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• W2100031693 abstract "Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than ∼5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow. Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than ∼5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow. The focal development of atherosclerosis has been linked to the local variations in blood flow that are observed near the irregular blood vessel geometries of bifurcations and bends.1Friedman MH O'Brien V Ehrlich LW Calculations of pulsatile flow through a branch: implications for the hemodynamics of atherogenesis.Circ Res. 1975; 36: 277-285Crossref PubMed Scopus (85) Google Scholar, 2Topper JN Gimbrone Jr, MA Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype.Mol Med Today. 1999; 5: 40-46Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar Continuous exposure of endothelial cells to flow in vivo generates a tangential force, shear stress, across their apical surfaces. A large number of studies support the hypothesized anti-atherosclerotic effect of shear stress on the endothelium, and are mainly based on the ability of shear stress to modulate endothelial gene expression.3Dewey Jr, CF Bussolari SR Gimbrone Jr, MA Davies PF The dynamic response of vascular endothelial cells to fluid shear stress.J Biomech Eng. 1981; 103: 177-185Crossref PubMed Scopus (915) Google Scholar Throughout the recent years, a collection of shear stress-responsive endothelial genes has been established.4Topper JN Cai J Falb D Gimbrone Jr, MA Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.Proc Natl Acad Sci USA. 1996; 93: 10417-10422Crossref PubMed Scopus (729) Google Scholar, 5Garcia-Cardena G Comander J Anderson KR Blackman BR Gimbrone Jr, MA Biomechanical activation of vascular endothelium as a determinant of its functional phenotype.Proc Natl Acad Sci USA. 2001; 98: 4478-4485Crossref PubMed Scopus (460) Google Scholar, 6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar, 7Wasserman SM Mehraban F Komuves LG Yang RB Tomlinson JE Zhang Y Spriggs F Topper JN Gene expression profile of human endothelial cells exposed to sustained fluid shear stress.Physiol Genom. 2002; 12: 13-23PubMed Google Scholar, 8McCormick SM Frye SR Eskin SG Teng CL Lu CM Russell CG Chittur KK McIntire LV Microarray analysis of shear stressed endothelial cells.Biorheology. 2003; 40: 5-11PubMed Google Scholar Usually no clear distinction is made between genes induced by prolonged shear and those induced by short-term shear (<24 hours), although the latter class typically represents a general stress response also observed with turbulent flow types and seems more related to endothelial dysfunction. Based on the rationale that only genes induced by prolonged shear would represent the healthy transcriptome, we previously identified a limited number of genes that are still highly induced after exposing human umbilical vein endothelial cells (HUVECs) to flow for 7 days, but which are not induced by various other (inflammatory) stimuli.6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar The expression of one of those genes, the transcription factor lung Krüppel-like factor (LKLF/KLF2), was restricted to the endothelium in the healthy adult human aorta. Furthermore, the inflammatory cytokine tumor necrosis factor-α repressed LKLF expression in HUVECs, making LKLF a potential marker for the resting, nonactivated state of the endothelial cell. The inverse regulation of LKLF by shear stress and cytokines was later confirmed by others and LKLF was shown to inhibit the induction of cell adhesion molecules by cytokines.9SenBanerjee S Lin Z Atkins GB Greif DM Rao RM Kumar A Feinberg MW Chen Z Simon DI Luscinskas FW Michel TM Gimbrone Jr, MA Garcia-Cardena G Jain MK KLF2 is a novel transcriptional regulator of endothelial proinflammatory activation.J Exp Med. 2004; 199: 1305-1315Crossref PubMed Scopus (556) Google Scholar These findings suggest that the combination of shear stress magnitude and inflammation can be the prime modulator of endothelial LKLF expression in vivo. Endothelial cell gene expression in vivo, however, is under the control of a complex combination of biomechanical, humoral, and various other biological stimuli. This necessitates isolation of these distinct stimuli in dedicated animal models to resolve the prime source of the regulation of the expression of single genes, such as LKLF, in vivo. One of the major, well-studied effects of shear stress on endothelial physiology is its ability to control vascular tone by regulating the transcription of genes that encode proteins with potent vasodilatory or vasoconstrictive properties.10Davies PF Flow-mediated endothelial mechanotransduction.Physiol Rev. 1995; 75: 519-560Crossref PubMed Scopus (2367) Google Scholar, 11Drexler H Hornig B Endothelial dysfunction in human disease.J Mol Cell Cardiol. 1999; 31: 51-60Abstract Full Text PDF PubMed Scopus (440) Google Scholar Well-known examples are the shear-repressed endothelin,12Sharefkin JB Diamond SL Eskin SG McIntire LV Dieffenbach CW Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells.J Vasc Surg. 1991; 14: 1-9Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar angiotensin-converting enzyme (ACE),13Rieder MJ Carmona R Krieger JE Pritchard Jr, KA Greene AS Suppression of angiotensin-converting enzyme expression and activity by shear stress.Circ Res. 1997; 80: 312-319Crossref PubMed Scopus (121) Google Scholar and adrenomedullin,14Shinoki N Kawasaki T Minamino N Okahara K Ogawa A Ariyoshi H Sakon M Kambayashi J Kangawa K Monden M Shear stress down-regulates gene transcription and production of adrenomedullin in human aortic endothelial cells.J Cell Biochem. 1998; 71: 109-115Crossref PubMed Scopus (32) Google Scholar and the shear-induced endothelial nitric oxide synthase (eNOS).11Drexler H Hornig B Endothelial dysfunction in human disease.J Mol Cell Cardiol. 1999; 31: 51-60Abstract Full Text PDF PubMed Scopus (440) Google Scholar It was recently demonstrated that LKLF potently induces functional eNOS expression by directly binding its promoter.9SenBanerjee S Lin Z Atkins GB Greif DM Rao RM Kumar A Feinberg MW Chen Z Simon DI Luscinskas FW Michel TM Gimbrone Jr, MA Garcia-Cardena G Jain MK KLF2 is a novel transcriptional regulator of endothelial proinflammatory activation.J Exp Med. 2004; 199: 1305-1315Crossref PubMed Scopus (556) Google Scholar In general, relatively little is known about the signaling pathways used by shear stress to regulate the transcription of these genes, and whether there is a common denominator in these routes and/or transcription factors that mediate their highly endothelial-specific response to flow. To date, expression of LKLF has been demonstrated in a limited number of cell types, ie, endothelial cells,6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar, 15Kuo CT Veselits ML Barton KP Lu MM Clendenin C Leiden JM The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis.Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (313) Google Scholar naive T cells,16Kuo CT Veselits ML Leiden JM LKLF: a transcriptional regulator of single-positive T cell quiescence and survival.Science. 1997; 277: 1986-1990Crossref PubMed Scopus (348) Google Scholar and preadipocytes.17Banerjee SS Feinberg MW Watanabe M Gray S Haspel RL Denkinger DJ Kawahara R Hauner H Jain MK The Kruppel-like factor KLF2 inhibits peroxisome proliferator-activated receptor-gamma expression and adipogenesis.J Biol Chem. 2003; 278: 2581-2584Crossref PubMed Scopus (242) Google Scholar High LKLF expression that is observed in naive T cells and preadipocytes is rapidly lost during cell activation/differentiation, thereby presenting LKLF as a marker for cell quiescence or a specific stage of cell differentiation.16Kuo CT Veselits ML Leiden JM LKLF: a transcriptional regulator of single-positive T cell quiescence and survival.Science. 1997; 277: 1986-1990Crossref PubMed Scopus (348) Google Scholar, 17Banerjee SS Feinberg MW Watanabe M Gray S Haspel RL Denkinger DJ Kawahara R Hauner H Jain MK The Kruppel-like factor KLF2 inhibits peroxisome proliferator-activated receptor-gamma expression and adipogenesis.J Biol Chem. 2003; 278: 2581-2584Crossref PubMed Scopus (242) Google Scholar Gene-knockout studies in mice have revealed that the expression of LKLF in the endothelium is essential for vasculogenesis in embryonic mice, which elaborated into the concept that downstream products of endothelial LKLF would have a critical role in stabilization of the (new) vessel wall.15Kuo CT Veselits ML Barton KP Lu MM Clendenin C Leiden JM The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis.Genes Dev. 1997; 11: 2996-3006Crossref PubMed Scopus (313) Google Scholar, 18Oettgen P Transcriptional regulation of vascular development.Circ Res. 2001; 89: 380-388Crossref PubMed Scopus (80) Google Scholar To gain more insight into the potential flow-mediated spatial expression of LKLF in endothelial cells, we have studied its detailed in vitro biomechanical regulation and in vivo expression in various human vascular tissues. Using a mouse model in which endothelial shear stress can be locally increased,19von der Thüsen JH van Berkel TJ Biessen EA Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.Circulation. 2001; 103: 1164-1170Crossref PubMed Scopus (210) Google Scholar the moderate basal expression of endothelial LKLF was shown to be elevated by shear stress in vivo. Overexpression and knockdown of LKLF in HUVECs revealed that the flow-responsive genes that are involved in the regulation of vascular tone are under transcriptional control of LKLF. HUVECs were isolated, cultured, and exposed to shear stress in a parallel plate-type flow chamber as described.6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar, 20Jaffe EA Nachman RL Becker CG Minick CR Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.J Clin Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (6029) Google Scholar The pulsatile flow of a peristaltic pump (Masterflex 7524-05 pump drive with a 7518-10 pump head; Cole-Parmer, Instrument Co., Chicago, IL) was dampened by placing two three-way taps with windkessels (∼80 ml of air) followed by a resistance cannula between the pump and flow cell. For the unidirectional pulsatile flow experiments, an independent 1.2-Hz flow-pulse with an amplitude of 5.7 dyne/cm2 was generated on top of this controllable steady flow (2 to 30 dyne/cm2) by placing a CellMax Quad positive displacement pump (Cellco, Germantown, MD) between the damper assembly and flow cell. For all pump settings, the steady and pulsatile flow patterns were recorded and used to calculate the mean, minimal, and maximal shear stress.6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar For the cell-stretch experiments, second-passage HUVEC cultures were grown to confluency on fibronectin-coated BioFlex collagen I plates (Flexcell Inc., Hillsborough, NC). Uniaxial cyclic strain of either 5% or 15% was applied for various time intervals, using a FX-3000 Flexercell Strain Unit (Flexcell) at a cycle frequency of 1 and 0.3 Hz, respectively. Human vascular tissue specimens were collected from multiorgan donors after obtaining informed consent (approved by the Academic Medical Center Medical Ethical Committee). For immunohistochemistry and in situ hybridization, vascular tissues were handled and pretreated as described.6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar Murine carotid arteries were stained immunohistochemically with antibodies directed against HAM-56 (dilution, 1:50), von Willebrand Factor (vWF) (rabbit anti-human; dilution, 1:250), proliferating cell nuclear antigen (PCNA) (monoclonal mouse anti-human; dilution, 1:100), AIA-31240 (rabbit anti-mouse; dilution, 1:5000), Ly-6G (dilution, 1:500). Secondary antibodies were biotin-labeled rat anti-mouse for PCNA (dilution, 1:400) and goat anti-rabbit for vWF and AIA (dilution, 1:200), which were detected using the StreptABComplex/HRP kit (DakoCytomation, Glostrup, Denmark). Carotid artery collar experiments were performed as described.19von der Thüsen JH van Berkel TJ Biessen EA Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.Circulation. 2001; 103: 1164-1170Crossref PubMed Scopus (210) Google Scholar For in situ hybridization, male Apo E−/− mice of 20 weeks of age were fed a semisynthetic Western-type diet. After 2 weeks, constrictive collars (diameter, 0.3 mm; length, 2 mm) were placed around both carotid arteries. Sham-operated mice were handled and operated identically, but no collar was placed. After continuing feeding the mice a Western-type diet for 2, 5, or 9 days, the carotid arteries were excised, fixed at 4°C for 24 hours in 4% (v/v) paraformaldehyde in phosphate-buffered saline, embedded in paraffin, and sectioned at 20 μm for in situ hybridization. Female wild-type C57BL/6 mice (normal diet) and male ApoE−/− mice (Western-type diet) of 8 weeks were used for laser microbeam microdissection (LMM). Collars were placed for a 4-day period, after which the carotid arteries were perfusion-fixed with methacarn (methanol-chloroform-glacial acetic acid at a 6:3:1 ratio), embedded in paraffin, and sectioned at 10 μm for LMM. Murine carotid artery sections were mounted onto slides for membrane-based microdissection (Leica Microsystems, Wetzlar, Germany) and subsequently deparaffinized in xylene, followed by washing in absolute ethanol. No staining was performed on these sections to ensure an optimal yield of RNA. Using LMM (P.A.L.M. Microlaser Technologies AG, Bernried, Germany), the endothelium/media was circumferentially excised from 18 proximal and 18 in-collar sections each from the same carotid arteries. Microdissected tissues were collected and stored at −80°C in proteinase K digestion buffer (Ambion, Austin, TX) until further processing. Total RNA was isolated and DNase I treated, using the paraffin block RNA isolation kit (Ambion). Real-time RT-PCR was performed on total RNA isolated using the Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA) as described.6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar Gene-specific primers for human and mouse LKLF, hypoxanthine phosphoribosyltransferase (HPRT), endothelin-1, adrenomedullin, ACE, eNOS, and CD31 were designed using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).21Rozen S Skaletsky HJ Primer3 on the WWW for general users and for biologist programmers.in: Krawetz S Misener S Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa2000: pp 365-386Google Scholar After correction for HPRT, the human LKLF mRNA levels were expressed as ratios compared to the control cultures. The in situ hybridization procedure was performed essentially as described.6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar, 22Moorman AF Houweling AC de Boer PA Christoffels VM Sensitive nonradioactive detection of RNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.J Histochem Cytochem. 2001; 49: 1-8Crossref PubMed Scopus (269) Google Scholar Riboprobes were derived from the following cDNA fragments: 460-bp BstNI-BstNI fragment of the human LKLF cDNA (GenBank: H28611), a 192-bp fragment of human vWF cDNA 8239 to 8442 bp (GenBank: X04385), 400-bp SalI-PacI fragment (complete 3′ untranslated region) of mouse LKLF cDNA (GenBank: AA184928), and a 200-bp fragment of mouse vWF cDNA (GenBank: W20754). All cDNA clones were obtained from the UK Human Genome Mapping Project Resource Centre (Cambridge, UK) as IMAGE-consortium cDNA clones.23Lennon G Auffray C Polymeropoulos M Soares MB The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression.Genomics. 1996; 33: 151-152Crossref PubMed Scopus (1089) Google Scholar Nuclear counterstaining was performed with nuclear fast red (Sigma, St. Louis, MO). Sections were examined using a Zeiss Axiophot microscope (Carl Zeiss, Jena, Germany) and photographed using a Sony DXC-950P digital camera (Sony Corp., Tokyo, Japan) operated with the Leica QWin software (Leica Imaging Systems Ltd., Cambridge, UK). Linear color corrections to the photomicrographs were made using Adobe Photoshop version 5.0 (Adobe Systems Inc., San Jose, CA). The entire human LKLF open reading frame was obtained by RT-PCR, cloned behind the human phosphoglycerate kinase (PGK) promoter of the pRRL-cPPT-PGK-MCS-PRE-SIN vector, and verified by sequencing. Lentiviruses were generated in HEK293T cells as described24Naldini L Blomer U Gallay P Ory D Mulligan R Gage FH Verma IM Trono D In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Scopus (4049) Google Scholar, 25Zufferey R Dull T Mandel RJ Bukovsky A Quiroz D Naldini L Trono D Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J Virol. 1998; 72: 9873-9880Crossref PubMed Google Scholar and virus-containing supernatants were titrated on HUVECs to determine the titers needed to transduce >95% of the cells. The otherwise identical vector, but without KLF2 cDNA, was used to generate mock viruses for control transductions. First passage HUVEC cultures were transduced at ∼50% confluency for 24 hours and grown to confluency in passage 2 within the next 7 days. KLF2 overexpression was confirmed by real-time RT-PCR, Western blotting, and fluorescence immunohistochemistry using antisera against two separate synthetic peptides of human LKLF, raised in rabbits by the Eurogentec Double-X program (Eurogentec, Seraing, Belgium). A stable knockdown of the KLF2 mRNA was achieved by lentiviral delivery of an expression cassette encoding an siRNA directed against the target sequence AAGACCTACACCAAGAGTTCG. This sequence is unique to KLF2, as determined by the Whitehead Institute (Cambridge, MA) siRNA selection program.26Yuan B Latek R Hossbach M Tuschl T Lewitter F siRNA selection server: an automated siRNA oligonucleotide prediction server.Nucl Acids Res. 2004; 32: W130-W134Crossref PubMed Scopus (264) Google Scholar The siRNA expression cassette was obtained by PCR amplification of the RNA polymerase III H1 promoter from the pSUPER vector,27Brummelkamp TR Bernards R Agami R A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3973) Google Scholar using the forward T3 primer and a reverse primer 5′-CTGTCTAGACAAAAAGACCTACACCAAGAGTTCGTCT-CTTGAACGAACTCTTGGTGTAGGTCGGGGATCTGTG-GTCTCATACA-3′. The reverse primer incorporates a 19-bp hairpin DNA sequence preceded by an XbaI restriction site. The PCR product was cloned into the pGEM-T easy vector (Promega, Madison, WI), digested with XbaI and SpeI restriction enzymes, and ligated into an NheI site of the lentiviral vector LV-CMV-GFP(dU3/NheI) (kindly provided by Dr. N.A. Kootstra, Sanquin Research at CLB, Amsterdam, The Netherlands).24Naldini L Blomer U Gallay P Ory D Mulligan R Gage FH Verma IM Trono D In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Scopus (4049) Google Scholar, 25Zufferey R Dull T Mandel RJ Bukovsky A Quiroz D Naldini L Trono D Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J Virol. 1998; 72: 9873-9880Crossref PubMed Google Scholar, 28Tiscornia G Singer O Ikawa M Verma IM A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA.Proc Natl Acad Sci USA. 2003; 100: 1844-1848Crossref PubMed Scopus (500) Google Scholar Viral constructs were packaged and transduced into HUVECs as described in the previous section. The otherwise identical lentiviral vector lacking the siRNA expression cassette was used as a control. First passage primary HUVEC cultures were transduced with the lentiviral KLF2 siRNA and cultured for an additional 2 days before seeding into the artificial capillaries, followed by a 4-day flow exposure as described.6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar Effective KLF2 silencing under prolonged flow was confirmed by real-time RT-PCR. Expression data are given as mean ± SEM for the indicated number of experiments. The unpaired Student's t-test was used to calculate the statistical significance of the expression ratios versus control cultures. P values less than 0.05 were considered statistically significant. Because a detailed description of the expression of LKLF in the human vasculature is still lacking, we first examined the expression of LKLF in human umbilical vessels by performing in situ hybridization on cross sections of a human umbilical cord. High levels of LKLF mRNA were observed in the endothelium of the umbilical arteries and vein (Figure 1, A and B). The qualitative spatial expression of LKLF in the vascular tree was further investigated by performing in situ hybridization on adult human vascular tissue specimens taken from three different positions in the aorta (aortic arch, abdominal aorta, and aorta bifurcation/iliac arteries) of donors of various ages (13 months to 76 years). Specimens with an intact, vWF-positive endothelium, as evaluated by in situ hybridization, were selected to study the expression of LKLF (Table 1). Moderate to high endothelial LKLF hybridization signals were found in all tested sections from donors of all ages (Figure 1; C to H). The LKLF mRNA was never detected in the vascular smooth muscle cells (SMCs). High endothelial expression was observed particularly in the aorta of a 13-month-old donor (Figure 1G). In some specimens, a LKLF hybridization signal was observed in medial and/or neointimal cells (eg, Figure 1C), possibly originating from LKLF-expressing infiltrated T cells16Kuo CT Veselits ML Leiden JM LKLF: a transcriptional regulator of single-positive T cell quiescence and survival.Science. 1997; 277: 1986-1990Crossref PubMed Scopus (348) Google Scholar or transdifferentiated endothelial cells.29DeRuiter MC Poelmann RE VanMunsteren JC Mironov V Markwald RR Gittenberger-de Groot AC Embryonic endothelial cells transdifferentiate into mesenchymal cells expressing smooth muscle actins in vivo and in vitro.Circ Res. 1997; 80: 444-451Crossref PubMed Scopus (302) Google ScholarTable 1Description of Human Aortic Specimens Analyzed by in Situ HybridizationAgeGenderVessel typeNeointimaFigure13 monthsFemaleDescending aorta (thoracic area)None1G12 yearsMaleCommon iliac artery (close to bifurcation)Minor1CAbdominal aorta1D13 yearsFemaleAortic arch-carotid bifurcationNone234 yearsFemaleCommon iliac artery (close to bifurcation)Medium3, B to D41 yearsFemaleCommon iliac arteryMinor1E49 yearsFemaleAbdominal aortaMedium1F57 yearsMaleCommon iliac artery (close to bifurcation)Medium3, E and F76 yearsMaleDescending aorta (thoracic area)None1H Open table in a new tab Our previous in vitro studies demonstrated that LKLF is exclusively induced in HUVECs that are exposed to laminar flow, ie, high-shear stress.6Dekker RJ van Soest S Fontijn RD Salamanca S de Groot PG VanBavel E Pannekoek H Horrevoets AJ Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).Blood. 2002; 100: 1689-1698Crossref PubMed Scopus (547) Google Scholar Sudden contrasting differences in endothelial shear stress levels can be observed in vivo near bifurcations, particularly those of the aorta.1Friedman MH O'Brien V Ehrlich LW Calculations of pulsatile flow through a branch: implications for the hemodynamics of atherogenesis.Circ Res. 1975; 36: 277-285Crossref PubMed Scopus (85) Google Scholar, 30Chandran KB Flow dynamics in the human aorta.J Biomech Eng. 1993; 115: 611-616Crossref PubMed Scopus (98) Google Scholar, 31Xu XY Long Q Collins MW Bourne M Griffith TM Reconstruction of blood flow patterns in human arteries.Proc Inst Mech Eng. 1999; 213: 411-421Crossref PubMed Scopus (28) Google Scholar Thus, from the preselected specimens described in the previous paragraph, the following selection was made: the abdominal aorta bifurcation, the common iliac artery, and the branch of the common carotid artery from the aortic arch. In the aortic arch of a 13-year-old donor" @default.
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• W2100031693 date "2005-08-01" @default.
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• W2100031693 title "Endothelial KLF2 Links Local Arterial Shear Stress Levels to the Expression of Vascular Tone-Regulating Genes" @default.
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