mTOR contributes to endothelium-dependent vasorelaxation by promoting eNOS expression and preventing eNOS uncoupling.

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.

Epoxyeicosatrienoic acid (EET)-stimulated angiogenesis is mediated by epoxy hydroxyeicosatrienoic acids (EHETs) formed from COX-2.

Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs promote angiogenesis linked to tumor growth in various cancer models that is attenuated in vivo by cyclooxygenase 2 (COX-2) inhibitors. This study further defines a role for COX-2 in mediating endothelial EET metabolism promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential in the presence and absence of a COX-2 inducer [phorbol 12,13-dibutyrate (PDBu)]. The angiogenic response to 8,9-EET in the presence of PDBu was 3-fold that elicited by 8,9-EET stabilized with a soluble epoxide hydrolase inhibitor (t-TUCB). Contributing to this response was the COX-2 metabolite of 8,9-EET, the 11-hydroxy-8,9-EET (8,9,11-EHET), which exogenously enhanced angiogenic responses in HAECs at levels comparable to those elicited by vascular endothelial growth factor (VEGF). In contrast, the 15-hydroxy-8,9-EET isomer was also formed but inactive. The 8,9,11-EHET also promoted expression of the VEGF family of tyrosine kinase receptors. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in the endothelium through the formation of 8,9,11-EHET. This alternative pathway for the metabolism of 8,9-EET may be particularly important in regulating angiogenesis under circumstances in which COX-2 is induced, such as in cancer tumor growth and inflammation.

Oxylipins in triglyceride-rich lipoproteins of dyslipidemic subjects promote endothelial inflammation following a high fat meal.

Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype.

Downregulation of GATA6 in mTOR-inhibited human aortic endothelial cells: effects on TNF-α-induced VCAM-1 expression and monocytic cell adhesion.

Increased expression of vascular cell adhesion molecule 1 (VCAM-1) on the aortic endothelium is an early marker of atherogenesis, promoted in part by elevated levels of inflammatory cytokines such as TNF-α. Mammalian target of rapamycin (mTOR) is a ubiquitous signaling molecule that has been considered to contribute to diverse cellular processes through mTOR complex 1 (mTORC1) or complex 2 (mTORC2). This study aimed to elucidate the role of mTOR signaling in TNF-α-induced VCAM-1 expression by the arterial endothelium. Primary human aortic endothelial cells (HAECs) were treated with low-dose (0.1 ng/ml) TNF-α, and VCAM-1 expression was measured by real-time quantitative PCR, Western blot analysis, and flow cytometry. Inhibition of mTOR through siRNA-mediated depletion or treatment with chemical inhibitors rapamycin or torin 1 suppressed VCAM1 transcription, which translated to inhibition of VCAM-1 surface expression by HAECs and concomitant decreased adhesion of monocytes. A promoter luciferase assay and chromatin immunoprecipitation indicated that mTOR regulated VCAM1 transcription through a mechanism involving transcription factor GATA6. Activation of PKC-α and an increase in miR-200a-3p expression, caused by mTOR inhibition but not disruption of mTORC1 or mTORC2 singly or together, decreased TNF-α-induced GATA6 expression and its enrichment at the VCAM1 promoter. In conclusion, mTOR inhibition activates PKC-α independently of disruption of mTORC1 and/or mTORC2, which challenges the conventional wisdom regarding mTOR signaling. Moreover, mTOR signals through transcriptional and posttranscriptional mechanisms to elicit maximal cytokine-induced endothelial inflammation that precedes atherosclerosis. NEW & NOTEWORTHY Both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 contribute to PKC-α activation in the human aortic endothelium. Inhibition of mTOR is not equivalent to disruption of mTORC1 and/or mTORC2 in affecting human aortic endothelial cell signaling. Specifically, inhibition of mTOR causes PKC-α activation and miR-200a-3p upregulation, which independently suppresses TNF-α-induced transcription factor GATA6 expression and subsequently inhibits VCAM-1 expression and monocytic cell adhesion onto the aortic endothelium.

Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation.

Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0-16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα.

Triglyceride-rich lipoprotein modulates endothelial vascular cell adhesion molecule (VCAM)-1 expression via differential regulation of endoplasmic reticulum stress.

Circulating triglyceride-rich lipoproteins (TGRL) from hypertriglyceridemic subjects exacerbate endothelial inflammation and promote monocyte infiltration into the arterial wall. We have recently reported that TGRL isolated from human blood after a high-fat meal can elicit a pro- or anti-atherogenic state in human aortic endothelial cells (HAEC), defined as up- or down-regulation of VCAM-1 expression in response to tumor necrosis factor alpha (TNFα) stimulation, respectively. A direct correlation was found between subjects categorized at higher risk for cardiovascular disease based upon serum triglycerides and postprandial production of TGRL particles that increased VCAM-1-dependent monocyte adhesion to inflamed endothelium. To establish how TGRL metabolism is linked to VCAM-1 regulation, we examined endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) pathways. Regardless of its atherogenicity, the rate and extent of TGRL internalization and lipid droplet formation by HAEC were uniform. However, pro-atherogenic TGRL exacerbated ER membrane expansion and stress following TNFα stimulation, whereas anti-atherogenic TGRL ameliorated such effects. Inhibition of ER stress with a chemical chaperone 4-phenylbutyric acid decreased TNFα-induced VCAM-1 expression and abrogated TGRL's atherogenic effect. Activation of ER stress sensors PKR-like ER-regulated kinase (PERK) and inositol requiring protein 1α (IRE1α), and downstream effectors including eukaryotic initiation factor-2α (eIF2α), spliced X-box-binding protein 1 (sXBP1) and C/EBP homologous protein (CHOP), directly correlated with the atherogenic activity of an individual's TGRL. Modulation of ER stress sensors also correlated with changes in expression of interferon regulatory factor 1 (IRF-1), a transcription factor of Vcam-1 responsible for regulation of its expression. Moreover, knockdown studies using siRNA defined a causal relationship between the PERK/eIF2α/CHOP pathway and IRF-1-mediated VCAM-1 expression. We conclude that ER stress and the UPR contribute to the regulation of Vcam-1 transcription as a function of the atherogenic nature of TGRL.

Shear stress modulates VCAM-1 expression in response to TNF-α and dietary lipids via interferon regulatory factor-1 in cultured endothelium.

Dyslipidemia is a primary risk factor for cardiovascular disease, but the specific mechanisms that determine the localization of atherosclerotic plaques in arteries are not well defined. Triglyceride-rich lipoproteins (TGRL) isolated from human plasma after a high-fat meal modulate TNF-α-induced VCAM-1 expression in cultured human aortic endothelial cells (HAECs) via an interferon regulatory factor (IRF)-1-dependent transcriptional mechanism. We examined whether fluid shear stress acts as a mediator of IRF-1-dependent VCAM-1 expression in response to cytokine and dietary lipids. IRF-1 and VCAM-1 were examined by immunofluorescence in TNF-α-stimulated HAEC monolayers exposed to TGRL and a linear gradient of shear stress ranging from 0 to 16 dyn/cm(2) in a microfluidic device. Shear stress alone modulated TNF-α-induced VCAM-1 expression, eliciting a 150% increase at low shear stress (2 dyn/cm(2)) and a 70% decrease at high shear stress (12 dyn/cm(2)) relative to static. These differences correlated with a 60% increase in IRF-1 expression under low shear stress and a 40% decrease under high shear stress. The addition of TGRL along with cytokine activated a fourfold increase in VCAM-1 expression and a twofold increase in IRF-1 expression. The combined effect of shear stress and TGRL on the upregulation of membrane VCAM-1 was abolished by transfection of HAECs with IRF-1-specific small interfering RNA. In a healthy swine model, elevated levels of endothelial IRF-1 were also observed within atherosusceptible regions of the aorta by Western blot analysis and immunohistochemistry, implicating arterial hemodynamics in the regulation of IRF-1 expression. These data demonstrate direct roles for fluid shear stress and postprandial TGRL from human serum in the regulation of IRF-1 expression and downstream inflammatory responses in HAECs.

On-chip endothelial inflammatory phenotyping.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers. These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-, lipid- and RAGE-induced inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-α. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

IRF-1 and miRNA126 modulate VCAM-1 expression in response to a high-fat meal.

RATIONALE: A high-fat diet accompanied by hypertriglyceridemia increases an individual's risk for development of atherosclerosis. An early event in this process is monocyte recruitment through binding to vascular cell adhesion molecule 1 (VCAM-1) upregulated on inflamed arterial endothelium. Diets high in polyunsaturated fatty acids (PUFAs) may provide athero-protection by ameliorating this effect. OBJECTIVE: We investigated the acute regulation of VCAM-1 expression in human aortic endothelial cells (HAEC) in response to triglyceride-rich lipoproteins (TGRL) isolated from subjects after consumption of a high-fat meal. METHODS AND RESULTS: Postprandial TGRL isolated from 38 subjects were categorized as proatherogenic or antiatherogenic according to their capacity to alter the inflammatory response of HAEC. Proatherogenic TGRL increased expression of VCAM-1, intercellular adhesion molecule 1 (ICAM-1), and E-selectin by ≈20% compared with stimulation with tumor necrosis factor-α alone, whereas antiatherogenic TGRL decreased VCAM-1 expression by ≈20% while still upregulating ICAM-1. The relative atherogenicity of TGRL positively correlated with particle density of TG, apolipoprotein (Apo)CIII, ApoE, and cholesterol. Ω3-PUFA mimicked the effect of antiatherogenic TGRL by downregulating VCAM-1 expression. TGRL exerted this differential regulation of VCAM-1 by reciprocally modulating expression and activity of the transcription factor interferon regulatory factor 1 (IRF-1) and expression of microRNA 126 (miR-126). Overexpression or silencing of IRF-1 or miR-126 expression recapitulated the proatherogenic or antiatherogenic regulation of VCAM-1. CONCLUSIONS: In response to a high-fat meal, TGRL bias the inflammatory response of endothelium via transcriptional and posttranscriptional editing of VCAM-1. Subjects with an anti-inflammatory response to a meal produced TGRL that was enriched in nonesterified fatty acids, decreased IRF-1 expression, increased miR-126 activity, and diminished monocyte arrest.

Shear stress modulates RAGE-mediated inflammation in a model of diabetes-induced metabolic stress.

Atherosclerosis occurs preferentially at sites of disturbed blood flow despite the influence of risk factors contributing to systemic inflammation. The receptor for advanced glycation endproducts (RAGE) is a prominent mediator of inflammation in diabetes that is upregulated in atherosclerotic plaques. Our goal was to elucidate a role for arterial hemodynamics in the regulation of RAGE expression and activity. Endothelial RAGE expression was elevated at sites of flow disturbance in the aortas of healthy swine. To demonstrate a direct role for physiological shear stress (SS) in modulating RAGE expression, human aortic endothelial cells (HAEC) were exposed to high SS (HSS; 15 dyn/cm(2)), which downregulated RAGE by fourfold, or oscillatory SS (OSS; 0 ± 5 dyn/cm(2)), which upregulated RAGE by threefold, compared with static culture at 4 h. In a model of diabetes-induced metabolic stress, HAEC were chronically conditioned under high glucose (25 mM) and then simultaneously stimulated with TNF-α (0.5 ng/ml) and the RAGE ligand high mobility group box 1 (HMGB1). A 50% increase in VCAM-1 expression over TNF-α was associated with increased cytoplasmic and mitochondrial reactive oxygen species and NF-κB activity. This increase was RAGE-specific and NADPH oxidase dependent. In activated HAEC, OSS amplified HMGB1-induced VCAM-1 (3-fold) and RAGE (1.6-fold) expression and proportionally enhanced monocyte adhesion to HAEC in a RAGE-dependent manner, while HSS mitigated these increases to the level of TNF-α alone. We demonstrate that SS plays a fundamental role in regulating RAGE expression and inflammatory responses in the endothelium. These findings may provide mechanistic insight into how diabetes accelerates the nonrandom distribution of atherosclerosis in arteries.

Endothelial inflammation correlates with subject triglycerides and waist size after a high-fat meal.

A rise in postprandial serum triglycerides (PP-sTG) can potentiate inflammatory responses in vascular endothelial cells (ECs) and thus serves as an independent risk factor for predicting increased cardiovascular morbidity. We examined postprandial triglyceride-rich lipoproteins (PP-TGRLs) in subjects ranging from normal to hypertriglyceridemic for their capacity to alter EC acute inflammatory responses. Cultured human aortic ECs (HAECs) were conditioned with PP-TGRLs isolated from human serum at the peak after a moderately high-fat meal. VLDL particle size increased postprandially and varied directly with the subject's PP-sTG level and waist circumference. PP-TGRL particles bound to HAECs and were internalized via LDL receptor-mediated endocytosis. PP-TGRL alone did not induce an inflammatory response over the range of individuals studied. However, combined with low-dose TNF-α stimulation (0.3 ng/ml), it elicited a net 10-15% increase above cytokine alone in the membrane expression of VCAM-1, ICAM-1, and E-selectin, which was not observed with fasting TGRLs. In contrast to upregulation of ICAM-1 and E-selectin, VCAM-1 transcription and expression varied in direct proportion with individual PP-sTG and waist circumference. The extent of monocyte arrest on inflamed HAECs under shear stress also correlated closely with VCAM-1 expression induced by conditioning with PP-TGRL and TNF-α stimulation. This ex vivo approach provides a quantitative means to assess an individual's inflammatory potential, revealing a greater propensity for endothelial inflammation in hypertriglyceridemic individuals with abdominal obesity.

Spatial regulation of inflammation by human aortic endothelial cells in a linear gradient of shear stress.

OBJECTIVE: Atherosclerosis is a focal disease that develops at sites of low and oscillatory shear stress in arteries. This study aimed to understand how endothelial cells sense a gradient of fluid shear stress and transduce signals that regulate membrane expression of cell adhesion molecules and monocyte recruitment. METHODS: Human aortic endothelial cells were stimulated with TNF-alpha and simultaneously exposed to a linear gradient of shear stress that increased from 0 to 16 dyne/cm2. Cell adhesion molecule expression and activation of NFkappa B were quantified by immunofluorescence microscopy with resolution at the level of a single endothelial cell. Monocyte recruitment was imaged using custom microfluidic flow chambers. RESULTS: VCAM-1 and E-selectin upregulation was greatest between 2-4 dyne/cm2 (6 and 4-fold, respectively) and above 8 dyne/cm2 expression was suppressed below that of untreated endothelial cells. In contrast, ICAM-1 expression and NFkappa B nuclear translocation increased with shear stress up to a maximum at 9 dyne/cm2. Monocyte recruitment was most efficient in regions where E-selectin and VCAM-1 expression was greatest. CONCLUSIONS: We found that the endothelium can sense a change in shear stress on the order of 0.25 dyne/cm2 over a length of approximately 10 cells, regulating the level of protein transcription, cellular adhesion molecule expression, and leukocyte recruitment during inflammation.

Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha.

High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-alpha. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor-associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-kappaB, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA.

Although mRNA amplification is necessary for microarray analyses from limited amounts of cells and tissues, the accuracy of transcription profiles following amplification has not been well characterized. We tested the fidelity of differential gene expression following linear amplification by T7-mediated transcription in a well-established in vitro model of cytokine [tumor necrosis factor alpha (TNFalpha)]-stimulated human endothelial cells using filter arrays of 13,824 human cDNAs. Transcriptional profiles generated from amplified antisense RNA (aRNA) (from 100 ng total RNA, approximately 1 ng mRNA) were compared with profiles generated from unamplified RNA originating from the same homogeneous pool. Amplification accurately identified TNFalpha-induced differential expression in 94% of the genes detected using unamplified samples. Furthermore, an additional 1,150 genes were identified as putatively differentially expressed using amplified RNA which remained undetected using unamplified RNA. Of genes sampled from this set, 67% were validated by quantitative real-time PCR as truly differentially expressed. Thus, in addition to demonstrating fidelity in gene expression relative to unamplified samples, linear amplification results in improved sensitivity of detection and enhances the discovery potential of high-throughput screening by microarrays.

Shear stress magnitude and directionality modulate growth factor gene expression in preconditioned vascular endothelial cells.

OBJECTIVE: The purpose of this study was to simultaneously monitor the transcriptional levels of 12 endothelial growth factor genes in response to alterations in wall shear stress (WSS) under conditions relevant to the development of intimal hyperplasia, a major cause of arterial bypass graft failure. METHODS: Human umbilical vein endothelial cells were preconditioned in vitro under steady flow (WSS, 15 dynes/cm(2)) for 24 hours before being subjected to WSS at 25 (Delta = +10), 15 (Delta = 0), 5 (Delta = -10), 2.5 (Delta = -12.5), and 0 (Delta = -15) dynes/cm(2) or low magnitude WSS reversal (-2.5 dynes/cm(2)) for 6 hours. A focused complementary DNA array was used to simultaneously measure messenger RNA expression levels for END1, endothelial nitric oxide synthase (NOS3), platelet-derived growth factor A, platelet-derived growth factor B (PDGFB), acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor-alpha, transforming growth factor-beta, vascular endothelial growth factor, insulin-like growth factor-1, epidermal growth factor, and angiotensin converting enzyme. RESULTS: Preconditioning significantly (P <.05) increased the fold expression of NOS3 (4.1 +/- 1.4), basic fibroblast growth factor (3.90 +/- 1.16), vascular endothelial growth factor (3.39 +/- 1.04), and insulin-like growth factor-1 (2.8 +/- 0.7) but decreased END1 (0.47 +/- 0.05) and PDGFB (0.70 +/- 0.04) messenger RNA expression levels relative to no-flow controls, an effect that was sustained on removal from flow for 6 hours. Notably, the ratio of END1/NOS3 expression was diminished (0.11 +/- 0.03) relative to that of cells maintained in static culture. Although few differences in gene expression from baseline (15 dynes/cm(2)) were measured in cells exposed to either constant (Delta = 0) or step decreases (Delta = -10, -12.5, or -15 dynes/cm(2)) in WSS, marked changes were seen in the group exposed to a step increase in WSS (Delta = +10) or to WSS reversal. Low magnitude retrograde WSS evoked significant (P <.05) transcriptional changes in multiple genes, including elevated END1 (4.1 +/- 0.5), platelet-derived growth factor A (1.5 +/- 0.2), PDGFB (2.3 +/- 0.3), and transforming growth factor-beta (1.5 +/- 0.2) levels, but depressed NOS3 (0.60 +/- 0.17) levels, and a marked increase in END1/NOS3 (6.7 +/- 1.6) when compared with equal magnitude antegrade WSS (2.5 dynes/cm(2)). CONCLUSION: These results support the implementation of a preconditioning phase for in vitro WSS studies to establish a physiologic baseline. Our findings complement previous macroscale findings and are consistent with a cellular mechanism involving increased END1 and PDGFB levels, but decreased NOS3 levels, leading to intimal hyperplasia at regions of low magnitude reversing WSS.