αvβ3 Integrins Mediate Flow-Induced NF-κB Activation, Proinflammatory Gene Expression, and Early Atherogenic Inflammation
2015; Elsevier BV; Volume: 185; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2015.05.013
ISSN1525-2191
AutoresJie Chen, Jonette Green, Arif Yurdagul, Patrick Albert, Marshall C. McInnis, A. Wayne Orr,
Tópico(s)Platelet Disorders and Treatments
ResumoEndothelial cell interactions with transitional matrix proteins, such as fibronectin, occur early during atherogenesis and regulate shear stress-induced endothelial cell activation. Multiple endothelial cell integrins bind transitional matrix proteins, including α5β1, αvβ3, and αvβ5. However, the role these integrins play in mediating shear stress-induced endothelial cell activation remains unclear. Therefore, we sought to elucidate which integrin heterodimers mediate shear stress-induced endothelial cell activation and early atherogenesis. We now show that inhibiting αvβ3 integrins (S247, siRNA), but not α5β1 or αvβ5, blunts shear stress-induced proinflammatory signaling (NF-κB, p21-activated kinase) and gene expression (ICAM1, VCAM1). Importantly, inhibiting αvβ3 did not affect cytokine-induced proinflammatory responses or inhibit all shear stress-induced signaling, because Akt, endothelial nitric oxide synthase, and extracellular regulated kinase activation remained intact. Furthermore, inhibiting αv integrins (S247), but not α5 (ATN-161), in atherosclerosis-prone apolipoprotein E knockout mice significantly reduced vascular remodeling after acute induction of disturbed flow. S247 treatment similarly reduced early diet-induced atherosclerotic plaque formation associated with both diminished inflammation (expression of vascular cell adhesion molecule 1, plaque macrophage content) and reduced smooth muscle incorporation. Inducible, endothelial cell-specific αv integrin deletion similarly blunted inflammation in models of disturbed flow and diet-induced atherogenesis but did not affect smooth muscle incorporation. Our studies identify αvβ3 as the primary integrin heterodimer mediating shear stress-induced proinflammatory responses and as a key contributor to early atherogenic inflammation. Endothelial cell interactions with transitional matrix proteins, such as fibronectin, occur early during atherogenesis and regulate shear stress-induced endothelial cell activation. Multiple endothelial cell integrins bind transitional matrix proteins, including α5β1, αvβ3, and αvβ5. However, the role these integrins play in mediating shear stress-induced endothelial cell activation remains unclear. Therefore, we sought to elucidate which integrin heterodimers mediate shear stress-induced endothelial cell activation and early atherogenesis. We now show that inhibiting αvβ3 integrins (S247, siRNA), but not α5β1 or αvβ5, blunts shear stress-induced proinflammatory signaling (NF-κB, p21-activated kinase) and gene expression (ICAM1, VCAM1). Importantly, inhibiting αvβ3 did not affect cytokine-induced proinflammatory responses or inhibit all shear stress-induced signaling, because Akt, endothelial nitric oxide synthase, and extracellular regulated kinase activation remained intact. Furthermore, inhibiting αv integrins (S247), but not α5 (ATN-161), in atherosclerosis-prone apolipoprotein E knockout mice significantly reduced vascular remodeling after acute induction of disturbed flow. S247 treatment similarly reduced early diet-induced atherosclerotic plaque formation associated with both diminished inflammation (expression of vascular cell adhesion molecule 1, plaque macrophage content) and reduced smooth muscle incorporation. Inducible, endothelial cell-specific αv integrin deletion similarly blunted inflammation in models of disturbed flow and diet-induced atherogenesis but did not affect smooth muscle incorporation. Our studies identify αvβ3 as the primary integrin heterodimer mediating shear stress-induced proinflammatory responses and as a key contributor to early atherogenic inflammation. Although traditional risk factors for atherosclerosis, such as hypercholesterolemia and hyperglycemia, are systemic throughout the circulation, atherosclerotic plaques form at discrete areas of the vasculature where vessel geometry results in altered hemodynamics.1Hahn C. Schwartz M.A. Mechanotransduction in vascular physiology and atherogenesis.Nat Rev Mol Cell Biol. 2009; 10: 53-62Crossref PubMed Scopus (812) Google Scholar, 2Zhou J. Li Y.S. Chien S. Shear stress-initiated signaling and its regulation of endothelial function.Arterioscler Thromb Vasc Biol. 2014; 34: 2191-2198Crossref PubMed Scopus (295) Google Scholar Endothelial cells respond to the frictional force generated by these flow patterns, termed shear stress, and convert them into intracellular biochemical signals that critically modulate endothelial cell function. In straight regions of arteries, shear stress generated by unidirectional, laminar flow promotes nitric oxide production and limits endothelial cell activation, consistent with the absence of atherosclerosis in these areas.1Hahn C. Schwartz M.A. Mechanotransduction in vascular physiology and atherogenesis.Nat Rev Mol Cell Biol. 2009; 10: 53-62Crossref PubMed Scopus (812) Google Scholar, 2Zhou J. Li Y.S. Chien S. Shear stress-initiated signaling and its regulation of endothelial function.Arterioscler Thromb Vasc Biol. 2014; 34: 2191-2198Crossref PubMed Scopus (295) Google Scholar In contrast, shear stress generated by disturbed flow patterns, such as those observed at sites of vessel branch points, bifurcations, and curvatures, results in endothelial cell activation with enhanced proinflammatory gene expression [intercellular adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1)] and permeability.1Hahn C. Schwartz M.A. Mechanotransduction in vascular physiology and atherogenesis.Nat Rev Mol Cell Biol. 2009; 10: 53-62Crossref PubMed Scopus (812) Google Scholar, 2Zhou J. Li Y.S. Chien S. Shear stress-initiated signaling and its regulation of endothelial function.Arterioscler Thromb Vasc Biol. 2014; 34: 2191-2198Crossref PubMed Scopus (295) Google Scholar In addition to flow patterns, cell matrix interactions can also affect local endothelial cell activation. Endothelial cells typically reside on basement membrane proteins (ie, collagen IV and laminin), which resist endothelial cell activation and promote a quiescent phenotype.3Funk S.D. Yurdagul Jr., A. Green J.M. Jhaveri K.A. Schwartz M.A. Orr A.W. Matrix-specific protein kinase A signaling regulates p21-activated kinase activation by flow in endothelial cells.Circ Res. 2010; 106: 1394-1403Crossref PubMed Scopus (48) Google Scholar, 4Orr A.W. Sanders J.M. Bevard M. Coleman E. Sarembock I.J. Schwartz M.A. The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.J Cell Biol. 2005; 169: 191-202Crossref PubMed Scopus (225) Google Scholar, 5Yurdagul Jr., A. Chen J. Funk S.D. Albert P. Kevil C.G. Orr A.W. Altered nitric oxide production mediates matrix-specific PAK2 and NF-kappaB activation by flow.Mol Biol Cell. 2013; 24: 398-408Crossref PubMed Scopus (37) Google Scholar However, during early atherogenesis, the subendothelial matrix transitions into a fibronectin and fibrinogen-rich matrix.4Orr A.W. Sanders J.M. Bevard M. Coleman E. Sarembock I.J. Schwartz M.A. The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.J Cell Biol. 2005; 169: 191-202Crossref PubMed Scopus (225) Google Scholar, 6Feaver R.E. Gelfand B.D. Wang C. Schwartz M.A. Blackman B.R. Atheroprone hemodynamics regulate fibronectin deposition to create positive feedback that sustains endothelial inflammation.Circ Res. 2010; 106: 1703-1711Crossref PubMed Scopus (86) Google Scholar Cell culture models suggest that transitional matrix proteins enhance the endothelial proinflammatory response to multiple atherogenic risk factors, including both shear stress and oxidized low-density lipoprotein (oxLDL).4Orr A.W. Sanders J.M. Bevard M. Coleman E. Sarembock I.J. Schwartz M.A. The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.J Cell Biol. 2005; 169: 191-202Crossref PubMed Scopus (225) Google Scholar, 7Yurdagul Jr., A. Green J. Albert P. McInnis M.C. Mazar A.P. Orr A.W. alpha5beta1 integrin signaling mediates oxidized low-density lipoprotein-induced inflammation and early atherosclerosis.Arterioscler Thromb Vasc Biol. 2014; 34: 1362-1373Crossref PubMed Scopus (68) Google Scholar Transitional matrix proteins enhance shear stress-induced endothelial cell activation by promoting p21-activated kinase 2 (PAK2) signaling, which activates the transcription NF-κB to drive proinflammatory gene expression (ie, ICAM1 and VCAM1).5Yurdagul Jr., A. Chen J. Funk S.D. Albert P. Kevil C.G. Orr A.W. Altered nitric oxide production mediates matrix-specific PAK2 and NF-kappaB activation by flow.Mol Biol Cell. 2013; 24: 398-408Crossref PubMed Scopus (37) Google Scholar, 8Orr A.W. Hahn C. Blackman B.R. Schwartz M.A. p21-activated kinase signaling regulates oxidant-dependent NF-kappa B activation by flow.Circ Res. 2008; 103: 671-679Crossref PubMed Scopus (74) Google Scholar Limiting fibronectin deposition, either genetically or through peptide inhibitors, blunts endothelial proinflammatory signaling (PAK2, NF-κB) and gene expression both in models of acute disturbed flow-induced vascular inflammation (partial carotid ligation) and models of diet-induced spontaneous atherosclerosis.7Yurdagul Jr., A. Green J. Albert P. McInnis M.C. Mazar A.P. Orr A.W. alpha5beta1 integrin signaling mediates oxidized low-density lipoprotein-induced inflammation and early atherosclerosis.Arterioscler Thromb Vasc Biol. 2014; 34: 1362-1373Crossref PubMed Scopus (68) Google Scholar, 9Chiang H.Y. Korshunov V.A. Serour A. Shi F. Sottile J. Fibronectin is an important regulator of flow-induced vascular remodeling.Arterioscler Thromb Vasc Biol. 2009; 29: 1074-1079Crossref PubMed Scopus (128) Google Scholar, 10Rohwedder I. Montanez E. Beckmann K. Bengtsson E. Duner P. Nilsson J. Soehnlein O. Fassler R. Plasma fibronectin deficiency impedes atherosclerosis progression and fibrous cap formation.EMBO Mol Med. 2012; 4: 564-576Crossref PubMed Scopus (84) Google Scholar The integrin family of matrix receptors consists of 18 α and 8 β subunits that assemble into 24 different integrin heterodimers with distinct matrix-binding affinities and signaling properties.11Hynes R.O. Integrins: bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6851) Google Scholar Fluid shear stress activates multiple endothelial cell integrins, including the collagen-binding integrin α2β1 and the transitional matrix-binding integrins α5β1 and αvβ3.12Orr A.W. Ginsberg M.H. Shattil S.J. Deckmyn H. Schwartz M.A. Matrix-specific suppression of integrin activation in shear stress signaling.Mol Biol Cell. 2006; 17: 4686-4697Crossref PubMed Scopus (124) Google Scholar Blocking the ligation of these transitional matrix-binding integrins by using fibronectin antibodies that mask the main integrin-binding sites significantly inhibits shear stress-induced proinflammatory responses.3Funk S.D. Yurdagul Jr., A. Green J.M. Jhaveri K.A. Schwartz M.A. Orr A.W. Matrix-specific protein kinase A signaling regulates p21-activated kinase activation by flow in endothelial cells.Circ Res. 2010; 106: 1394-1403Crossref PubMed Scopus (48) Google Scholar, 4Orr A.W. Sanders J.M. Bevard M. Coleman E. Sarembock I.J. Schwartz M.A. The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.J Cell Biol. 2005; 169: 191-202Crossref PubMed Scopus (225) Google Scholar, 13Tzima E. Del Pozo M.A. Kiosses W.B. Mohamed S.A. Li S. Chien S. Schwartz M.A. Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression.EMBO J. 2002; 21: 6791-6800Crossref PubMed Scopus (293) Google Scholar However, the specific role of these transitional matrix-binding integrins in shear stress-induced endothelial cell activation remains undefined. Both α5β1 and αvβ3 show enhanced expression in the endothelial layer overlying the atherosclerotic plaque,7Yurdagul Jr., A. Green J. Albert P. McInnis M.C. Mazar A.P. Orr A.W. alpha5beta1 integrin signaling mediates oxidized low-density lipoprotein-induced inflammation and early atherosclerosis.Arterioscler Thromb Vasc Biol. 2014; 34: 1362-1373Crossref PubMed Scopus (68) Google Scholar, 14Hoshiga M. Alpers C.E. Smith L.L. Giachelli C.M. Schwartz S.M. Alpha-v beta-3 integrin expression in normal and atherosclerotic artery.Circ Res. 1995; 77: 1129-1135Crossref PubMed Scopus (211) Google Scholar and both α5β1 and αvβ3 are implicated in endothelial NF-κB activation in other systems.7Yurdagul Jr., A. Green J. Albert P. McInnis M.C. Mazar A.P. Orr A.W. alpha5beta1 integrin signaling mediates oxidized low-density lipoprotein-induced inflammation and early atherosclerosis.Arterioscler Thromb Vasc Biol. 2014; 34: 1362-1373Crossref PubMed Scopus (68) Google Scholar, 15Scatena M. Almeida M. Chaisson M.L. Fausto N. Nicosia R.F. Giachelli C.M. NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival.J Cell Biol. 1998; 141: 1083-1093Crossref PubMed Scopus (446) Google Scholar Therefore, we sought to elucidate which integrin heterodimer regulates shear stress-induced endothelial cell activation and early atherogenesis. Human aortic endothelial cells [HAECs; purchased from Lonza (Baltimore, MD) at passage 3] were cultured in MCDB-131 that contained 10% fetal bovine serum, 60 μg/mL heparin, 24 μg/mL bovine brain extract, 10 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO, Carlsbad, CA). HAECs were used between passages 6 through 10. To perform in vitro shear stress experiments, glass slides were coated with 10 μg/mL fibronectin with or without 10 μg/mL vitronectin overnight and blocked with 0.2% bovine serum albumen. HAECs were then plated onto the slides in low serum media (0.5% to 1% fetal bovine serum) and subjected to either laminar flow (12 dynes/cm2) or oscillatory flow (±0.5 dynes/cm2 superimposed with 1 dyne/cm2) by using parallel-plate flow apparatus with the environment maintained at 37°C and 5% CO2 as previously described.8Orr A.W. Hahn C. Blackman B.R. Schwartz M.A. p21-activated kinase signaling regulates oxidant-dependent NF-kappa B activation by flow.Circ Res. 2008; 103: 671-679Crossref PubMed Scopus (74) Google Scholar HAECs at 70% confluence were transfected with siRNA oligos to α5, αv, β3, or β5 (SMARTpool; Dharmacon, Lafayette, CO) by using Lipofectamine 2000 for 2.5 hours on two consecutive days. Cell lysis and immunoblotting were performed as previously described.4Orr A.W. Sanders J.M. Bevard M. Coleman E. Sarembock I.J. Schwartz M.A. The subendothelial extracellular matrix modulates NF-kappaB activation by flow: a potential role in atherosclerosis.J Cell Biol. 2005; 169: 191-202Crossref PubMed Scopus (225) Google Scholar Lysates separated by SDS-PAGE were transferred to polyvinylidene difluoride membranes, and membranes were blocked in 5% nonfat dry milk before addition of primary antibodies. Antibodies include rabbit anti–phospho-Akt Thr473, rabbit anti–phospho-endothelial nitric oxide synthase (eNOS) Ser1177, rabbit anti–phospho-eNOS Thr495, rabbit anti–phospho-extracellular regulated kinase (ERK1/2), rabbit anti–phospho-focal adhesion kinase (FAK) Tyr397, rabbit anti–glyceraldehyde-3-phosphate dehydrogenase, rabbit anti–ICAM-1, rabbit anti-integrin αv, rabbit anti-integrin β3, rabbit anti-integrin β5, rabbit anti–phospho–NF-κB (p65 subunit, Ser536), rabbit anti–NF-κB (p65), rabbit anti-PAK2 (Cell Signaling Technology Inc., Danvers, MA), goat anti-Akt, rabbit anti-ERK1/2, rabbit anti-FAK, rabbit anti-integrin α5, rabbit anti–VCAM-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti–phospho-eNOS Ser633 (Millipore Corporation, Billerica, MA), mouse anti-eNOS (Becton Dickinson, Franklin Lakes, NJ), and rabbit anti–phospho-PAK (Ser141; Invitrogen, Carlsbad, CA). Densitometry was performed with ImageJ software version 1.45s (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Endothelial cells were fixed in formaldehyde, permeabilized, and stained for the NF-κB p65 subunit (dilution 1:200; Santa Cruz Biotechnology, Inc.). Staining was visualized with Alexa-conjugated secondary antibodies on a TiU epifluorescence microscope (Nikon, Melville, NY), and images were collected with the CoolSNAP120 ES2 camera (Photometrics, Tucson, AZ). Cells were scored as positive or negative for nuclear NF-κB staining with 100 cells counted per condition for each experiment. mRNA isolated from tissues and cultured endothelial cells was extracted with TRIzol (Life Technologies, Inc., Carlsbad, CA). cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative real-time PCR (qPCR) was performed with a Bio-Rad iCycler with the use of SYBR Green Master mix (Bio-Rad). Primers were designed with the online Primer3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and then validated by sequencing the PCR products (Table 1). Results were expressed as fold change by using the 2ΔΔCT method.Table 1Quantitative Real-Time PCR PrimersGeneForward primerReverse primerHuman GAPDH5′-GAAGGTGAAGGTCGGAGTC-3′5′-GAAGATGGTGATGGGATTTC-3′ B2M5′-AGCATTCGGGCCGAGATGTCT-3′5′-CTGCTGGATGACGTGAGTAAACCT−3′ ITGA15′-GCCAACCCAAAAACAGAAC-3′5′-CACAAGCCAGAAATCCTCCA-3′ ITGA25′-GGATTTGTTTGGCTGACTGG-3′5′-TGAAGGAGTGGGAACTTTGG-3′ ITGA35′-CTTCAAACGGAACCAGAGGA-3′5′-ATGCCAGACTCACCCATCAC-3′ ITGA45′-TCCAGAGCCAAATCCAAGAG-3′5′-GCCAGCCTTCCACATAACAT-3′ ITGA55′-TAGTGCCTCCCTCACCATCT-3′5′-TACCCCTCCCTTCTGCTTCT-3′ ITGA65′-GATAAACTGCGTCCCATTCC-3′5′-TCGTCTCCACATCCCTCTTT-3′ ITGA75′-CCTTTGATGGTGATGGGAAA-3′5′-CAGCAGGTCAGGGTATTGGT-3′ ITGA85′-GATGGTGGTGTGTGACCTTG-3′5′-TGCTGTCTGGATTGTCCTTG-3′ ITGA95′-AAGGCATCGGCAAGGTTTAT-3′5′-AGGGCTCCATTTCCTCTGTT-3′ ITGA105′-GCTCAGATGGGACAAGAAGC-3′5′-CCAGTGTTGGCTATGGAACC-3′ ITGA115′-GAAGGGTGGAAGGAAAGGAG-3′5′-GGGTCACTGGCGATGTATTT-3′ ITGAV5′-TGGTCTTCGTTTCAGTGTGC-3′5′-TCTCCTTGTGCTCCCAGTTT-3′ ITGB15′-CTGATTGGCTGGAGGAATGT-3′5′-TTTCTGGACAAGGTGAGCAA-3′ ITGB25′-GGTCATCTGGAAGGCTCTGA-3′5′-CACCAAGTGCTCCTAACTCTCA-3′ ITGB35′-CCTCCTGTCCCTCATCCATA-3′5′-TCCATCCTGGCACTTATTCC-3′ ITGB45′-GGAGTGTGTCCGTGTGGATA-3′5′-GGTGGTGTCAATCTGGGTCT-3′ ITGB55′-CAGGTTACATCGGGGACAAC-3′5′-ACGCAATCTCTCTTGGTGCT-3′ ITGB65′-ATGGGGATTGAACTGCTTTG-3′5′-AAGGTTTGCTGGGGTATCAC-3′ ITGB75′-GTCGCAGCACAGAGTTTGAC-3′5′-CCAGGAGGGAGTGAAGAGTG-3′ ITGB85′-CGAGGAGTTTGTGTTTGTGG-3′5′-CACTGAAGCATTGGCATCTG-3′ VCAM15′-ATGAGGGGACCACATCTACG-3′5′-CACCTGGATTCCTTTTTCCA-3′ ICAM15′-TGTCCCCCTCAAAAGTCATC-3′5′-TAGGCAACGGGGTCTCTATG-3′Mouse B2m5′-TTCTGGTGCTTGTCTCACTGA-3′5′-CAGTATGTTCGGCTTCCCATTC-3′ Vcam15′-TCAAAGAAAGGGAGACTG-3′5′-GCTGGAGAACTTCATTATC-3′ Icam15′-GTGATGCTCAGGTATCCATCCA-3′5′-CACAGTTCTCAAAGCACAGCG-3′ Itga55′-CGTTGAGTCATTCGCCTCTGG-3′5′-GTGCCCGCTCTTCCCTGTC-3′ Itgav5′-TCCATTGTACCACTGGAGGA-3′5′-TTTGACCTGCATGGAGCATA-3′ Klf25′-AGAATGCACCTGAGCCTGCTAG-3′5′-AATTTCCCCGAAAGCCTGC-3′ Open table in a new tab The Louisiana State University Health Sciences Center-Shreveport Animal Care and Use Committee approved all animal protocols, and all animals were cared for according to the NIH Guide for the Care and Use of Laboratory Animals.16Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar Male Apoe−/− mice on the C57Bl/6J genetic background were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice that contained the αvfl/fl allele (a gift from Dr. Richard Hynes, MIT, Cambridge, MA) and mice that contained the vascular endothelial (VE)-cadherinCreERT2 transgene (a gift of Dr. Luisa Iruela-Arispe, UCLA, Los Angeles, CA), both on the C57Bl/6J background, were crossed with Apoe−/−.17Lacy-Hulbert A. Smith A.M. Tissire H. Barry M. Crowley D. Bronson R.T. Roes J.T. Savill J.S. Hynes R.O. Ulcerative colitis and autoimmunity induced by loss of myeloid alphav integrins.Proc Natl Acad Sci U S A. 2007; 104: 15823-15828Crossref PubMed Scopus (180) Google Scholar, 18Monvoisin A. Alva J.A. Hofmann J.J. Zovein A.C. Lane T.F. Iruela-Arispe M.L. VE-cadherin-CreERT2 transgenic mouse: a model for inducible recombination in the endothelium.Dev Dyn. 2006; 235: 3413-3422Crossref PubMed Scopus (178) Google Scholar Genotype was determined by PCR reactions by using DNA isolated from tail snips and genotyping primers (Apoe forward common, 5′-GCCTAGCCGAGGGAGAGCCG-3′; Apoe wild-type reverse, 5′-GCCGCCCCGACTGCATCT-3′; Apoe mutant reverse, 5′-TGTGACTTGGGAGCTCTGCAGC-3′; ITGAV forward, 5′-TTCAGGACGGCACAAAGACCGTTG-3′; ITGAV reverse, 5′-CACAAATCAAGGATGACCAAACTGAG-3′; Cf, 5′-ACTGGGATCTTCGAACTCTTTGGAC-3′; Cr, 5′-GATGTTGGGGCACTGCTCATTCACC-3′; Cref, 5′-CCATCTGCCACCAGCCAG-3′; Crer, 5′-TCGCCATCTTCCAGCAGG-3′) as previously described.17Lacy-Hulbert A. Smith A.M. Tissire H. Barry M. Crowley D. Bronson R.T. Roes J.T. Savill J.S. Hynes R.O. Ulcerative colitis and autoimmunity induced by loss of myeloid alphav integrins.Proc Natl Acad Sci U S A. 2007; 104: 15823-15828Crossref PubMed Scopus (180) Google Scholar Male inducible endothelial cell (iEC)-αv knockout (KO) mice (Apoe−/−, VE-cadherinCreERT2tg/?, αvfl/fl) and iEC-Control (Apoe−/−, VE-cadherinCreERT2tg/?) mice were treated with 1 mg/kg tamoxifen i.p. (Sigma-Aldrich, St. Louis, MO) every other day for five total injections to induce Cre expression and αv excision. Eight- to 10-week-old mice were fed a high-fat, Western diet (TD 88137; Harlan-Teklad, Madison, WI) that contained 21% fat by weight (0.15% cholesterol and 19.5% casein without sodium cholate) for 8 weeks. Mice were then euthanized by pneumothorax under isoflurane anesthesia, and blood was collected. Total cholesterol, high-density lipoprotein cholesterol (Wako Bioproducts, Richmond, VA), and triglycerides (Pointe Scientific, Canton MI) were analyzed with commercially available kits. LDL cholesterol was calculated with the Friedewald equation. Hearts were then perfused with phosphate-buffered saline to remove residual blood from the circulation. The lungs were collected for enzymatic digestion and endothelial cell isolation by using magnetic beads coupled to ICAM2 antibodies (eBiosource, San Diego, CA).19Sobczak M. Dargatz J. Chrzanowska-Wodnicka M. Isolation and culture of pulmonary endothelial cells from neonatal mice.J Vis Exp. 2010; : 2316PubMed Google Scholar The left common carotid was collected for mRNA isolation by TRIzol flush as previously described.20Nam D. Ni C.W. Rezvan A. Suo J. Budzyn K. Llanos A. Harrison D. Giddens D. Jo H. Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.Am J Physiol Heart Circ Physiol. 2009; 297: H1535-H1543Crossref PubMed Scopus (339) Google Scholar Briefly, carotids were cleaned of perivascular adipose tissue and flushed with 150 μL TRIzol from an insulin syringe. The remaining media/adventitia were then placed in 150 μL TRIzol and sonicated to lyze the tissue. Samples were then frozen until analysis by qPCR. The aortic root, aorta, and carotid sinus were excised, placed in 3.7% phosphate-buffered saline–buffered formaldehyde, and analyzed for plaque size and composition. After 4 weeks of feeding a Western diet to the Apoe−/− mice, Alzet (Cuperino, CA) pumps (Micro-Osmotic Pump, Model 1004) that contained either saline or 40 mg/kg/day S247 were implanted under isoflurane anesthesia (5% on induction; 2% for maintenance during surgery), and the Western diet feeding was resumed for an additional 4 weeks. To analyze endothelial activation by low flow, partial ligation of the left carotid artery was performed as previously described.20Nam D. Ni C.W. Rezvan A. Suo J. Budzyn K. Llanos A. Harrison D. Giddens D. Jo H. Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.Am J Physiol Heart Circ Physiol. 2009; 297: H1535-H1543Crossref PubMed Scopus (339) Google Scholar Briefly, a 4- to 6-mm vertical incision on the skin was made, and blunt dissection was used to expose the left carotid artery. Subsequently, the external, internal, and occipital arteries were ligated with 7-0 silk suture. The incision was sutured and then closed with a small amount of tissue glue followed by suturing the incision. For the integrin inhibitor studies, mice were implanted with Alzet pumps (Micro-Osmotic pump, Model 1007D) that contained either saline or S247 immediately after the ligation or given 5 mg/kg ATN-161 three times per week by i.p. injection. At the start of surgery, a single injection of 0.1 mg/kg buprenorphine or 5 mg/kg carprofen was given, and recovery of the mice was monitored on a heating pad. All ultrasound measurements were taken with a VEVO 770 high-resolution in vivo microimaging ultrasound system with a 30-MHz mouse probe (VisualSonics, Toronto, ON, Canada). Echocardiography was performed on the left and right carotid arteries 1 day after the partial ligation surgery. Mice were euthanized after 48 hours for mRNA analysis after TRIzol flush of the left and right carotid and after 7 days for immunohistochemical analysis of the left and right carotids excised for analysis. All tissue was fixed in 4% formaldehyde, embedded in paraffin, and cut into 5-μm sections. Immunohistochemistry and Russell-Movat Pentachrome staining was performed as previously described.7Yurdagul Jr., A. Green J. Albert P. McInnis M.C. Mazar A.P. Orr A.W. alpha5beta1 integrin signaling mediates oxidized low-density lipoprotein-induced inflammation and early atherosclerosis.Arterioscler Thromb Vasc Biol. 2014; 34: 1362-1373Crossref PubMed Scopus (68) Google Scholar Antibodies used for mouse tissues included rabbit anti–VCAM-1 (dilution 1:40 or 1:100; Santa Cruz Biotechnology, Inc.), rat anti-Mac2 (dilution 1:10,000; Accurate Chemical, Westbury, NY), and mouse anti-smooth muscle actin (SMA; dilution 1:400; Sigma-Aldrich). Staining was visualized with Alexa-conjugated secondary antibodies. Images were collected with the Photometrics CoolSNAP120 ES2 camera and the NIS Elements 3.00 by using SP5 imaging software. Data were tested for normality (Kolmogorov-Smirnov test) and significance by using Prism software version 5.02 (GraphPad Inc., San Diego, CA). Data that passed the normality assumption were analyzed with t-test or two-way analysis of variance with Bonferroni post-tests. Data that failed the normality assumption were analyzed with the nonparametric U-test and the Kruskal Wallis test with post hoc analysis. Data are expressed as means ± SEM. Differences were considered statistically significant at a value of P < 0.05. Although chronic exposure to laminar flow reduces inflammatory signaling,1Hahn C. Schwartz M.A. Mechanotransduction in vascular physiology and atherogenesis.Nat Rev Mol Cell Biol. 2009; 10: 53-62Crossref PubMed Scopus (812) Google Scholar acute onset of laminar flow stimulates proinflammatory signaling pathways and ICAM-1/VCAM-1 expression similar to disturbed flow models. Therefore, we first sought to determine how individual transitional matrix integrins contribute to endothelial cell activation by acute onset of flow. Consistent with previous reports,21Short S.M. Talbott G.A. Juliano R.L. Integrin-mediated signaling events in human endothelial cells.Mol Biol Cell. 1998; 9: 1969-1980Crossref PubMed Scopus (137) Google Scholar qPCR analysis of HAECs indicated the expression of multiple transitional matrix-binding integrins, including the binding partners α5β1, αvβ3, and αvβ5 (Supplemental Figure S1). To test which of these transitional matrix-binding integrins mediate flow-induced endothelial cell activation, we used specific small-molecule inhibitors or competitive peptides to block integrin signaling. HAECs plated on the transitional matrix protein fibronectin were treated with either 50 μmol/L α5β1 signaling inhibitor ATN-1617Yurdagul Jr., A. Green J. Albert P. McInnis M.C. Mazar A.P. Orr A.W. alpha5beta1 integrin signaling mediates oxidized low-density lipoprotein-induced inflammation and early atherosclerosis.Arterioscler Thromb Vasc Biol. 2014; 34: 1362-1373Crossref PubMed Scopus (68) Google Scholar or 1 μmol/L αv inhibitor S247,22Shannon K.E. Keene J.L. Settle S.L. Duffin T.D. Nickols M.A. Westlin M. Schroeter S. Ruminski P.G. Griggs D.W. Anti-metastatic properties of RGD-peptidomimetic agents S137 and S247.Clin Exp Metastasis. 2004; 21: 129-138Crossref PubMed Scopus (71) Google Scholar and shear stress-induced signaling was assessed. Although an RGD peptidomimetic, S247 shows a 160-fold higher affinity for αvβ3 than for α5β1 and a 950-fold higher affinity for αvβ3 than for the platelet integrin αIIbβ3.22Shannon K.E. Keene J.L. Settle S.L. Duffin T.D. Nickols M.A. Westlin M. Schroeter S. Ruminski P.G. Griggs D.W. Anti-metastatic properties of RGD-peptidomimetic agents S137 and S247.Clin Exp Metastasis. 2004; 21: 129-138Crossref PubMed Scopus (71) Google Scholar Inhibiting αv integrins with S247 completely suppressed shear-induced activation of FAK (Figure 1, A and B), PAK2 (Figure 1, A and C), and NF-κB (Figure 1, A and D), whereas ATN-161 did not. In contrast, neither inhibitor affected shear stress-induced activation of ERK1/2 (Figure 1, A and E) or Akt (Supplemental Figure S2, A and B), and neither affected shear-induced eNOS Ser1177 phosphorylation (Figure 1A and Supplemental Figure S2C) or Thr495 dephosphorylation (Supplemental Figure S2, A and D). However, shear stress-induced eNOS phosphorylation on Ser633 showed significant reductions with S247 treatment but not ATN-161 (Supplemental Figure S2, A and E). To confirm these results, endothelial cells were transfected with α5 and αv siRNA to selectively deplete these integrins. Knockdown efficiencies were similar with an approximately 80% knockdown of each integrin, and no compensation was observed after knockdown of either integrin (Supplem
Referência(s)