Shear Stress Response Element Research Articles

Shear stress switches the association of endothelial enhancers from ETV/ETS to KLF transcription factor binding sites

Endothelial cells (ECs) lining blood vessels are exposed to mechanical forces, such as shear stress. These forces control many aspects of EC biology, including vascular tone, cell migration and proliferation. Despite a good understanding of the genes responding to shear stress, our insight into the transcriptional regulation of these genes is much more limited. Here, we set out to study alterations in the chromatin landscape of human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress. To do so, we performed ChIP-Seq for H3K27 acetylation, indicative of active enhancer elements and ATAC-Seq to mark regions of open chromatin in addition to RNA-Seq on HUVEC exposed to 6 h of laminar shear stress. Our results show a correlation of gained and lost enhancers with up and downregulated genes, respectively. DNA motif analysis revealed an over-representation of KLF transcription factor (TF) binding sites in gained enhancers, while lost enhancers contained more ETV/ETS motifs. We validated a subset of flow responsive enhancers using luciferase-based reporter constructs and CRISPR-Cas9 mediated genome editing. Lastly, we characterized the shear stress response in ECs of zebrafish embryos using RNA-Seq. Our results lay the groundwork for the exploration of shear stress responsive elements in controlling EC biology.

In Vivo Function of Flow-Responsive Cis-DNA Elements of eNOS Gene: A Role for Chromatin-Based Mechanisms.

eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Wild type mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.

Identification of atheroprone shear stress responsive regulatory elements in endothelial cells.

Oscillatory shear stress (OSS) is an atheroprone haemodynamic force that occurs in areas of vessel irregularities and is implicated in the pathogenesis of atherosclerosis. Changes in signalling and transcriptional programme in response to OSS have been vigorously studied; however, the underlying changes in the chromatin landscape controlling transcription remain to be elucidated. Here, we investigated the changes in the regulatory element (RE) landscape of endothelial cells under atheroprone OSS conditions in an in vitro model. Analyses of H3K27ac chromatin immunoprecipitation-Seq enrichment and RNA-Seq in primary human umbilical vein endothelial cells 6 h after onset of OSS identified 2806 differential responsive REs and 33 differentially expressed genes compared with control cells kept under static conditions. Furthermore, gene ontology analyses of putative RE-associated genes uncovered enrichment of WNT/HIPPO pathway and cytoskeleton reorganization signatures. Transcription factor (TF) binding motif analysis within RE sequences identified over-representation of ETS, Zinc finger, and activator protein 1 TF families that regulate cell cycle, proliferation, and apoptosis, implicating them in the development of atherosclerosis. Importantly, we confirmed the activation of EGR1 as well as the YAP/TAZ complex early (6 h) after onset of OSS in both cultured human vein and artery endothelial cells and, by undertaking luciferase assays, functionally verified their role in RE activation in response to OSS. Based on the identification and verification of specific responsive REs early upon OSS exposure, we propose an expanded mechanism of how OSS might contribute to the development of atherosclerosis.

Strain-induced mechanotransduction through primary cilia, extracellular ATP, purinergic calcium signaling, and ERK1/2 transactivates CITED2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes

To determine the strain-induced signaling pathways involved in regulating the transactivation of the transcription regulator Cbp/p300 Interacting Transactivator with ED-rich tail 2 (CITED2) and downstream targets in chondrocytes. Primary human chondrocytes or C28/I2 chondrocytic cells were subjected to various strain regimes. C57BL/6 mice were subjected to treadmill running. Loss-of-function was carried out using siRNA or inhibitors specific for targeted molecules. mRNA levels were assayed by RT-qPCR, and proteins by western blotting, immunofluorescence, and/or immunohistochemical staining. CITED2 promoter activity was assayed in chondrocytes using wild-type or mutant constructs. Cyclic strain at 5%, 1 Hz induced CITED2 expression and suppressed expression of matrix metalloproteinase (MMP)-1 and -13 at the messenger RNA (mRNA) and protein levels in human chondrocytes. Abolishing primary cilia through knockdown of intraflagellar transport protein (IFT88) attenuated CITED2 gene expression and decreased protein levels. Similar effects were observed with inhibitors of extracellular adenosine triphosphate (ATP) or P2 purinergic receptors, or antagonists of Ca(2+) signaling. Knockdown of IFT88 in articular chondrocytes in vivo diminished treadmill induced-CITED2 expression and upregulated MMPs. Knockdown of hypoxia-inducible factor (HIF)1α, specificity protein 1 (Sp1), or deletion of the shear stress response element (SSRE) in the CITED2 promoter limited cyclic strain-induced transactivation of CITED2. However, the strain induced-transactivation of CITED2 was abolished only on knockdown of HIF1α, Sp1, and SSRE or by loss-of-function of IFT88 or extracellular-signal-regulated kinases (ERK)1/2. CITED2 transactivation is a critical event in signaling generated by strain and transduced by primary cilia, extracellular ATP, P2 purinergic receptors, and Ca(2+) signaling. Strain-induced CITED2 transactivation requires HIF1α, Sp1, and an intact SSRE and leads to the downregulation of MMPs such as MMP-1 and MMP-13.

Growing Collateral Arteries On Demand

Recent studies have significantly advanced our understanding of arteriogenesis, raising hope that therapies to increase collateral arterial formation may become important new tools in the treatment of ischemic disease. The most important initiating trigger for arteriogenesis is the marked increase in shear stress which is sensed by the endothelium and leads to characteristic changes. Intracellularly, it was shown that platelet endothelial cell adhesion molecule (PECAM-1) becomes tyrosine-phosphorylated in response to increased shear stress, suggesting a role as a possible mechanoreceptor for dynamic and continual monitoring of shear stress. The signal generated by PECAM-1 leads to the activation of the Rho pathway among others. More than 40 genes have been shown to have a shear stress responsive element. The Rho pathway is activated early and appears to be essential to the arteriogenic response as inhibiting it abolished the effect of fluid shear stress. Overexpression of a Rho pathway member, Actin-binding Rho protein (Abra), led to a 60% increase in collateral perfusion over simple femoral artery occlusion. A patent for the Abra gene has been filed recently. It may be a harbinger of a future where collateral arteries grown on demand may become an effective treatment for ischemic vascular disease.

Interplay of CCR2 signaling and local shear force determines vein graft neointimal hyperplasia in vivo

Leukocytes play a central role in vein graft neointimal hyperplasia, which is significantly augmented under low shear conditions. The current concept is that shear force regulates leukocyte adhesion predominately through up-regulation of chemokines and growth factors within the graft wall. Using rabbit and murine vein graft models, we demonstrate that CC chemokine receptor 2/monocyte chemoattractant protein-1 mediated monocyte recruitment and a low shear environment act synergistically to augment neointimal hyperplasia development and removal of either of the conditions leads to a significant reduction in neointimal thickening. We propose a novel concept that the shear stress response element phenotypically stems from the complex interplay of the biological and physical microenvironments.

Shear stress-induced transcriptional regulation via hybrid promoters as a potential tool for promoting angiogenesis

Among the key effects of fluid shear stress on vascular endothelial cells is modulation of gene expression. Promoter sequences termed shear stress response elements (SSREs) mediate the responsiveness of endothelial genes to shear stress. While previous studies showed that shear stress responsiveness is mediated by a single SSRE, these endogenous promoters often encode for multiple SSREs. Moreover, hybrid promoters encoding a single SSRE rarely respond to shear stress at the same magnitude as the endogenous promoter. Thus, to better understand the interplay between the various SSREs, and between SSREs and endothelial-specific sequences (ESS), we generated a series of constructs regulated by SSREs cassettes alone, or in combination with ESS, and tested their response to shear stress and endothelial specific expression. Among these constructs, the most responsive promoter (NR1/2) encoded a combination of two GAGACC/SSREs, the Sp1/Egr1 sequence, as well as a TPA response element (TRE). This construct was four- to five-fold more responsive to shear stress than a promoter encoding a single SSRE. The expression of constructs containing other SSRE combinations was unaffected or suppressed by shear stress. Addition of ESS derived from the Tie2 promoter, either 5' or 3' to NR1/2 resulted in shear stress transcriptional suppression, yet retained endothelial specific expression. Thus, the combination and localization order of the various SSREs in a single promoter is crucial in determining the pattern and degree of shear stress responsiveness. These shear stress responsive cassettes may prove beneficial in our attempt to time the expression of an endothelial transgene in the vasculature.

Regulation of human bone sialoprotein gene transcription by platelet-derived growth factor-BB

Platelet-derived growth factor (PDGF) is produced by mesenchymal cells and released by platelets following aggregation and is synthesized by osteoblasts. In bone, PDGF stimulates proliferation and differentiation of osteoblasts. PDGF also increases bone resorption, most likely by increasing the number of osteoclasts. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone, selectively expressed by differentiated osteoblast. To determine the molecular mechanisms PDGF regulation of human BSP gene transcription, we have analyzed the effects of PDGF-BB on osteoblast-like Saos2 and ROS17/2.8 cells. PDGF-BB (5 ng/ml) increased BSP mRNA and protein levels at 12 h in Saos2 cells, and induced BSP mRNA expression at 3 h, reached maximal at 12 h in ROS17/2.8 cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of Saos2 cells with PDGF-BB (5 ng/ml, 12 h) increased luciferase activities of all constructs between – 184LUC to – 2672LUC including the human BSP gene promoter. Effects of PDGF-BB abrogated in constructs included 2 bp mutations in the two cAMP response elements (CRE1 and CRE2), activator protein 1(3) (AP1(3)) and shear stress response element 1 (SSRE1). Luciferase activities induced by PDGF-BB were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel mobility shift analyses showed that PDGF-BB increased binding of CRE1, CRE2, AP1(3) and SSRE1 elements. CRE1- and CRE2-protein complexes were supershifted by CREB1 and phospho-CREB1 antibodies. Notably, AP1(3)-protein complexes were supershifted by c-Fos and JunD, and disrupted by CREB1, phospho-CREB1, c-Jun and Fra2 antibodies. These studies, therefore, demonstrate that PDGF-BB stimulates human BSP transcription by targeting the CRE1, CRE2, AP1(3) and SSRE1 elements in the human BSP gene promoter.

Fluid shear stress induces arterial differentiation of endothelial progenitor cells

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood and contribute to angiogenesis in tissues. In the process, EPCs are exposed to the shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress promotes differentiation of EPCs into mature endothelial cells. In this study, we investigated whether EPCs differentiate into arterial or venous endothelial cells in response to shear stress. When cultured EPCs derived from human peripheral blood were exposed to controlled levels of shear stress in a flow-loading device, the mRNA levels of the arterial endothelial cell markers ephrinB2, Notch1/3, Hey1/2, and activin receptor-like kinase 1 increased, but the mRNA levels of the venous endothelial cell markers EphB4 and neuropilin-2 decreased. Both the ephrinB2 increase and the EphB4 decrease were shear stress dependent rather than shear rate dependent. EphrinB2 protein was increased in shear-stressed EPCs, and the increase in ephrinB2 expression was due to activated transcription and not mRNA stabilization. Deletion analysis of the ephrinB2 promoter indicated that the cis-element (shear stress response element) is present within 106 bp 5' upstream from the transcription initiation site. This region contains the Sp1 consensus sequence, and a mutation in its sequence decreased the basal level of transcription and abolished shear stress-induced ephrinB2 transcription. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that shear stress markedly increased binding of Sp1 to its consensus sequence. These results indicate that shear stress induces differentiation of EPCs into arterial endothelial cells by increasing ephrinB2 expression in EPCs through Sp1 activation.

Influence of a GT repeat element on shear stress responsiveness of the VWF gene promoter.

Plasma von Willebrand factor (VWF) is mainly derived from endothelial cells, cells that express a large repertoire of genes that are transcriptionally regulated by fluid shear stress. Endothelial VWF expression is not uniform throughout the vasculature, and levels are increased at regions associated with disturbed blood flow and steep gradients of shear stress. It is, however, unknown whether shear stress influences the regulation of VWF gene expression. Our objective was to evaluate the effect of shear stress on endogenous endothelial VWF mRNA expression and VWF promoter (-2722 to -1224) activity and to determine whether genetic elements modulate this flow-induced expression. A parallel plate flow chamber was used to expose endothelial cells to a shear level of 15 dynes cm(-2) for 24 or 6 h. VWF mRNA expression was analyzed. Various VWF promoter constructs that each contain either SNP haplotypes 1 or 2 and either a 17-GT or a 23-GT repeat element were transfected into endothelial cells, and flow-induced promoter activation was assessed. When endothelial cells were exposed to shear stress, endogenous VWF mRNA expression increased 1.84-fold and average VWF promoter activity was enhanced 3.4-fold. Single nucleotide polymorphisms at -2708 and -2525, and the shear stress-response element at -1585, are not responsible for the shear stress-induced increase. Rather a GT repeat element at -2124 mediates the increase in activity, and the length of this polymorphic repeat element influences the magnitude of induction. Shear stress enhances VWF promoter activity and a polymorphic GT repeat element mediates the stress-induced transactivation.

Activation of p300 Histone Acetyltransferase Activity Is an Early Endothelial Response to Laminar Shear Stress and Is Essential for Stimulation of Endothelial Nitric-oxide Synthase mRNA Transcription

Previous studies have shown that the acute stimulation of endothelial nitric-oxide synthase (eNOS) mRNA transcription by laminar shear stress is dependent on nuclear factor κ B (NFκB) subunits p50 and p65 binding to a shear stress response element (SSRE) in the human eNOS promoter and that mutation of the SSRE abrogates the shear-stimulated increase in eNOS promoter activity. In the present study, we found that although shear markedly increased eNOS mRNA, the increase in nuclear translocation of p50 and p65 caused by shear was only 2-fold, suggesting that shear has additional effects on NFκB cofactor activity beyond nuclear translocation. Chromatin immunoprecipitation assays showed that virtually no p50 or p65 was bound to the eNOS promoter at base line but that shear increased the binding of these subunits to the human eNOS SSRE by 10- to 20-fold. Co-immunoprecipitation studies demonstrated during the first 30 min of shear p300 bound to p65. Shear also increased p300 histone acetyltransferase (HAT) activity by 2.5-fold and increased acetylation of p65. The increase in eNOS mRNA caused by shear was completely blocked by pharmacological inhibition of p300/HAT activity with curcumin or by p300 small interfering RNA. Chromatin immunoprecipitation assays also showed that shear stimulated acetylation of histones 3 and 4 at the region of the eNOS promoter SSRE and extended 3′ toward the eNOS coding region. This was associated with opening of chromatin at the SSRE. In conclusion, these studies reveal a previously unknown role of p300/HAT activation as a very early response to shear that is essential for increasing eNOS mRNA levels.

Phosphorothioate oligonucleotide inhibits tissue factor expression in endothelial cells induced by blood flow shear stress in rats

Objective To determine the effect of antiparallel phosphorothioate triplex-forming oligonucleotide (apsTFO), which was designed according to shear stress response element (SSRE) in tissue factor (TF) gene promoter region, on the expression of endothelial TF in carotid artery stenosis rats. Methods Rat model of severe carotid artery stenosis were inflicted by silica gel tube ligation. Half an hour before the model infliction, GT20-apsTFO, GT20-psTFO and GT21-apsTFO labeled with green fluorescence (FITC) were injected into the vena caudalis of rat at a dose of 0.5 mg/kg. Half an hour, 4 or 9 h after the ligation, the distribution of TFO in the common carotid artery, the liver and the kidney was detected with aid of fluorescence microscopy. And the mRNA and protein expressions of TF, Egr-1 and Sp1 in the above-mentioned organs were determined with i n situ hybridization and immunohistochemical assay respectively in 6 h after the model establishment, and the results were analyzed with an image analysis system. Results Only in 1 h after TFO injection, fluorescent granules appeared in the liver, the kidney and the vascular wall and lumen of carotid artery, and then in 4.5 h, they still deposited in above sites except the vascular lumen. GT20-apsTFO and GT21-apsTFO significant down-regulated the mRNA and protein expressions of TF compared to the rats without treatment ( P<0.05), and the former apsTFO had a more stronger effect than the later ( P<0.05). GT20-psTFO had no such effect ( P>0.05). The 3 TFOs had no inhibition on the mRNA and protein expressions of Egr-1 and Sp1. Conclusion Pretreated apsTFO can partly come into the vascular endothelial cells, and inhibit TF expression induced by shear stress, but had no effect on Egr-1 and Sp1 gene expressions.

Physics Meets Molecules

See related article, pages 538–545 Arteries and veins are permanently exposed to hemodynamic forces because of the pulsatile nature of blood pressure and flow. Hence, the endothelium is constantly detecting different biomechanical forces, cyclic stretch and shear stress in particular,1–2 and converts the latter stimuli into intra- and extracellular signals. Endothelial cells thereby modulate multiple of physiological and pathophysiological processes: production of growth-promoting and growth-inhibiting hormones, enzymes, cytokines, etc; mediation of inflammatory responses through the expression of chemotactic and adhesion molecules on the endothelial surface; modulation of hemostasis and thrombosis via secretion of procoagulant, anticoagulant, and fibrinolytic agents; and the regulation of vascular smooth muscle cell contraction through the release of vasodilators and vasoconstrictors.3–5 This being the case, the equilibrium between physiological levels of blood flow (shear stress) and the endothelium is tightly counterbalanced. Thereby, the lumen radius of an artery is the most important denominator, which signifies that the smaller the lumen the higher the shear stress. However, once physiological shear forces are reduced, several pathological conditions may arise: proatherogenic and/or prothrombotic states and hence atherosclerosis and/or thrombosis.6–7 Inversely, high levels of shear forces play a key role in adaptive phases of arteriogenesis (collateral artery growth), the most clinically relevant mechanism of vascular development.8 In case of an arterial stenosis, these arterial/arteriolar anastomoses are the only anastomosis to the low-pressure territory and perfuse the periphery with nutrient blood flow. Elevation of shear stress and concomitant cyclic stretch are currently discussed to be the strongest inducers of arteriogenesis. In a rabbit model of femoral artery ligation, Schaper and colleagues induced an abrupt increase in shear stress by means of an arterial–venous shunt (pressure drop across collateral arteries) and accelerated the speed of arteriogenesis, surpassing the conductance values of the occluded artery when shear …

Atheroprone shear‐stress upregulates LOX‐1 expression in endothelial cells

Distribution of atherosclerosis suggests that the local environment of susceptible sites harbors the factors for atherogenesis. Geometry of brachpoints, ostia, and curvatures, where atheromas usually develop, implies a role for disturbed flow. It is not clear what specific alterations are caused by disturbed shear-stress in the susceptible sites that are pro-atherogenic. We hypothesized that disturbed flow leads to increase in expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells, since the promoter region of its gene was shown to contain two shear-stress response elements (Aoyama, Biochem. J. 339:177, 1999). Endothelial cells in artificial capillary tubes were subjected to atheroprone, oscillatory flow using a computer controlled pump for 24 h. Atherogenic flow pulse simulating carotid bifurcation shear-stress pattern (Dai, PNAS 101:14871, 2004) was generated by the DasyLab software and transmitted to the pump using the analog output of an Iotech data acquisition and control card. Disturbed flow resulted in significant upregulation of LOX-1 protein expression compared with atheroprotective flow as determined by Western blot analysis. Increased expression of LOX-1 under disturbed flow might lead to increased uptake of oxidized LDL and thus facilitate atherogenesis.

Molecular mechanisms of the vascular responses to haemodynamic forces

Blood vessels are permanently subjected to mechanical forces in the form of stretch, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow and shear stress. Significant variations in mechanical forces, of physiological or physiopathological nature, occur in vivo. These are accompanied by phenotypical modulation of smooth muscle cells and endothelial cells, producing structural modifications of the arterial wall. In all the cases, vascular remodelling can be allotted to a modification of the tensional strain or shear, and underlie a trend to reestablish baseline mechanical conditions. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by pressure and shear stress. The cytoskeleton and other structural components have an established role in mechanotransduction, being able to transmit and modulate tension within the cell via focal adhesion sites, integrins, cellular junctions and the extracellular matrix. Mechanical forces also initiate complex signal transduction cascades, including nuclear factor-kappaB and mitogen-activated protein kinase pathways, leading to functional changes within the cell.

Activator Protein-1 Mediates Shear Stress–Induced Prostaglandin D Synthase Gene Expression in Vascular Endothelial Cells

We attempted to determine the molecular mechanism of fluid shear stress-induced lipocalin-type prostaglandin D synthase (l-PGDS) expression in vascular endothelial cells. We examined the promoter region of the l-PGDS gene by loading laminar shear stress (20 dyne/cm2), using a parallel-plate flow chamber, on endothelial cells transfected with luciferase reporter vectors containing the 5'-flanking regions of the human l-PGDS gene. A deletion mutant analysis revealed that a shear stress-responsive element resided in the region between -2607 and -2523 bp. A mutation introduced into the putative binding site for activator protein-1 (AP-1) within this region eliminated the response to shear stress. In an electrophoretic mobility shift assay, shear stress stimulated nuclear protein binding to the AP-1 binding site, which was supershifted by antibodies to c-Fos and c-Jun. Shear stress elevated the c-Jun phosphorylation level in a time-dependent manner, similar to that of l-PGDS gene expression. SP600125, a c-Jun N-terminal kinase inhibitor, decreased the c-Jun phosphorylation, DNA binding of AP-1, and l-PGDS expression induced by shear stress. Additionally, an mRNA chase experiment using actinomycin D demonstrated that shear stress did not stabilize l-PGDS mRNA. Shear stress induces l-PGDS expression by transcriptional activation through the AP-1 binding site.

Molecular regulation of the brain natriuretic peptide gene

After brain natriuretic peptide (BNP) was isolated in 1988, rapid progress was made in cloning its cDNA and gene, facilitating studies of tissue-specific expression and molecular regulation of gene expression. This review focuses on the molecular determinants of regulation of the rat and human BNP genes, including signaling pathways that impact on changes in gene expression and cis regulatory elements responsive to these signaling pathways. For both rat and human genes, elements in the proximal promoter (−124 to −80), including GATA, MCAT, and AP-1-like, have been shown to contribute to basal and inducible regulation. More distal elements in the human BNP gene respond to calcium signals (an NF-AT site at −927), thyroid hormone (a thyroid-responsive element at −1000), and mechanical stretch (shear stress-responsive elements at −652 and −162). Understanding how BNP is regulated by signaling molecules that are activated in the hypertrophied and ischemic heart should be useful in understanding the underlying pathology. This may lead to therapeutic strategies that prevent hypertrophy while allowing for the beneficial effects of BNP production.

Effects of turbulent flow on cell adhesion molecule expression in endothelial cells

Introduction: Turbulent flow induces atherosclerosis and neointimal hyperplasia to a greater degree than arterial levels of laminar flow. Little is known regarding the effects of turbulent flow on endothelial cells (EC), including the regulation of cell adhesion molecule expression. We have previously observed that orbital flow yields two populations of cells, in the center and periphery of the culture well, corresponding to turbulent and laminar components, respectively, and suggesting the utility of this model to study differential effects of flow types. Since expression of some EC cell adhesion molecules, ICAM, are regulated by the Shear Stress Response Element (SSRE), whereas others, E-selectin, are not, we hypothesized that laminar and turbulent flow would differentially affect EC E-selectin and ICAM expression. Methods: Human umbilical vein EC were differentially seeded in either the center or periphery of culture wells. Cells were treated with or without TNF-alpha (10ng/ml) and orbital flow (210rpm) for 6 hours. E-selectin and ICAM expression were analyzed with FACS. Results: EC exposed only to orbital flow demonstrated an up-regulation of ICAM expression, despite the area of seeding. However, TNF induced E-selectin expression was down-regulated in the periphery, whereas in the center, TNF induced E-selectin expression was similar to those cells exposed to TNF but no flow (n = 3, p < 0.0001, ANOVA). Conclusions: ICAM expression is stimulated by both laminar and turbulent flow, whereas E-selectin expression is downregulated by laminar, but not turbulent flow. This suggests that laminar and turbulent flow may differentially regulate EC function both through SSRE-dependent and independent pathways.

Identification of two novel shear stress responsive elements in rat angiotensin I converting enzyme promoter.

Mechanical forces contribute to maintenance of cardiovascular homeostasis via the control of release and production of vasoactive substances. We demonstrated previously that shear stress decreases rat ACE activity and expression. Using a reporter gene approach and mutagenesis, we show now that the classic shear stress responsive element or SSRE (GAGACC) contained within 1,274 bp of this promoter is not functional in response to shear stress (15 dyn/cm2, 18 h) [for the wild-type ACE promoter (WLuc), static control (C) = 107 +/- 6.5%, shear stress (SS) = 65.9 +/- 9.4%, n = 8; for the promoter with the classic SSRE mutated (WSS-mut), C = 100 +/- 8.2%, SS = 60.2 +/- 5.2%, n = 10, respectively]. Analysis of progressive deletion mutants unraveled a 57-bp fragment, position -251 to -195, from the transcription start site, containing functional SSRE (for WLuc, C = 107 +/- 6.5%, SS = 65.9 +/- 9.4%, n = 8; for 378, C = 100 +/- 6.4%, SS = 60.4 +/- 4.3%, n = 11; for 251, C = 99.7 +/- 2.6%, SS = 63.2 +/- 5.5%, n = 7; for 194, C = 104.6 +/- 8.1%, SS = 92.4 +/- 6.9%, n = 9). This fragment responded to shear stress even in the context of a heterologous promoter. Finally, functional analysis of mutated candidate regulatory elements identified by gel shift, DNase I footprint, and conservation of aligned sequences revealed that only the double mutant (Barbie/GAGA-mut) but not isolated disruption of the Barbie (WBarbie-mut) or the GAGA (WGAGA-mut) prevented the shear-stress-induced response (for Barbie/GAGA-mut, C = 97.9 +/- 5%, SS = 99.4 +/- 7.2%, n = 6; for WBarbie-mut, C = 106.1 +/- 8.6%, SS = 65.9 +/- 9.4%, n = 6; for WGAGA-mut, C = 100.1 +/- 2.9%, SS = 66.7 +/- 1.6, n = 6;). Taken together, these data provide direct evidence for the new role of Barbie and GAGA boxes in mediating the shear-stress-induced downregulation of rat ACE expression and demonstrate that the classic SSRE (GAGACC) is not functional under the experimental conditions tested.

The Shear Stress Response Element of Endothelium and the expression of TF Gene(Cardiovascular Mechanics)

The Shear Stress Response Element of Endothelium and the expression of TF Gene(Cardiovascular Mechanics)