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• W2005184523 abstract "Elastic laminae are extracellular matrix constituents that not only contribute to the stability and elasticity of arteries but also play a role in regulating arterial morphogenesis and pathogenesis. We demonstrate here that an important function of arterial elastic laminae is to prevent monocyte adhesion, which is mediated by the inhibitory receptor signal regulatory protein (SIRP) α and Src homology 2 domain-containing protein-tyrosine phosphatase (SHP)-1. In a matrix-based arterial reconstruction model in vivo, elastic laminae were resistant to leukocyte adhesion and transmigration compared with the collagen-dominant arterial adventitia. The density of leukocytes within the elastic lamina-dominant media was about 58-70-fold lower than that within the adventitia from 1 to 30 days. An in vitro assay confirmed the inhibitory effect of elastic laminae on monocyte adhesion. The exposure of monocytes to elastic laminae induced activation of SIRP α, which in turn activated SHP-1. Elastic lamina degradation peptides extracted from arterial specimens could also activate SIRP α and SHP-1. The knockdown of SIRP α and SHP-1 by specific small interfering RNA diminished the inhibitory effect of elastic laminae, resulting in a significant increase in monocyte adhesion. These observations suggest that SIRP α and SHP-1 potentially mediate the inhibitory effect of elastic laminae on monocyte adhesion. Elastic laminae are extracellular matrix constituents that not only contribute to the stability and elasticity of arteries but also play a role in regulating arterial morphogenesis and pathogenesis. We demonstrate here that an important function of arterial elastic laminae is to prevent monocyte adhesion, which is mediated by the inhibitory receptor signal regulatory protein (SIRP) α and Src homology 2 domain-containing protein-tyrosine phosphatase (SHP)-1. In a matrix-based arterial reconstruction model in vivo, elastic laminae were resistant to leukocyte adhesion and transmigration compared with the collagen-dominant arterial adventitia. The density of leukocytes within the elastic lamina-dominant media was about 58-70-fold lower than that within the adventitia from 1 to 30 days. An in vitro assay confirmed the inhibitory effect of elastic laminae on monocyte adhesion. The exposure of monocytes to elastic laminae induced activation of SIRP α, which in turn activated SHP-1. Elastic lamina degradation peptides extracted from arterial specimens could also activate SIRP α and SHP-1. The knockdown of SIRP α and SHP-1 by specific small interfering RNA diminished the inhibitory effect of elastic laminae, resulting in a significant increase in monocyte adhesion. These observations suggest that SIRP α and SHP-1 potentially mediate the inhibitory effect of elastic laminae on monocyte adhesion. Arterial elastic laminae have long been considered a structure that determines the strength and elasticity of blood vessels (1Hinek A. Biol. Chem. 1996; 377: 471-480PubMed Google Scholar, 2Mecham R.P. Broekelmann T. Davis E.C. Gibson M.A. Brown-Augsburger P. CIBA Found. Symp. 1995; 192: 172-181PubMed Google Scholar, 3Robert L. Connect. Tissue Res. 1999; 40: 75-82Crossref PubMed Scopus (30) Google Scholar, 4Urry D.W. Hugel T. Seitz M. Gaub H.E. Sheiba L. Dea J. Xu J. Parker T. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2002; 357: 169-184Crossref PubMed Scopus (242) Google Scholar, 5Vrhovski B. Weiss A.S. Eur. J. Biochem. 1998; 258: 1-18Crossref PubMed Scopus (394) Google Scholar, 6Wong L.C. Langille B.L. Circ. Res. 1996; 78: 799-805Crossref PubMed Scopus (121) Google Scholar). Recent studies, however, have demonstrated that arterial elastic laminae also participate in the regulation of arterial morphogenesis and pathogenesis (7Brooke B.S. Karnik S.K. Li D.Y. Trends Cell Biol. 2003; 13: 51-56Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8Karnik S.K. Brooke B.S. Bayes-Genis A. Sorensen L. Wythe J.D. Schwartz R.S. Keating M.T. Li D.Y. Development (Camb.). 2003; 130: 411-423Crossref PubMed Scopus (370) Google Scholar, 9Li D.Y. Brooke B Davis E.C. Mecham R.P. Sorensen L.K. Boak B.B. Eichwald E. Keating M.T. Nature. 1998; 393: 276-280Crossref PubMed Scopus (633) Google Scholar, 10Liu S.Q. Tieche C. Alkema P.K. Biomaterials. 2004; 25: 1869-1882Crossref PubMed Scopus (33) Google Scholar, 11Merrilees M.J. Lemire J. Fischer J. Braun K. Kinsella M. Clowes A. Wight T.N. Circ. Res. 2002; 90: 481-487Crossref PubMed Scopus (83) Google Scholar, 12Urban Z. Riazi S. Seidl T.L. Katahiram J. Smoot L.B. Chitayat D. Boyd C.D. Hinek A. Am. J. Hum. Genet. 2002; 71: 30-44Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). An important contribution of elastic laminae is to confine smooth muscle cells (SMCs) 2The abbreviations used are:SMCssmooth muscle cellsSIRPsignal regulatory proteinSHPSH2 domain-containing protein-tyrosine phosphatasesiRNAsmall interfering RNAFITCfluorescein isothiocyanateELDPselastic lamina degradation peptides to the arterial media by inhibiting SMC proliferation (8Karnik S.K. Brooke B.S. Bayes-Genis A. Sorensen L. Wythe J.D. Schwartz R.S. Keating M.T. Li D.Y. Development (Camb.). 2003; 130: 411-423Crossref PubMed Scopus (370) Google Scholar, 9Li D.Y. Brooke B Davis E.C. Mecham R.P. Sorensen L.K. Boak B.B. Eichwald E. Keating M.T. Nature. 1998; 393: 276-280Crossref PubMed Scopus (633) Google Scholar) and migration (10Liu S.Q. Tieche C. Alkema P.K. Biomaterials. 2004; 25: 1869-1882Crossref PubMed Scopus (33) Google Scholar), thus preventing intimal hyperplasia under physiological conditions. Arterial elastic laminae also exhibit thrombosis-resistant properties. When implanted in an artery, elastic lamina scaffolds are associated with significantly lower leukocyte adhesion and thrombosis compared with collagen matrix scaffolds (10Liu S.Q. Tieche C. Alkema P.K. Biomaterials. 2004; 25: 1869-1882Crossref PubMed Scopus (33) Google Scholar). These observations suggest an inhibitory role for elastic laminae relative to collagen matrix. Although such a role is well documented, the mechanisms remain poorly understood. smooth muscle cells signal regulatory protein SH2 domain-containing protein-tyrosine phosphatase small interfering RNA fluorescein isothiocyanate elastic lamina degradation peptides Leukocytes are known to express the inhibitory receptor SIRP α (also known as Src homology 2 domain-containing tyrosine phosphatase substrate-1), a transmembrane glycoprotein receptor that exerts an inhibitory effect on cell mitogenic (13Adams S. van der Laan L.J. Vernon-Wilson E. Renardel de Lavalette C. Dopp E.A. Dijkstra C.D. Simmons D.L. van den Berg T.K. J. Immunol. 1998; 161: 1853-1859PubMed Google Scholar, 14Berg K.L. Carlberg K. Rohrschneider L.R. Siminovitch K.A. Stanley E.R. Oncogene. 1998; 17: 2535-2541Crossref PubMed Scopus (39) Google Scholar, 15Kharitonenkov A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (549) Google Scholar, 16Gardai S.J. Xiao Y.O. Dickinson M. Nick J.A. Voelker D.R. Greene K.E. Henson P.M. Cell. 2003; 115: 13-23Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 17Lienard H. Bruhns P. Malbec O. Fridman W.H. Daeron M. J. Biol. Chem. 1999; 274: 32493-32499Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18Stofega M.R. Argetsinger L.S. Wang H. Ullrich A. Carter-Su C. J. Biol. Chem. 2000; 275: 28222-28229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and inflammatory (19Oldenborg P.A. Gresham H.D. Lindberg F.P. J. Exp. Med. 2001; 193: 855-862Crossref PubMed Scopus (342) Google Scholar, 20Yamao T. Noguchi T. Takeuchi O. Nishiyama U. Morita H. Hagiwara T. Akahori H. Kato T. Inagaki K. Okazawa H. Hayashi Y. Matozaki T. Takeda K. Akira S. Kasuga M. J. Biol. Chem. 2002; 277: 39833-39839Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) activities. Upon ligand binding, SIRP α transmits inhibitory signals through tyrosine phosphorylation of its intracellular immunoreceptor tyrosine-based inhibitory motif (15Kharitonenkov A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (549) Google Scholar, 16Gardai S.J. Xiao Y.O. Dickinson M. Nick J.A. Voelker D.R. Greene K.E. Henson P.M. Cell. 2003; 115: 13-23Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 17Lienard H. Bruhns P. Malbec O. Fridman W.H. Daeron M. J. Biol. Chem. 1999; 274: 32493-32499Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18Stofega M.R. Argetsinger L.S. Wang H. Ullrich A. Carter-Su C. J. Biol. Chem. 2000; 275: 28222-28229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 21Oshima K. Ruhul Amin A.R. Suzuki A. Hamaguchi M. Matsuda S. FEBS Lett. 2002; 519: 1-7Crossref PubMed Scopus (94) Google Scholar, 22Veillette A. Thibaudeau E. Latour S. J. Biol. Chem. 1998; 273: 22719-22728Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The phosphorylation of the immunoreceptor tyrosine-based inhibitory motif initiates the recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase (SHP)-1 to SIRP α, which is known as a substrate of SHP-1 (21Oshima K. Ruhul Amin A.R. Suzuki A. Hamaguchi M. Matsuda S. FEBS Lett. 2002; 519: 1-7Crossref PubMed Scopus (94) Google Scholar, 22Veillette A. Thibaudeau E. Latour S. J. Biol. Chem. 1998; 273: 22719-22728Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The recruitment of SHP-1 also localizes and activates SHP-1 (23Neel B.G. Gu H. Pao L. Bradshaw R.A. Dennis E.A. Handbook of Cell Signaling. Vol. 1. Academic Press, Amsterdam2004: 707-728Google Scholar), which in turn dephosphorylates protein kinases, possibly including receptor tyrosine kinases (23Neel B.G. Gu H. Pao L. Bradshaw R.A. Dennis E.A. Handbook of Cell Signaling. Vol. 1. Academic Press, Amsterdam2004: 707-728Google Scholar, 24Timms J.F. Carlberg K. Gu H. Chen H. Kamatkar S. Nadler M.J. Rohrschneider L.R. Neel B.G. Mol. Cell. Biol. 1998; 18: 3838-3850Crossref PubMed Scopus (174) Google Scholar, 25Tran K.T. Rusu S.O. Satish L. Wells A. Exp. Cell Res. 2003; 289: 359-367Crossref PubMed Scopus (40) Google Scholar), the Src family protein-tyrosine kinases (26Roach T.I. Slater S.E. White L.S. Zhang X. Majerus P.W. Brown E.J. Thomas M.L. Curr. Biol. 1998; 8: 1035-1038Abstract Full Text Full Text PDF PubMed Google Scholar), phosphatidylinositol 3-kinase (26Roach T.I. Slater S.E. White L.S. Zhang X. Majerus P.W. Brown E.J. Thomas M.L. Curr. Biol. 1998; 8: 1035-1038Abstract Full Text Full Text PDF PubMed Google Scholar), and the Janus family tyrosine kinases (27David M. Chen H.E. Goelz S. Larner A.C. Neel B.G. Mol. Cell. Biol. 1995; 15: 7050-7058Crossref PubMed Scopus (318) Google Scholar, 28Klingmuller U. Lorenx U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (842) Google Scholar). These activities potentially suppress inflammatory and mitogenic responses (29Zhang J. Somani A.K. Siminovitch K.A. Semin. Immunol. 2000; 12: 361-378Crossref PubMed Scopus (290) Google Scholar, 30Dong Q. Siminovitch K.A. Fialkow L. Fukushima T. Downey G.P. J. Immunol. 1999; 162: 3220-3230PubMed Google Scholar, 31Kamata T. Yamashita M. Kimura M. Murata K. Inami M. Shimizu C. Sugaya K. Wang C.R. Taniguchi M. Nakayama T. J. Clin. Investig. 2003; 111: 109-119Crossref PubMed Scopus (92) Google Scholar, 32Daigle I. Yousefi S. Colonna M. Green D.R. Simon H.U. Nat. Med. 2002; 8: 61-67Crossref PubMed Scopus (160) Google Scholar). Because the inhibitory effect of elastic laminae coincides with the activity of the inhibitory receptor in leukocytes, it is conceivable that, upon contacting leukocytes, elastic laminae may interact with SIRP α and activate SHP-1, leading to the inhibition of leukocyte adhesion. In this study, we test the possibility that SIRP α and SHP-1 mediate the inhibitory effect of elastic laminae. Matrix-based Aortic Reconstruction—We have established a matrix-based aortic reconstruction model to test the inhibitory effect of elastic laminae on leukocyte transmigration relative to collagen matrix. Aortic substitutes were constructed with three types of aortic matrix scaffold as follows: NaOH-treated matrix scaffolds with an elastic lamina blood-contacting surface, untreated matrix scaffolds with a basal lamina blood-contacting surface, and NaOH-treated matrix scaffolds with an adventitial blood-contacting surface. The first type of aortic substitute was created by treating fresh rat aortic specimens with 0.1 m NaOH at 20 °C for 2 h, followed by washing in distilled water for 12 h with vigorous agitation. Such a treatment removes the cellular components, basal lamina, and medial collagen matrix, leaving an aortic matrix scaffold with medial elastic laminae and collagen-dominant adventitia, as detected by immunohistochemistry (10Liu S.Q. Tieche C. Alkema P.K. Biomaterials. 2004; 25: 1869-1882Crossref PubMed Scopus (33) Google Scholar). The aortic substitutes with untreated matrix scaffolds and a basal lamina blood-contacting surface were constructed by freezing (-76 °C) and thawing (20 °C) fresh aortic specimens for three cycles, followed by washing in distilled water with vigorous agitation for 12 h. Such a treatment removes endothelial cells and destroys other cell types, leaving a matrix scaffold with intact matrix and a basal lamina blood-contacting surface. The removal of endothelial cells was verified by immunohistochemistry with an anti-factor VIII antibody (10Liu S.Q. Tieche C. Alkema P.K. Biomaterials. 2004; 25: 1869-1882Crossref PubMed Scopus (33) Google Scholar). The exposure of the basal lamina was verified by using an anti-collagen type IV antibody (10Liu S.Q. Tieche C. Alkema P.K. Biomaterials. 2004; 25: 1869-1882Crossref PubMed Scopus (33) Google Scholar). To create aortic substitutes with an adventitial blood-contacting surface, fresh rat aortic specimens were treated with 0.1 m NaOH at 20 °C for 2 h, followed by washing in distilled water for 12 h with vigorous agitation, which removes cells and proteoglycans in the adventitia. The aortic specimens were then turned outside in, resulting in aortic substitutes with an adventitial blood-contacting surface. The presence of the collagen blood-contacting surface was verified by immunohistochemistry with an anti-collagen type III antibody (10Liu S.Q. Tieche C. Alkema P.K. Biomaterials. 2004; 25: 1869-1882Crossref PubMed Scopus (33) Google Scholar). To create an aortic reconstruction model, a rat (Sprague-Dawley, male, 300-350 g) was anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital. Two aortic substitutes, either a pair with elastic lamina (NaOH-treated) and basal lamina (untreated) blood-contacting surfaces or a pair with elastic lamina (NaOH-treated) and adventitial surfaces, were anastomosed together in a series and grafted into the host rat abdominal aorta by using a method established previously (33Liu S.Q. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2630-2639Crossref PubMed Scopus (39) Google Scholar). Such an arrangement ensured that each matrix substitute within the pair was in contact with the host aorta at one end. It should be pointed out that it is possible to graft all three types of matrix substitutes in a series in each animal. With such an arrangement, however, one of the three substitutes will not be in contact with the host aorta. Because cell migration from the host aorta to the substitutes contribute to intimal hyperplasia (34Liu S.Q. Atherosclerosis. 1998; 140: 365-377Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), a model with a series of three aortic substitutes will result in conditions of inconsistent controls. Observations were carried out at 1, 5, 10, 20, and 30 days with five rats at each observation time. Experimental procedures were approved by the Animal Care and Use Committee of Northwestern University. Measurement of Leukocyte Transmigration in Vivo—To measure leukocyte transmigration in matrix substitutes, a rat was anesthetized as described above at each observation time. Specimens were collected from all types of arterial substitutes, fixed in 4% formaldehyde in phosphate-buffered saline, cut into transverse cryosections of 10 μm in thickness, incubated with phycoerythrin-conjugated anti-CD11b/c antibody (Caltag) and Hoechst 33258 (for cell nucleus labeling), and observed by fluorescence microscopy. The density of CD11b/c-positive cells in the elastic lamina-dominant media and collagen-dominant adventitia was measured and compared between different specimens. Measurement of Monocyte Adhesion to Elastic Lamina, Basal Lamina, and Adventitia in Vitro—We used an in vitro monocyte adhesion assay to demonstrate the mechanisms of the inhibitory effect of elastic laminae. To collect monocytes for the in vitro assay, rats were anesthetized as described above. A blood sample of ∼10 ml was collected from the vena cava of each rat and mixed with 20% acid/citrate/dextrose (120 mmol/liter sodium citrate, 110 mmol/liter glucose, and 80 mmol/liter citric acid). Monocytes were collected by using a monocyte enrichment kit (Stemcell Technologies Inc., Vancouver, British Columbia, Canada) according to the manufacturer's instructions. Enriched monocytes were suspended in 15% fetal bovine serum/Dulbecco's modified Eagle's medium at a cell count ∼6 × 106 cells/ml, supplemented with 100 μg/ml streptomycin and 100 units/ml penicillin. Aortic matrix specimens were prepared with four different surfaces for interacting with monocytes as follows: NaOH-treated elastic lamina untreated elastic lamina, basal lamina, and adventitia. Specimens with NaOH-treated elastic lamina, basal lamina, and adventitial surfaces were prepared as described under “Matrix-based Aortic Reconstruction.” Matrix specimens with an untreated elastic lamina surface were prepared as follows. Fresh aortic specimens were collected and separated between the media and adventitia with fine tweezers under a surgical microscope. The media were further separated between interior elastic laminae to expose the surface of elastic laminae. Separated medial specimens were frozen (-76 °C) and thawed (20 °C) for three cycles. The surface of exposed elastic laminae was scraped with a fine metal wire along the elastic fiber direction to remove cell debris and other matrix components, including medial collagen and proteoglycans. The medial specimens were washed in distilled water for 12 h with vigorous agitation. Medial specimens with an elastic lamina surface were selected by immunohistochemistry. All prepared elastic lamina specimens were incubated consecutively with four antibodies, including anti-collagen type III (Chemicon), anti-collagen type IV (Chemicon), anti-vascular proteoglycans (US Biological), and anti-SMC α-actin (Chemicon) antibodies. Hoechst 33258 was used for labeling cell nuclei. Specimens without collagen type III, collagen type IV, proteoglycans, SMC α-actin filaments, and cell nuclei at the elastic lamina surface were selected by fluorescence microscopy and used as untreated elastic laminae. Fig. 1 shows the presence of elastic lamina and the absence of collagen matrix and SMCs at the surface of prepared specimens. Enriched monocytes were incubated in culture media at 37 °C in the presence of the four types of matrix specimen for 3, 6, 12, and 24 h with gentle agitation. At each observation time, samples from the four types of matrix specimen were collected, fixed in 4% formaldehyde in phosphate-buffered saline, incubated with an anti-CD 14 antibody and Hoechst 33258, and observed with a fluorescence microscope for measuring the density of monocytes adhered to the matrix specimens. Specimens from five rats were used for statistical analyses at each time point. Detection of the Role of Elastic Laminae in Regulating the Activity of SIRP α and SHP-1—To test whether the exposure of monocytes to elastic laminae induces activation of SIRP α and SHP-1, enriched monocytes were cultured in dishes coated with four types of matrix specimens as follows: NaOH-treated elastic lamina, untreated elastic lamina, basal lamina, and adventitia, which were prepared as described above. At culture times of 3, 6, and 12 h, monocytes were collected from each of the four types of matrix coatings and prepared for detecting the expression and phosphorylation of SIRP α and SHP-1. Collected monocytes were lysed in lysis buffer containing 1% Triton X-100, 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm sodium orthovanadate, 1 mm EDTA, and a protease inhibitor mixture (1 μg/ml aprotinin, 1 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride). The lysates were measured for total protein concentration, precleaned with protein A-conjugated agarose beads (10% packed beads, Upstate), immunoprecipitated with an anti-SIRP α antibody (4 μg/ml, sc-17803; Santa Cruz Biotechnology) at 4 °C for 4 h, and incubated with protein A-agarose beads (10% packed beads) at 4 °C for 4 h. The agarose beads were collected and treated with protein sample buffer at ∼100 °C for 5 min. Immunoprecipitates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with the anti-SIRP α antibody (0.5 μg/ml). The relative level of protein was examined by secondary peroxidase-IgG labeling and chemiluminescent detection of peroxidase activity (35Liu S.Q. Tieche C. Tang D. Alkema P. Am. J. Physiol. 2003; 285: H1081-H1090Crossref PubMed Scopus (25) Google Scholar). The nitrocellulose membrane was stripped and immunoblotted consecutively with an anti-SHP-1 antibody (sc-287; Santa Cruz Biotechnology) and an anti-phosphotyrosine antibody (clone 4G10, 05-321; Upstate). Detection of the Role of SIRP α and SHP-1 in Regulating Monocyte Adhesion to Elastic Laminae—To test the role of SIRP α and SHP-1 in regulating monocyte adhesion to elastic laminae, small interfering RNA (siRNA) specific to SIRP α and SHP-1 mRNA was used to degrade these mRNAs and thus knock down the expression of SIRP α and SHP-1, respectively. Briefly, SIRP α- and SHP-1-specific siRNAs were prepared according to the provider's instruction (Santa Cruz Biotechnology). Enriched monocytes were transfected with siRNA for SIRP α (0.05 μm) or siRNA for SHP-1 (0.05 μm) by the mediation of siRNA transfection reagent (0.5%, Santa Cruz Biotechnology) at 37 °C for 48 h. Monocytes transfected with a nontargeting scrambled siRNA control, which does not degrade known mRNAs, were used as a control (36Schoenemeyer A. Barnes B.J. Mancl M.E. Latz E. Goutagny N. Pitha P.M. Fitzgerald K.A. Golenbock D.T. J. Biol. Chem. 2005; 280: 17005-17012Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 37Hoashi T. Watabe H. Muller J. Yamaguchi Y. Vieira W. Hearing V.J. J. Biol. Chem. 2005; 280: 14006-14016Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Note that the transfection medium contains 8.8% fetal bovine serum without antibiotics. Following siRNA transfection, fetal bovine serum was added to the culture to make the final serum concentration of 15%. Matrix specimens with NaOH-treated and untreated elastic lamina, basal lamina, and adventitia, prepared as described above, were applied to the culture with siRNA-transfected monocytes. Cells were subsequently cultured with the matrix specimens for 3, 6, 12, and 24 h. At each time, five matrix specimens were collected and prepared for measuring the density of monocytes adhered to the matrix surface by using methods described above. The effectiveness of siRNA treatment was tested by immunoprecipitation and immunoblotting for each type of siRNA. At 48 h of culture with an siRNA, monocytes were collected and prepared for detecting the expression of SIRP α and SHP-1 by immunoprecipitation and immunoblotting with anti-SIRP α and SHP-1 antibodies, respectively, as described above (two assays done for each siRNA). Furthermore, monocytes were transfected with a fluorescein-conjugated nontargeting siRNA and observed by fluorescence microscopy for the verification of positive siRNA transfection. In addition, cell viability was tested by estimating the total cell number in each culture dish before and after the siRNA treatment (four samples tested for each siRNA and the control siRNA). Note that after 48 h of culture, a fraction of cells adhered to the culture base. The total cell number after the siRNA treatment was the sum of the adherent and suspended cells. Detection of Elastic Lamina Degradation Peptide Binding to SIRP α—To test the possibility that elastic laminae interact with SIRP α in monocytes, a reasonable approach is to detect whether components from elastic laminae bind to SIRP α. To achieve such a goal, we prepared elastic lamina degradation peptides from rat aortic specimens by using a method established previously (38Mecham R.P. Lange G. Methods Enzymol. 1982; 82: 744-759Crossref PubMed Scopus (35) Google Scholar). Briefly, aortic specimens were collected from rats. The media of the aorta were manually separated from the adventitia under a surgical microscope with a pair of fine surgical forceps. The media were collected, minced, and treated with 1 n KOH/ethanol (80:20, v/v) at 37 °C for 1 h. The resulting mixture was centrifuged, and the supernatant was discarded. Samples were randomly selected from the remaining insoluble fraction and used for verifying the removal of medial cells, collagen fibers, and proteoglycans by immunohistochemistry as described above. Note that the KOH treatment removed all medial components except the elastic laminae. The insoluble elastic lamina fraction was treated with the KOH/ethanol mixture at 37 °C for 1 h. The supernatant was collected and neutralized with perchloric acid to pH 7.4, and the resulting precipitate was discarded. Elastic lamina degradation peptides were collected from the remaining supernatant. Monocytes were cultured in the presence of 10 μg/ml elastic lamina degradation peptides, collected at 0.5, 1, and 3 h, and lysed in lysis buffer as described above. Lysates were processed for immunoprecipitation with an anti-elastin antibody (Elastin Products), resolved by SDS-PAGE, and probed with an anti-SIRP α antibody (sc-17803; Santa Cruz Biotechnology) and an anti-phosphotyrosine antibody (clone 4G10; Upstate) as described above. Detection of SIRP α and SHP-1 Phosphorylation in the Presence of Elastic Lamina Degradation Peptides—To detect whether the binding of elastic lamina degradation peptides induces SIRP α phosphorylation and whether SIRP α phosphorylation induces SHP-1 recruitment, monocytes were cultured in the presence and absence of elastic lamina degradation peptides (10 μg/ml), collected at 0.5, 1, 3, and 6 h, and lysed in lysis buffer. Lysates were processed for immunoprecipitation with an anti-SIRP α antibody (sc-17803; Santa Cruz Biotechnology) and for consecutive immunoblotting by using anti-SIRP α, anti-SHP-1 (sc-287; Santa Cruz Biotechnology), and anti-phosphotyrosine (clone 4G10; Upstate) antibodies with antibody stripping after each immunoblotting reaction. Cytometry Confirmation of Elastic Lamina Degradation Peptide Binding to SIRP α—To confirm the binding of elastic lamina degradation peptides to SIRP α, the influence of an anti-SIRP α antibody (sc-17803; Santa Cruz Biotechnology), developed with the extracellular domain of SIRP α1 (1-300 amino acids) as an antigen, on the relative binding of elastic lamina degradation peptides was detected by flow cytometry. Monocytes were treated with 0, 5, and 10 μg/ml anti-SIRP α antibody separately at 37 °C for 1 h and subsequently incubated with 10 μg/ml fluorescein-conjugated elastic lamina degradation peptides at 37 °C for 1 h. Monocytes incubated with an unrelated fluorescein-conjugated secondary antibody (10 μg/ml) without elastic lamina degradation peptides were used as controls. In addition, an anti-CD11b antibody (10 μg/ml, MLDP5; R & D Systems) was used as a control in the presence of 10 μg/ml elastic lamina degradation peptides. Monocytes were detected for fluorescent intensity by flow cytometry (Beckman Coulter Epics XL-MCL). Statistical Analyses—Means and standard deviations were calculated for each measured parameter at each observation time. The Student's t test was used for difference comparisons between two groups. A difference is considered statistically significant at p < 0.05. Role of Elastic Laminae in Preventing Leukocyte Transmigration in Vivo—We used an in vivo matrix-based arterial reconstruction model to observe the role of elastic laminae in preventing leukocyte transmigration through the arterial media. As shown in Fig. 2, many cells found in the matrix of aortic substitutes were CD11 b/c-positive leukocytes (predominantly monocytes/macrophages and granulocytes), especially during the early period. Although a large number of leukocytes migrated into the collagen-dominant adventitia, few leukocytes were found within the elastic lamina-dominant media of the matrix-based aortic substitutes. The density of leukocytes in the media was 58-70-fold lower than that in the adventitia from 1 to 30 days after surgery, whereas no significant difference was detected in the media between NaOH-treated (with an elastic lamina blood-contacting surface) and untreated matrix (with a basal lamina blood-contacting surface) substitutes (Fig. 2). At the end of the elastic lamina-dominant media, leukocytes were not able to migrate into the gaps between the elastic laminae, even though the gaps were apparently larger than the diameter of leukocytes (Fig. 2A, Day 10*). However, at locations with aneurysm-like changes (induced possibly by excessive mechanical stretch because of surgical damage to the adventitia), leukocytes migrated into the medial wall, where elastic laminae were largely destroyed (Fig. 2A, Day 10**). These observations demonstrate that in" @default.
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• W2005184523 date "2005-11-01" @default.
• W2005184523 modified "2023-09-26" @default.
• W2005184523 title "Negative Regulation of Monocyte Adhesion to Arterial Elastic Laminae by Signal Regulatory Protein α and Src Homology 2 Domain-containing Protein-Tyrosine Phosphatase-1" @default.
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