 1) is upregulated intracellularly by chronic LS, compared to OS and static (ST) culture. In addition, PRX 1‐knockdown attenuated LS‐dependent ROS reduction in EC. We hypothesize that shear‐dependent PRX 1 expression is regulated by ARE through the transcription factor nuclear factor (erythroid‐derived 2)‐related factor 2 (Nrf2). Quantitative, real‐time PCR indicated that chronic LS increased PRX 1 mRNA, compared to OS and ST. Immunoblots also showed increased PRX 1 protein expression in response to Nrf2 adenovirus infection of EC. These results indicate that shear‐dependent PRX 1 expression is regulated at the transcriptional level, and Nrf2 may play an important role in this mechanism. Through this pathway, PRX 1 acts as a mechanosensitive antioxidant implementing anti‐inflammatory and anti‐atherogenic effects of shear stress.Supported by NIH HL71014, HL75209, and HL70531 (HJ), Korean Ministry of Science & Technology FPR05C2‐510 (SWK), and AHA pre‐doctoral fellowship (ALM).

KLF‐2‐dependent, shear stress‐induced expression of CD59: a novel cytoprotective mechanism against complement‐mediated injury in the vasculature

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress (LSS) led us to explore its effects on endothelial cell (EC) expression of complement inhibitory proteins CD59 and decay‐accelerating factor (DAF). Human umbilical vein and aortic EC were exposed to LSS (12 dynes/cm2), or disturbed flow (DF) (+/−5 dynes/cm2at 1Hz), in a parallel‐plate flow chamber. LSS induced a flow rate‐dependent increase in steady‐state CD59 mRNA, reaching 4‐fold at 12 dynes/cm2(p<0.01). Following 24–48h of LSS, cell surface CD59 was upregulated by 100% (p<0.01), while DAF expression was unchanged. The increase in CD59 following LSS was functionally significant, reducing C9 deposition and complement‐mediated lysis of flow‐conditioned EC by 50% (p<0.05). While CD59 induction was independent of PI‐3K, ERK1/2 and nitric oxide, an siRNA approach demonstrated dependence upon an ERK5/KLF2 signalling pathway. In contrast to LSS, DF failed to induce EC CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional LSS, when compared to atheroprone areas exposed to DF (p<0.05). We propose that upregulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement‐mediated injury, and protects from atherogenesis in regions of LSS.

Gene Expression in Endothelial Cells and Intimal Smooth Muscle Cells in Atherosclerosis-Prone or Atherosclerosis-Resistant Regions of the Human Aorta

Background/Aims: We compared the atherogenic gene expression in the intimas of atherosclerosis-prone regions (proximal walls), which are exposed to disturbed shear stress, and atherosclerosis-resistant regions (apices), which are exposed to unidirectional laminar shear stress, at the orifices of the intercostal arteries of human aortas. Methods and Results: Expression of mRNAs, detected by in situ RT-PCR, for IL-1β, TNF-α, VCAM-1, PAF receptor and GRP in endothelial cells (ECs), and of PDGF receptor β (PDGFR-β), MCP-1, GRP and collagen type-1 by smooth muscle cells (SMCs) in the proximal walls, was significantly enhanced, while seldom observed in the elastic-hyperplastic layer of the apices. Protein expression of PDGFR-β, IL-1β and TNF-α was also observed on the proximal walls. SMC growth in the apices was inhibited. Cultured SMC growth and their expression of PDGFR-β were also significantly inhibited by elastin. Conclusion: These results suggest that the construction of the elastic-hyperplastic layer and the subsequent inhibition of SMC growth by elastin, with stabilized ECs under unidirectional laminar shear stress, resulted in atherosclerosis-resistant regions at the apices of human aortas, and that the continuous induction of atherogenic gene expression by ECs activated by disturbed shear stress inhibits the formation of atherosclerosis-resistant intima along the proximal walls.

NFKB1 promoter variation implicates shear-induced NOS3 gene expression and endothelial function in prehypertensives and stage I hypertensives

In endothelial cells, NF-kappaB is an important intracellular signaling molecule by which changes in wall shear stress are transduced into the nucleus to initiate downstream endothelial nitric oxide synthase (NOS3) gene expression. We investigated whether NF-kappa light-chain gene enhancer in B cells 1 (NFKB1) promoter polymorphism ((-94)NFKB1 I/D, where I is the insertion allele and D is the deletion allele) was associated with 1) NOS3 gene expression in endothelial cells under physiological levels of unidirectional laminar shear stress (LSS) and 2) endothelial function in prehypertensive and stage I hypertensive individuals before and after a 6-mo supervised endurance exercise intervention. Competitive EMSAs revealed that proteins present in the nuclei of endothelial cells preferentially bound to the I allele NFKB1 promoter compared with the D allele. Reporter gene assays showed that the I allele promoter had significantly higher activity than the D allele. In agreement with these observations, homozygous II genotype cells had higher p50 expression levels than homozygous DD genotype cells. Cells with the homozygous II genotype showed a greater increase in NOS3 protein expression than did homozygous DD genotype cells under LSS. Functional experiments on volunteers confirmed higher baseline reactive hyperemic forearm blood flow, and, furthermore, the subgroup analysis revealed that DD homozygotes were significantly less prevalent in the exercise responder group compared with II and ID genotypes. We conclude that the (-94)NFKB1 I/D promoter variation contributes to the modulation of vascular function and adaptability to exercise-induced flow shear stress, most likely due to differences in NFKB1 gene transactivity.