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Vascular endothelial cells are exposed to shear stresses with disturbed vs. laminar flow patterns, which lead to proinflammatory vs. antiinflammatory phenotypes, respectively. Effective treatment against endothelial inflammation and the consequent atherogenesis requires the identification of new therapeutic molecules and the development of drugs targeting these molecules. Using Connectivity Map, we have identified vitexin, a natural flavonoid, as a compound that evokes the gene-expression changes caused by pulsatile shear, which mimics laminar flow with a clear direction, vs. oscillatory shear (OS), which mimics disturbed flow without a clear direction. Treatment with vitexin suppressed the endothelial inflammation induced by OS or tumor necrosis factor-α. Administration of vitexin to mice subjected to carotid partial ligation blocked the disturbed flow-induced endothelial inflammation and neointimal formation. In hyperlipidemic mice, treatment with vitexin ameliorated atherosclerosis. Using SuperPred, we predicted that apurinic/apyrimidinic endonuclease1 (APEX1) may directly interact with vitexin, and we experimentally verified their physical interactions. OS induced APEX1 nuclear translocation, which was inhibited by vitexin. OS promoted the binding of acetyltransferase p300 to APEX1, leading to its acetylation and nuclear translocation. Functionally, knocking down APEX1 with siRNA reversed the OS-induced proinflammatory phenotype, suggesting that APEX1 promotes inflammation by orchestrating the NF-κB pathway. Animal experiments with the partial ligation model indicated that overexpression of APEX1 negated the action of vitexin against endothelial inflammation, and that endothelial-specific deletion of APEX1 ameliorated atherogenesis. We thus propose targeting APEX1 with vitexin as a potential therapeutic strategy to alleviate atherosclerosis.

Pulsatile shear (PS) and oscillatory shear (OS) elicit distinct mechanotransduction signals that maintain endothelial homeostasis or induce endothelial dysfunction, respectively. A subset of microRNAs (miRs) in vascular endothelial cells (ECs) are differentially regulated by PS and OS, but the regulation of the miR processing and its implications in EC biology by shear stress are poorly understood. From a systematic in silico analysis for RNA binding proteins that regulate miR processing, we found that nucleolin (NCL) is a major regulator of miR processing in response to OS and essential for the maturation of miR-93 and miR-484 that target mRNAs encoding Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS). Additionally, anti-miR-93 and anti-miR-484 restore KLF2 and eNOS expression and NO bioavailability in ECs under OS. Analysis of posttranslational modifications of NCL identified that serine 328 (S328) phosphorylation by AMP-activated protein kinase (AMPK) was a major PS-activated event. AMPK phosphorylation of NCL sequesters it in the nucleus, thereby inhibiting miR-93 and miR-484 processing and their subsequent targeting of KLF2 and eNOS mRNA. Elevated levels of miR-93 and miR-484 were found in sera collected from individuals afflicted with coronary artery disease in two cohorts. These findings provide translational relevance of the AMPK-NCL-miR-93/miR-484 axis in miRNA processing in EC health and coronary artery disease.

Blood flow and vascular shear stress patterns play a significant role in inducing and modulating physiological responses of endothelial cells (ECs). Pulsatile shear (PS) is associated with an atheroprotective endothelial phenotype, while oscillatory shear (OS) is associated with an atheroprone endothelial phenotype. Although mechanisms of endothelial shear response have been extensively studied, most studies focus on characterization of single molecular pathways, mainly at fixed time points after stress application. Here, we carried out a longitudinal time-series study to measure the transcriptome after the application of PS and OS. We performed systems analyses of transcriptional data of cultured human vascular ECs to elucidate the dynamics of endothelial responses in several functional pathways such as cell cycle, oxidative stress, and inflammation. By combining the temporal data on differentially expressed transcription factors and their targets with existing knowledge on relevant functional pathways, we infer the causal relationships between disparate endothelial functions through common transcriptional regulation mechanisms. Our study presents a comprehensive temporally longitudinal experimental study and mechanistic model of shear stress response. By comparing the relative endothelial expressions of genes between OS and PS, we provide insights and an integrated perspective into EC function in response to differential shear. This study has significant implications for the pathogenesis of vascular diseases.

Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application.

The earliest atherosclerotic lesions preferentially develop in arterial regions experienced disturbed blood flow, which induces endothelial expression of pro-atherogenic genes and the subsequent endothelial dysfunction. Our previous study has demonstrated an up-regulation of DNA methyltransferase 1 (DNMT1) and a global hypermethylation in vascular endothelium subjected to disturbed flow. Here, we determined that DNMT1-specific inhibition in arterial wall ameliorates the disturbed flow-induced atherosclerosis through, at least in part, targeting cell cycle regulator cyclin A and connective tissue growth factor (CTGF). We identified the signaling pathways mediating the flow-induction of DNMT1. Inhibition of the mammalian target of rapamycin (mTOR) suppressed the DNMT1 up-regulation both in vitro and in vivo. Together, our results demonstrate that disturbed flow influences endothelial function and induces atherosclerosis in an mTOR/DNMT1-dependent manner. The conclusions obtained from this study might facilitate further evaluation of the epigenetic regulation of endothelial function during the pathological development of atherosclerosis and offer novel prevention and therapeutic targets of this disease.

Colorectal cancer (CRC) patients with KRAS mutations do not benefit from epidermal growth factor receptor (EGFR) targeted therapy. In clinical practice, identifying patients with KRAS mutations is critical prior to EGFR targeting therapy, and gene testing is generally performed using the DNA extracted from tumor tissue. The aim of this study was to compare the presence of KRAS mutations in circulating cell-free DNA (cfDNA) and primary tumor tissue using a peptide nucleic acid mediated polymerase chain reaction. We extracted and analyzed the DNA from plasmas and corresponding primary tumor samples from 52 patients with CRC. The results demonstrated that the detection rate of KRAS sequence variations was 50% (26 of 52) in plasma samples and 28.8% (15 of 52) in resected primary tumor tissue samples. The majority of KRAS mutations detected in tumors were also found in matched plasma specimens with an agreement rate of 78.8%. Eleven plasma cfDNA were found positive for KRAS mutation but not in their corresponding tissue. In conclusion, our results suggest that circulating cfDNA provides a better representation of the malignant disease as a whole and could be a reliable source of diagnostic DNA to replace the tumor tissue in a diagnostic setting.

ADSCs are a great cell source for tissue engineering and regenerative medicine. However, the development of methods to appropriately manipulate these cells invitro remains a challenge. Here the proliferation and differentiation of ADSCs on microfabricated surfaces with varying geometries were investigated. To create the patterned substrates, a maskless biofabrication method was developed based on dynamic optical projection stereolithography. Proliferation and early differentiation of ADSCs were compared across three distinct multicellular patterns, namely stripes (ST), symmetric fork (SF), and asymmetric fork (AF). The ST pattern was designed for uniaxial cell alignment while the SF and AF pattern were designed with altered cell directionality to different extents. The SF and AF patterns generated similar levels of regional peak stress, which were both significantly higher than those within the ST pattern. No significant difference in ADSC proliferation was observed among the three patterns. In comparison to the ST pattern, higher peak stress levels of the SF and AF patterns were associated with up-regulation of the chondrogenic and osteogenic markers SOX9 and RUNX2. Interestingly, uniaxial cell alignment in the ST pattern seemed to increase the expression of SM22α and smooth muscle α-actin, suggesting an early smooth muscle lineage progression. These results indicate that geometric cues that promote uniaxial alignment might be more potent for myogenesis than those with increased peak stress. Overall, the use of these patterned geometric cues for modulating cell alignment and form-induced stress can serve as a powerful and versatile technique towards controlling differentiation in ADSCs. © 2013 Elsevier Ltd.

Rationale: Endothelial microRNA-126 (miR-126) modulates vascular development and angiogenesis. However, its role in the regulation of smooth muscle cell (SMC) function is unknown. Objective: To elucidate the role of miR-126 secreted by endothelial cells (ECs) in regulating SMC turnover in vitro and in vivo, as well as the effects of shear stress on the regulation. Methods and Results: Coculture of SMCs with ECs or treatment of SMCs with conditioned media from static EC monoculture (EC-CM) increased SMC miR-126 level and SMC turnover; these effects were abolished by inhibition of endothelial miR-126 and by the application of laminar shear stress to ECs. SMC miR-126 did not increase when treated with EC-CM from ECs subjected to inhibition of miR biogenesis, or with CM from sheared ECs. Depletion of extracellular/secreted vesicles in EC-CM did not affect the increase of SMC miR-126 by EC-CM. Biotinylated miR-126 or FLAG (DYKDDDDK epitope)-tagged Argonaute2 transfected into ECs was detected in the cocultured or EC-CM–treated SMCs, indicating a direct EC-to-SMC transmission of miR-126 and Argonaute2. Endothelial miR-126 represses forkhead box O3, B-cell lymphoma 2, and insulin receptor substrate 1 mRNAs in the cocultured SMCs, suggesting the functional roles of the transmitted miR-126. Systemic depletion of miR-126 in mice inhibited neointimal lesion formation of carotid arteries induced by cessation of blood flow. Administration of EC-CM or miR-126 mitigated the inhibitory effect. Conclusions: Endothelial miR-126 acts as a key intercellular mediator to increase SMC turnover, and its release is reduced by atheroprotective laminar shear stress.

Little is known regarding the interplays between the mechanical and molecular bases for vein graft restenosis. We elucidated the stenosis initiation using a high-frequency ultrasonic (HFU) echogenicity platform and estimated the endothelium yield stress from von-Mises stress computation to predict the damage locations in living rats over time. The venous-arterial transition induced the molecular cascades for autophagy and apoptosis in venous endothelial cells (ECs) to cause neointimal hyperplasia, which correlated with the high echogenicity in HFU images and the large mechanical stress that exceeded the yield strength. The ex vivo perfusion of arterial laminar shear stress to isolated veins further confirmed the correlation. EC damage can be rescued by inhibiting autophagy formation using 3-methyladenine (3-MA). Pretreatment of veins with 3-MA prior to grafting reduced the pathological increases of echogenicity and neointima formation in rats. Therefore, this platform provides non-invasive temporal spatial measurement and prediction of restenosis after venous-arterial transition as well as monitoring the progression of the treatments.

Little is known regarding the interplays between the mechanical and molecular bases for vein graft restenosis. We elucidated the stenosis initiation using a high-frequency ultrasonic (HFU) echogenicity platform and estimated the endothelium yield stress from von-Mises stress computation to predict the damage locations in living rats over time. The venous-arterial transition induced the molecular cascades for autophagy and apoptosis in venous endothelial cells (ECs) to cause neointimal hyperplasia, which correlated with the high echogenicity in HFU images and the large mechanical stress that exceeded the yield strength. The ex vivo perfusion of arterial laminar shear stress to isolated veins further confirmed the correlation. EC damage can be rescued by inhibiting autophagy formation using 3-methyladenine (3-MA). Pretreatment of veins with 3-MA prior to grafting reduced the pathological increases of echogenicity and neointima formation in rats. Therefore, this platform provides non-invasive temporal spatial measurement and prediction of restenosis after venous-arterial transition as well as monitoring the progression of the treatments.

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.

The Eph family of receptor tyrosine kinases and their cognate ligands, the Ephrins, form a coordinated program of cell contact-mediated migration control, polarity establishment, and tissue architecture development. Specifically, the ligand Ephrin-A1 has been shown to regulate cell morphology and motility through the activation of EphA receptors, which signal to the PI3K pathway to induce cell retraction. MEFs with PI3K subunit p85β knockout (p85β−/−) exhibited markedly reduced cell retraction following Ephrin-A1 stimulation. Ephrin-A1 also serves as an inhibitory substrate for cell spreading and migration. Moreover, Ephrin-A1 treatment results in the dephosphorylation of paxillin and induces the reorganization of phospho-paxillin-containing focal adhesions. The Ephrin-A1 regulated paxillin dephosphorylation is phosphatase dependent, but p85β independent. The present study serves to demonstrate a novel molecular signaling pathways that regulate Ephrin-A1-regulated cell retraction and interaction to the substrate.

The traction forces exerted by an adherent cell on a substrate have been studied only in the two-dimensions (2D) tangential to substrate surface (Txy). We developed a novel technique to measure the three-dimensional (3D) traction forces exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate. On 3D images acquired by confocal microscopy, displacements were determined with image-processing programs, and traction forces in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated traction force in normal direction (Tz) with an order of magnitude comparable to Txy. Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. The method was evaluated regarding accuracy and precision of displacement measurements, effects of FE mesh size, displacement noises, and simple bootstrapping. These results provide new insights into cell-matrix interactions in terms of spatial and temporal variations in traction forces in 3D. This technique can be applied to study live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, for investigating cellular functions in health and disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12195-009-0082-6) contains supplementary material, which is available to authorized users.

Background and Purpose— Endovascular thrombectomy has shown promise for the treatment of acute strokes resulting from large-vessel occlusion. Reperfusion-related injury may contribute to the observed decoupling of angiographic and clinical outcomes. Iatrogenic disruption of the endothelium during thrombectomy is potentially a key mediator of this process that requires further study. Methods— An in vitro live-cell platform was developed to study the effect of various commercially available endovascular devices on the endothelium. In vivo validation was performed using porcine subjects. Results— This novel in vitro platform permitted high-resolution quantification and characterization of the pattern and timing of endothelial-cell injury among endovascular thrombectomy devices and vessel diameters. Thrombectomy devices displayed heterogeneous effects on the endothelium; the device performance assessed in vitro was substantiated by in vivo findings. Conclusions— In vitro live-cell artificial vessel modeling enables a detailed study of the endothelium after thrombectomy and may contribute to future device design. Large animal studies confirm the relevance of this in vitro system to investigate endothelial physiology. This artificial vessel model may represent a practical, scalable, and physiologically relevant system to assess new endovascular technologies.

Vascular endothelial cells are continuously exposed to mechanical (e.g., shear stress) and chemical (e.g., growth factors) stimuli. It is important to elucidate the mechanisms by which cells perceive and integrate these different stimuli to regulate the downstream signaling pathways. We ( 50 ) have previously reported the shear-induced interplay between two membrane receptors, integrins and Flk-1. In the present study, we investigated the molecular mechanisms regulating the downstream IκB kinase (IKK) pathway in response to shear stress and VEGF. Both shear stress and VEGF induced a transient increase of IKK activity. These effects were inhibited by SU-1498, a specific Flk-1 inhibitor, and by a negative mutant of Casitas B-lineage lymphoma (Cbl) with tyrosine-to-phenylalanine mutations at sites 700, 731, and 774 (Cblnm). Because Flk-1 and Cbl form a complex upon shearing or VEGF applications ( 50 ), these results suggest that shear stress and VEGF activate IKK via the receptor Flk-1 and its recruitment of the adapter protein Cbl. The inhibition of the shear- and VEGF-induced IKK activities by a negative mutant of Akt indicates that Akt acts upstream to IKK in response to shear stress and VEGF. Furthermore, SU-1498 and Cbl-nmabolished the shear- and VEGF-induced Akt activity, indicating that Akt acts at a level downstream to Flk-1 and Cbl. Therefore, our results indicate that the signaling events induced by shear stress and VEGF converge at the membrane receptor Flk-1 and that these stimuli share the Flk-1/Cbl/Akt pathway in activating IKK activation.

Osteoblast growth and differentiation are critical processes for bone development and maintenance, and are regulated by both humoral and mechanical factors. Humoral (hormonal) factors can affect gene transcription via MAPkinases, e.g., extracellular signal-regulated kinase (ERK). We studied whether the ERK pathway is also involved in processing mechanical inputs in human bone cells. Exposing MG63 cells to physiologically relevant levels of fluid flow resulted in ERK phosphorylation. Genistein blocked this response, indicating that it is dependent on tyrosine phosphorylation. Furthermore, alpha v beta 3 integrins were activated in response to fluid flow, as shown by recruitment of adaptor molecule Shc and clustering of alpha v beta 3 in focal adhesion-like structures. Antibodies blocking formation of beta 1 or beta 3 integrin-matrix interactions or RGD peptides could not inhibit fluid flow-induced ERK phosphorylation, suggesting that formation of new integrin-matrix interactions is not essential for this response and that other upstream mechanosensors regulate induction of ERK phosphorylation in response to fluid flow in human bone cells.

Ephrins, the Eph family of receptor tyrosine kinases and their ligands, have been shown to be key players in guiding cell migration, polarization, and cell-matrix and cell-cell adhesions. Specifica...

Vascular endothelium transduces the temporal gradients in shear stress (tau) originating from unsteady blood flow into functional responses. We measured the effects of step-tau and ramp-tau (i.e., t with different temporal shear gradients) on the lipid lateral diffusion coefficient (D) in the apical membranes of confluent cultured bovine aortic endothelial cells by using fluorescence recovery after photobleaching. A step-tau of 10 dynes/cm2 elicited a rapid (5 s) increase of D in the portion of the cell upstream of the nucleus and a concomitant decrease in the downstream portion. A ramp-tau with a rate of 20 dynes/cm2 per min elicited a rapid (5 s) decrease of D in both the upstream and the downstream portions. The mitogen-activated protein kinases (MAPKs) ERK and JNK were activated by step-tau but not by ramping to the same tau level. Benzyl alcohol, which increases D, enhanced the activities of both MAPKs; cholesterol, which reduces D, diminished these activities. We conclude that the lipid bilayer can sense the temporal features of the applied tau with spatial discrimination and that the tau-induced membrane perturbations can be transduced into MAPK activation. These results have implications for understanding the role of t in modulating vascular functions in health and disease.

This study was designed to elucidate the mechanism underlying the inhibition of endothelial cell growth by laminar shear stress. Tumor suppressor gene p53 was increased in bovine aortic endothelial cells subjected to 24 h of laminar shear stress at 3 dynes (1 dyne = 10 μN)/cm 2 or higher, but not at 1.5 dynes/cm 2 . One of the mechanisms of the shear-induced increase in p53 is its stabilization after phosphorylation by c-Jun N-terminal kinase. To investigate the consequence of the shear-induced p53 response, we found that prolonged laminar shear stress caused increases of the growth arrest proteins GADD45 (growth arrest and DNA damage inducible protein 45) and p21 cip1 , as well as a decrease in phosphorylation of the retinoblastoma gene product. Our results suggest that prolonged laminar shear stress causes a sustained p53 activation, which induces the up-regulation of GADD45 and p21 cip1 . The resulting inhibition of cyclin-dependent kinase and hypophosphorylation of retinoblastoma protein lead to endothelial cell cycle arrest. This inhibition of endothelial cell proliferation by laminar shear stress may serve an important homeostatic function by preventing atherogenesis in the straight part of the arterial tree that is constantly subjected to high levels of laminar shearing.

© 2014 American Heart Association, Inc. Atherosclerosis develops preferentially at branches and curvatures of the arterial tree, where blood flow pattern is disturbed rather than being laminar, and wall shear stress has an irregular distribution without defined directions. The endothelium in the atherosusceptible regions, in comparison to that in atheroresistant regions, shows activation of proproliferative and proinflammatory gene expressions, reduced production of nitric oxide (NO), increased leukocyte adhesion, and permeability, as well as other atheroprone phenotypes. Differences in gene expressions and cell phenotypes have been detected in endothelia residing in native atherosusceptible and atheroresistant regions of the arteries, or in arteries from animal models with artificial creation of disturbed flow. Similar results have also been shown in in vitro systems that apply controlled shear stresses with or without clear directions to cultured endothelial cells in fluid-dynamically designed flow-loading devices. The available evidence indicates that the coordination of multiple signaling networks, rather than individual separate pathways, links the mechanical signals to specific genetic circuitries in orchestrating the mechanoresponsive networks to evoke comprehensive genetic and functional responses.

The forces exerted by an adherent cell on a substrate were studied previously only in the two-dimensions (2D) tangential to the substrate surface. We used a novel technique to measure the three-dimensional (3D) stresses exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate, with particular emphasis on the 3D forces of focal adhesions. On 3D images acquired by confocal microscopy, displacements were determined with imageprocessing programs, and stresses in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated stress in normal direction (Tz) with an order of magnitude comparable to that in tangential direction (Txy). Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. With the use of green fluorescent protein (GFP) labeled paxillin, the dynamics of this intracellular molecule were studied concurrently with the measurement of 3D forces. In the dynamic region, including the new lamellapodium forming region in the front and the retracting region in the rear, the tangential forces (Fxy) are correlated with the size of the focal adhesions (FAs) much more strongly than those in the stable region under the nucleus. In the dynamic region, normal force (Fz) was upward and positively correlated with FA size, while Fz in the stable region was downward and negatively correlated with FA size. These findings show the influence of the size of FAs on the 3D forces they exert on the substrate. This technique can be applied to study any adherent type of live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, thus elucidating cellular functions in health and disease.

Shear stress imposed by blood flow is crucial for maintaining vascular homeostasis. We examined the role of shear stress in regulating SIRT1, an NAD + -dependent deacetylase, and its functional relevance in vitro and in vivo. The application of laminar flow increased SIRT1 level and activity, mitochondrial biogenesis, and expression of SIRT1-regulated genes in cultured endothelial cells (ECs). When the effects of different flow patterns were compared in vitro, SIRT1 level was significantly higher in ECs exposed to physiologically relevant pulsatile flow than pathophysiologically relevant oscillatory flow. These results are in concert with the finding that SIRT1 level was higher in the mouse thoracic aorta exposed to atheroprotective flow than in the aortic arch under atheroprone flow. Because laminar shear stress activates AMP-activated protein kinase (AMPK), with subsequent phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser-633 and Ser-1177, we studied the interplay of AMPK and SIRT1 on eNOS. Laminar flow increased SIRT1-eNOS association and eNOS deacetylation. By using the AMPK inhibitor and eNOS Ser-633 and -1177 mutants, we demonstrated that AMPK phosphorylation of eNOS is needed to prime SIRT1-induced deacetylation of eNOS to enhance NO production. To verify this finding in vivo, we compared the acetylation status of eNOS in thoracic aortas from AMPKα2 −/− mice and their AMPKα2 +/+ littermates. Our finding that AMPKα2 −/− mice had a higher eNOS acetylation indicates that AMPK phosphorylation of eNOS is required for the SIRT1 deacetylation of eNOS. These results suggest that atheroprotective flow, via AMPK and SIRT1, increases NO bioavailability in endothelium.

Objective— Vascular endothelial cells (ECs) confer atheroprotection at locations of the arterial tree where pulsatile laminar flow (PS) exists with a high shear stress and a large net forward direction. We investigated whether the PS-induced expression of the transcription factor Krüppel-Like Factor 2 (KLF2) in cultured ECs and its expression in the mouse aorta is regulated by AMP-activated protein kinase (AMPK). Methods and Results— AMPK inhibition by Compound C or siRNA had a significant blocking effect on the PS-induced KLF2 expression. The induction of KLF2 by PS led to the increase in eNOS and the suppression of ET-1, which could be reversed by KLF2 siRNA. In addition, PS induced the phosphorylation of ERK5 and MEF2 which are necessary for the KLF2 expression. These mechanotransduction events were abrogated by the blockade of AMPK. Furthermore, the phosphorylation levels of ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in the aorta of AMPKα2 knockout mice when compared with wild-type control mice. Conclusion— The flow-mediated AMPK activation is a newly defined KLF2 regulatory pathway in vascular endothelium that acts via ERK5/MEF2.

We investigated the effects of oscillatory flow in regulating the gene expressions of type I collagen (COL1, the main component of human bone tissues) and osteopontin (OPN, the key gene for calcium deposition) in human osteoblast-like (MG-63) cells, and the roles of mitogen-activated protein kinases (MAPKs) in this regulation. The cells were subjected to oscillatory flow (0.5 +/- 4 dyn/cm(2)) or kept under static condition for various time periods (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h). Oscillatory flow caused significant up-regulations of both COL1 and OPN gene expressions over the 16 h of study, and a transient activation of MAPKs was starting at 15 min and declining to basal level in 2 h. The flow-induction of COL1 was blocked by an ERK inhibitor (PD98059) and reduced by a JNK inhibitor (SP600125), whereas that of OPN was abolished by PD98059. Analysis of the cis-elements in the COL1 and OPN promoters suggests the involvement of transacting factors Elk-1 and AP-1 in the transcription regulation. The ERK inhibitor (PD98059) blocked Elk-1 phosphorylation, as well as COL1 and OPN gene expression. The JNK inhibitor (SP600125) abolished c-jun phosphorylation and COL1 expression. These results suggest that the flow-induction of OPN was mediated through the ERK-Elk1-OPN pathway, and that COL1 was regulated by both the ERK-Elk1-COL1 and JNK-c-JUN-COL1 pathway.

Flow patterns in blood vessels contribute to focal distribution of atherosclerosis; the underlying mechanotransduction pathways remain to be investigated. We demonstrate that different flow patterns elicit distinct responses of Kruppel-like factor-2 (KLF2) in endothelial cells (ECs) in vitro and in vivo. While pulsatile flow with a significant forward direction induced sustained expression of KLF2 in cultured ECs, oscillatory flow with little forward direction caused prolonged suppression after a transient induction. The suppressive effect of oscillatory flow was Src-dependent. Immunohistochemical studies on ECs at arterial branch points revealed that KLF2 protein levels were related to local hemodynamics. Such flow-associated expression patterns were also demonstrated in a rat aortic restenosis model. Inhibition of KLF2 with siRNA sensitized ECs to oxidized LDL-induced apoptosis, indicating a protective role of KLF2. In conclusion, differential regulation of KLF2 may mediate the distinct vascular effects induced by various patterns of shear stress.

Objective— The long-term efficacy of stent implantation is affected by in-stent restenosis (ISR). Multiple factors can contribute to ISR, and the underlying mechanism remains elusive. We investigated the possible role of mechanical stretch and the associated molecular signaling in ISR. Methods and Results— Stent implantation in rat abdominal aortas induced neointima formation. Immunohistochemical studies revealed the activation of Akt in the media and neointima of the stented vessels. Western blotting showed increased phosphorylation of Akt at both Thr308 and Ser473 and phosphorylation of GSK-3β in the stented vessels. A stretch device applying static equibiaxial stretch on cultured vascular smooth muscle cells was used to delineate the molecular mechanism underlying the stretch activation of Akt. Static mechanical stretch induced the sustained activation of Akt and its upstream phosphoinositide 3-kinase (PI3K) and the phosphorylation of GSK-3β, its downstream effector in vascular smooth muscle cells. LY294002, a PI3K inhibitor, and N-acetylcysteine, a scavenger of reactive oxygen species, inhibited the stretch activation of Akt. Furthermore, N -acetylcysteine and wortmannin, another PI3K inhibitor, reduced the neointima formation after stent implantation. Conclusions— Mechanical stretch of the vascular wall during stent deployment may contribute to ISR by activating the Akt pathway.

Abnormal mechanical loading of joints may induce degeneration of articular cartilage. Shear stress is one mode of mechanical loading that may regulate chondrocyte metabolism. We investigated the mechanism by which shear stress induces the gene encoding matrix metalloproteinase-9, a mediator of the progressive degradation of articular cartilage in osteoarthritis. In vitro experiments using passaged rabbit chondrocytes in monolayer culture subjected to a shear stress of 16 dyn/cm2 (1.6 Pa) in a flow channel showed increased expression of the matrix metalloproteinase-9 gene. The induction of matrix metalloproteinase-9 appeared to depend on a region in the 5' promoter of the gene that contains a 12-0-tetradecanoylphorbol 13-acetate-responsive element. Transfection experiments using a construct containing a luciferase reporter driven by a 12-0-tetradecanoylphorbol 13-acetate-responsive element indicated that shear stress activated a 12-0-tetradecanoylphorbol 13-acetate-responsive element-mediated transcription in chondrocytes. Similar experiments showed that shear stress induced a matrix metalloproteinase-9 promoter construct (matrix metalloproteinase-9-luciferase). Shear stress activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38. Transfection of matrix metalloproteinase-9-luciferase together with the dominant negative mutant of c-Jun NH2-terminal kinase, but not with that of extracellular signal-regulated kinase or p38, attenuated the shear-induced matrix metalloproteinase-9 promoter activity. In addition, transfection of constructs encoding dominant negative mutants of Ras, Rac, and Cdc42 attenuated the induction of c-Jun transcriptional activity by shear stress. Thus. shear stimulation of chondrocytes stimulates Ras, Rac, and Cdc42, which subsequently activate c-Jun NH2-terminal kinase to induce a 12-0-tetradecanoylphorbol 13-acetate-responsive element-mediated expression of matrix metalloproteinase-9.

Background. Ras protein is a key signal transducer in the cause of cell proliferation. We studied the effects of active and negative mutants of the Ras gene on arterial neointimal formation in rats, with the aim of elucidating the molecular mechanisms regulating restenosis following percutaneous transluminal coronary angioplasty. Materials and methods. AdRasV12 and AdRasN17, the recombinant adenoviruses containing a constitutively active mutant and a dominant negative mutant of Ras, respectively, were used to determine whether Ras is necessary and sufficient to modulate the smooth muscle cell proliferation and neointima formation. Following balloon injury, rat common carotid arteries were treated in their distal half with AdRasV12, AdRasN17, or AdLacZ, with the proximal half used as uninfected control. Results. In rat arteries subjected to balloon injury, either uninfected or treated with AdLacZ, there were pronounced SMC proliferation and neointima formation. These changes were markedly augmented by AdRasV12 and reduced by AdRasN17. Conclusion. Ras is necessary and sufficient for SMC proliferation and neointima formation and may play a critical role in restenosis following balloon angioplasty.

The monocyte chemotactic protein-1 (MCP-1) is a 76 amino acid protein that specifically attracts monocytes. The expression of MCP-1 gene can be induced by lipopolysaccharides (LPS), phorbol esters (TPA) and several cytokines. However, how they regulate MCP-1 gene expression is not known. We tested whether the two putative TPA-responsive elements (TREs) and one κB enhancer-like region found in the MCP-1 promoter region, are involved in this regulation of MCP-1 gene expression. The 5′ untranslated region of MCP-1 gene was linked to chloramphenicol acetyl transferase (CAT) reporter gene and transfected into human glioblastoma cells in which endogenous MCP gene expression was found to be stimulated by TPA and tumor necrosis factor-α (TNF-α). The 128 bp 5′-flanking region containing one TRE was adequate for basal promoter activity but the presence of both TREs in the MCP-1 promoter region were needed to give TPA responsive enhancement (2.5 fold) of expression of the marker gene. Mutations in either of the TRE's could abolish the TPA induction of CAT expression. Replacement of the κB enhancer-like element with a TRE-like sequence caused a 10-fold enhancement of CAT expression by TPA treatment. Random mutation of κB enhancer-like element did not affect CAT expression or its TPA induction. None of the MCP promoter constructs showed significant increase in CAT expression by treatment with tumor necrosis factor-α (TNF-α). This result suggested that the TNF regulation of MCP-1 gene involves other parts of the gene besides the proximal 5′ flanking region. (Mol Cell Biochem141: 121–128, 1994)

A monocyte chemotactic protein (MCP-1) is thought to play a major role in recruiting monocytes to the vascular endothelium where the adherence of monocytes is one of the earliest events in atherogenesis. We cloned MCP-1 cDNA from a lambda gt 11 cDNA library constructed from human aortic endothelial mRNA to test whether MCP-1 expressed in arterial endothelium is identical to those from other sources. A approximately 670 bp MCP-1 cDNA clone was identified and showed the identical sequence with the ones from other cell lines. Northern blot analysis using this cloned MCP-1 cDNA as probe revealed two hybridizing bands of RNA at 0.68 and 0.77 kb in human aortic, human pulmonary arterial, and human umbilical vein endothelial cell cultures. Primer extension analysis showed that the difference in size (approximately 90 bp) between the two transcripts is not due to a difference at the 5'-noncoding region. The amount of MCP-1 transcripts increased dramatically in aortic endothelial cells when stimulated with recombinant IL-1 alpha (100 units/ml), IL-1 beta (100 units/ml), or TNF-alpha (200 ng/ml). Northern blot and slot blot analysis of RNA isolated from both the endothelium and the underlying vessel wall of freshly removed human arteries and veins showed MCP-1 transcripts. This observation demonstrates for the first time that MCP-1 is expressed not only in atherosclerotic human arteries but also in symptom free arteries and veins in vivo.

Multiple signal transduction pathways including protein kinase C, tyrosine phosphorylation, and an independent third signaling mechanism are involved in the activation of monocyte chemotactic protein-1 gene. Northern blot analysis showed that the incubation of endothelial cell with dioctanoylglycerol induced maximum level of monocyte chemotactic protein-1 transcripts. The TPA-induced monocyte chemotactic protein-1 expression was abolished by treating the cells with both staurosporine and genistein; however, only a portion of the LPS-induced expression was inhibited by staurosporine/genistein. This is in accordance with the observation that LPS induced the expression of monocyte chemotactic protein-1 in desensitized cells. Thus, a third signal transduction pathway other than protein kinase C or tyrosine kinase is involved in the LPS-induced monocyte chemotactic protein-1 expression in human endothelial cell.

Monocyte chemotactic protein released by endothelium plays an important role in inflammation, immune reactions and probably in atherogenesis. To elucidate the regulation of synthesis of this protein, the human gene encoding its synthesis was cloned and its nucleotide sequence was determined. This gene is composed of 3 exons of 145, 118 and 478 bp in length with 2 introns of 800 and 385 bp in length. Phorbol ester responsive elements (TRE) were found 129 and 157 bp upstream from the translation initiation site and the phorbol ester treatment of endothelial cell cultures elevated the transcript level of this gene.

We studied the response of porcine vascular smooth muscle cells (PVSMCs) to cyclic sinusoidal stretch at a frequency of 1 Hz. Cyclic stretch with an area change of 25% caused an increase in PVSMC apoptosis, which was accompanied by sustained activation of c-Jun NH2-terminal kinases (JNK) and the mitogen-activated protein kinase p38. Cyclic stretch with an area change of 7% had no such effect. Infection of PVSMCs with recombinant adenoviruses expressing constitutively active forms of upstream molecules that activate JNK and p38 also led to apoptosis. The simultaneous blockade of both JNK and p38 pathways with adenovirus-mediated expression of dominant-negative mutants of c-Jun and p38 caused a significant decrease (to 1/2) of the apoptosis induced by 25% cyclic stretch. The 25% stretch also caused sustained clustering of tumor necrosis factor-α (TNF-α) receptor-1 and its association with TNF-α receptor-associated factor-2 (TRAF-2). Overexpressing the wild-type TRAF-2 in PVSMCs caused an increase in apoptosis. In contrast, the expression of a dominant-negative mutant of TRAF-2 attenuated stretch-induced apoptois. These results support the hypothesis that circumferential overload under hypertensive conditions induces a clustering of death receptors that cause vascular smooth muscle cell apoptosis.