Bone Morphogenic Protein 4 Produced in Endothelial Cells by Oscillatory Shear Stress Stimulates an Inflammatory Response
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m300703200
ISSN1083-351X
AutoresGeorge P. Sorescu, Michelle Sykes, Daiana Weiss, Manu O. Platt, Aniket Saha, Jinah Hwang, Nolan L. Boyd, Yong Chool Boo, J. David Vega, W. Robert Taylor, Hanjoong Jo,
Tópico(s)Bone Metabolism and Diseases
ResumoAtherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFκB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas. Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFκB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas. Endothelial cells are constantly exposed to shear stress (a dragging force generated by blood flow), which controls cellular structure and function such as regulation of vascular tone and diameter, vessel wall remodeling, hemostasis, and inflammatory responses (1Davies P.F. Polacek D.C. Shi C. Helmke B.P. Biorheology. 2002; 39: 299-306PubMed Google Scholar). The importance of various types of shear stress is highlighted by the focal development of atherosclerosis (2Zarins C.K. Giddens D.P. Bharadvaj B.K. Sottiurai V.S. Mabon R.F. Glagov S. Circ. Res. 1983; 53: 502-514Crossref PubMed Scopus (1225) Google Scholar). Atherosclerosis preferentially occurs in the arterial regions exposed to unstable shear stress conditions in branched or curved arteries, whereas straight arteries exposed to unidirectional laminar shear (LS) 1The abbreviations used are: LS, laminar shear; BMP4, bone morphogenic protein-4; OS, oscillatory shear; ICAM-1, intercellular adhesion molecule-1; VCAM1, vascular cell adhesion molecule-1; MAEC, mouse aortic endothelial cells; HAEC, human aortic endothelial cells; FACS, fluorescence-activated cytometry sorting; ALK, activin-like kinase. are relatively lesion-free (1Davies P.F. Polacek D.C. Shi C. Helmke B.P. Biorheology. 2002; 39: 299-306PubMed Google Scholar, 2Zarins C.K. Giddens D.P. Bharadvaj B.K. Sottiurai V.S. Mabon R.F. Glagov S. Circ. Res. 1983; 53: 502-514Crossref PubMed Scopus (1225) Google Scholar, 3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar). Atherosclerosis is now known as an inflammatory disease caused by endothelial dysfunction (3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar, 4Libby P. Ridker P.M. Maseri A. Circulation. 2002; 105: 1135-1143Crossref PubMed Scopus (5922) Google Scholar). One of the first visible markers of endothelial dysfunction in the lesion-prone areas is up-regulation of inflammatory adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 (3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar, 4Libby P. Ridker P.M. Maseri A. Circulation. 2002; 105: 1135-1143Crossref PubMed Scopus (5922) Google Scholar, 5Cybulsky M.I. Iiyama K. Li H. Zhu S. Chen M. Iiyama M. Davis V. Gutierrez-Ramos J.C. Connelly P.W. Milstone D.S. J. Clin. Invest. 2001; 107: 1255-1262Crossref PubMed Scopus (973) Google Scholar, 6Endres M. Laufs U. Merz H. Kaps M. Stroke. 1997; 28: 77-82Crossref PubMed Scopus (70) Google Scholar). These endothelial adhesion molecules play essential roles in adhesion and recruitment of monocytes to the subendothelial layer (3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar, 4Libby P. Ridker P.M. Maseri A. Circulation. 2002; 105: 1135-1143Crossref PubMed Scopus (5922) Google Scholar). How do unstable shear conditions such as low and oscillating shear stress (OS) cause inflammation in those lesion-prone areas, whereas LS exerts athero-protective effects? The opposite effects of LS and OS may be determined by differential expression of genes and proteins, ultimately inducing anti- and pro-inflammatory and atherogenic responses. Recently, several studies (7Garcia-Cardena G. Comander J. Anderson K.R. Blackman B.R. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4478-4485Crossref PubMed Scopus (458) Google Scholar, 8Chen B.P. Li Y.S. Zhao Y. Chen K.D. Li S. Lao J. Yuan S. Shyy J.Y. Chien S. Physiol. Genomics. 2001; 9: 55-63Google Scholar, 9McCormick S.M. Eskin S.G. McIntire L.V. Teng C.L. Lu C.M. Russell C.G. Chittur K.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98 (8860): 8955Crossref PubMed Scopus (335) Google Scholar, 10Brooks A.R. Lelkes P.I. Rubanyi G.M. Physiol. Genomics. 2002; 9: 27-41Crossref PubMed Scopus (241) Google Scholar) have begun to address the initial question to determine the expression profiles of mechanosensitive genes. However, the functional importance of those genes has not been clearly established. Here, we report identification of a mechanosensitive gene, BMP4, by DNA microarray analyses and subsequent verification by a variety of additional approaches in both cultured endothelial cells and human coronary arteries. More importantly, we discovered a novel role of BMP4 as an inflammatory cytokine, providing a potential mechanistic link from shear forces to inflammatory responses and atherogenesis. Endothelial Cells—Mouse aortic endothelial cells (MAEC) were cultured and used at passages 4–8 as described by us (11Cai H. Li Z. Dikalov S. Hwang J. Jo H. Dudley S.C. Harrison D. J. Biol. Chem. 2002; 107: 48311-48317Abstract Full Text Full Text PDF Scopus (165) Google Scholar). Human aortic endothelial cells (HAEC) purchased from Clonetics were cultured using the EGM-2 bullet kit (Clonetics) and used at passages 4–8. Shear Stress Studies—Confluent endothelial monolayers grown in 100-mm tissue culture dishes were exposed to an arterial level of unidirectional LS (15 dyn/cm2) in the growth medium by rotating a Teflon cone (0.5° cone angle) as described previously by us (12Go Y.M. Boo Y.C. Park H. Maland M.C. Patel R. Pritchard Jr., K.A. Fujio Y. Walsh K. Darley-Usmar V. Jo H. J. Appl. Physiol. 2001; 91: 1574-1581Crossref PubMed Scopus (88) Google Scholar). To mimic unstable shear conditions in vivo, endothelial cells were exposed to OS with directional changes of flow at 1 Hz cycle (±5 dyn/cm2) by rotating the cone back and forth using a stepping motor (Servo Motor) and a computer program (DC Motor Company, Atlanta, GA). In some studies, 5 dyn/cm2 unidirectional LS was used for comparison to OS (±5 dyn/cm2). Preparation of Cell Lysates and Immunoblotting—Following experimental treatments, endothelial cell lysates were prepared and analyzed by Western blot analysis as described by us (13Boo Y.C. Hwang J. Sykes M. Michell B.J. Kemp B.E. Lum H. Jo H. Am J. Physiol. Heart Circ. Physiol. 2002; 283: H1819-H1828Crossref PubMed Scopus (209) Google Scholar, 14Boo Y.C. Sorescu G. Boyd N. Shiojima I. Walsh K. Du J. Jo H. J. Biol. Chem. 2002; 277: 3388-3396Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Briefly, cells were washed in ice-cold phosphate-buffered saline and lysed in 0.1 ml of boiling lysis buffer A (10 mm Tris-HCl, pH 7.6, 1 mm sodium vanadate, and 1% SDS). The lysate was further homogenized by repeated aspiration through a 25-gauge needle. Protein content of each sample was measured by using a Bio-Rad DC assay (15Jo H. Sipos K. Go Y.M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). To detect secreted BMP4 in conditioned media, endothelial monolayers were first washed in serum-free Dulbecco's modified Eagle's medium supplemented with minimum non-essential amino acids and pyruvic acid and exposed to OS, LS, or static conditions for 1 day. The conditioned media were then centrifuged at 1,000 × g for 10 min. Aliquots (2 ml) of the supernatant were collected and placed on ice with 10 ml of ice-cold acetone to precipitate protein for 30 min. Samples were pelleted by centrifugation (15,000 × g for 10 min) and resuspended in 100 μl of sample buffer for SDS-PAGE (13Boo Y.C. Hwang J. Sykes M. Michell B.J. Kemp B.E. Lum H. Jo H. Am J. Physiol. Heart Circ. Physiol. 2002; 283: H1819-H1828Crossref PubMed Scopus (209) Google Scholar, 14Boo Y.C. Sorescu G. Boyd N. Shiojima I. Walsh K. Du J. Jo H. J. Biol. Chem. 2002; 277: 3388-3396Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Aliquots of cell lysates (20 μg of protein each) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with a primary antibody overnight at 4 °C and then with a secondary antibody conjugated with alkaline phosphatase (1 h at room temperature), which were detected by a chemiluminescence method (15Jo H. Sipos K. Go Y.M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The intensities of immunoreactive bands in Western blots were analyzed by using the NIH Image program. The following primary antibodies were used: a monoclonal BMP4 antibody, rabbit ICAM1 antibody, goat VCAM1 antibody, and goat actin antibody (Santa Cruz Biotechnology). DNA Microarray Analysis—DNA microarray analyses were performed with an Affymetrix murine gene chip containing 12,000 genes (U74Av2; Affymetrix) and a Motorola murine genome chip containing 10,000 genes, according to the protocols provided by each manufacturer (16Napoli C. de Nigris F. Welch J.S. Calara F.B. Stuart R.O. Glass C.K. Palinski W. Circulation. 2002; 105: 1360-1367Crossref PubMed Scopus (152) Google Scholar). Affymetrix chips were scanned and analyzed at the DNA core facility at Emory University School of Medicine; studies with Motorola chips, the entire process from reverse transcription to hybridization, scanning to initial data analysis, were performed by using the manufacturer's protocol and laboratory (Chicago, IL). Quantitative Real-time PCR—Real-time PCR for BMP4 was carried out as previously described (17Sorescu D. Weiss D. Lassegue B. Clempus R.E. Szocs K. Sorescu G.P. Valppu L. Quinn M.T. Lambeth J.D. Vega J.D. Taylor W.R. Griendling K.K. Circulation. 2002; 105: 1429-1435Crossref PubMed Scopus (776) Google Scholar). Briefly, 4 μg of total RNA was reverse-transcribed by using random primers and a Superscript-II kit (Invitrogen) to synthesize first-strand cDNA. The cDNA was purified using a microbiospin 30 column (Bio-Rad) in Tris buffer and stored at –20 °C until used. The cDNA was amplified using a LightCycler (Roche Applied Science) RT-PCR machine. The mRNA copy numbers were determined based on standard curves generated with murine BMP4 and 18 S templates. The 18 S primers (50 nm at 61 °C annealing temperature; Ambion) were used as an internal control for real-time PCR using a LightCycler and capillaries (Roche Applied Science), recombinant Taq polymerase (Invitrogen), and Taq start antibody (Clontech). A quantitative RT-PCR using BMP4 primer pair (forward, 5′-CTGCGGGACTTCGAGGCGACACTTCT-3′, reverse, 5′-TCTTCCTCCTCCTCCTCCCCAGACTG-3′) using endothelial RNA sample yielded a 130-base pair fragment on agarose gel electrophoresis. This pair of primers was verified by a nested PCR using other BMP4 primer pairs (forward, 5′-ATGGACTGTTATTATGCCTTGTTTTCTGTCAACACCATGATTC-3′, reverse, 5′-CCACGTATAGTGAATGGCGACGGCAGTTCTT-3′, and forward, 5′-GTCAACACCATGATTCCTGGTAACCGAATGCTGA-3′, reverse, 5′-TTATACGGTGGAAGCCCTGTTCCCAGTCAG-3′) and by running DNA gels. Real-time PCR for BMP4 was carried out using the annealing temperature 65 °C and extension time for 7 s in the PCR buffer (20 mm Tris-Cl, pH 8.4, at 25 °C, 4mm MgCl2 to which was added 250 μg/ml bovine serum albumin, 200 μm deoxynucleotides) containing SYBR green (1:84,000 dilution), 0.05 unit/μl Taq DNA polymerase, and Taq Start antibody (1:100 dilution). Fluorescence-activated Cytometry Sorting (FACS) Analysis—Treated cells were dissociated into single-cell suspensions using 0.25% trypsin-EDTA and resuspended in a FACS buffer (Hank's buffered solution containing 5% fetal bovine serum). Aliquots of cell suspensions were incubated with ICAM1 antibody (R&D Systems) for 20 min on ice, washed twice with FACS buffer, and incubated with secondary antibody (fluorescein-5-isothiocyanate- or phycoerytherin-conjugated; Chemicon) for 20 min on ice in the dark. Then samples were washed again, fixed in 1% paraformaldehyde, and analyzed by FACS (Calibur; Becton-Dickinson) using CellQuest software. The fluorescence intensity of ICAM1 and forward cell scattering of 30,000 cells were measured, and the geometric means calculated from histograms were shown. In some studies, HAEC were transfected with either BMP4 (Dr. Elizabeth J. Robertson, Harvard University) cloned in a bicistronic pAdTrack CMV vector (Dr. Bert Vogelstein, The Johns Hopkins University) or an empty vector, both expressing green fluorescent protein (GFP), using LipofectAMINE 2000. In these experiments, ICAM1 expression was measured in the red phycoerytherin channel, while the green channel was used to monitor GFP expression. GFP expression was determined by FACS analysis and fluorescence microscopy (20–30% transfection efficiency). Because expression of GFP was similar among different treatment groups within the same experiment, we did not need to normalize ICAM1 expression data to the GFP level. Human recombinant noggin was either a kind gift from Dr. Arturo Alvarez-Buylla (University of California at San Francisco) or purchased from R&D Systems and used in all experiments at 50 ng/ml (18Zimmerman L.B. De Jesus-Escobar J.M. Harland R.M. Cell. 1996; 86: 599-606Abstract Full Text Full Text PDF PubMed Scopus (1335) Google Scholar, 19Brunet L.J. McMahon J.A. McMahon A.P. Harland R.M. Science. 1998; 280: 1455-1457Crossref PubMed Scopus (689) Google Scholar, 20Dale L. Jones C.M. Bioessays. 1999; 21: 751-760Crossref PubMed Scopus (150) Google Scholar). Immunohistochemical Study—Frozen sections of human coronary arteries obtained from patients undergoing heart transplants were prepared (17Sorescu D. Weiss D. Lassegue B. Clempus R.E. Szocs K. Sorescu G.P. Valppu L. Quinn M.T. Lambeth J.D. Vega J.D. Taylor W.R. Griendling K.K. Circulation. 2002; 105: 1429-1435Crossref PubMed Scopus (776) Google Scholar) and stained with antibodies specific for BMP4 (1:1,000 dilution, goat antibody, Santa Cruz Biotechnology), ICAM-1 (1:50 dilution, mouse antibody; R&D), or von Willebrand factor (1:100 dilution, mouse antibody; Dako) for 2 h at room temperature, washed, and followed by incubation with secondary antibodies (anti-goat IgG or anti-mouse IgG) conjugated to alkaline phosphatase. Then the slides were washed and developed with a DAKO kit (DAKO). Photographs of the slides were taken using a Zeiss microscope. Fifteen different human coronary arteries, containing various stages of atherosclerosis from minimally diseased to fatty streak to advanced atheroma stage, from six different patients were examined. Monocyte Adhesion—Monocyte binding was determined under no-flow conditions using THP-1 monocytes (ATCC) by the method described by Chappel et al. (21Chappell D.C. Varner S.E. Nerem R.M. Medford R.M. Alexander R.W. Circ. Res. 1998; 82: 532-539Crossref PubMed Scopus (471) Google Scholar). Briefly, THP-1 cells (5 × 105 cells/ml) were labeled with a fluorescent dye 2′,7′-bis(carboxyethyl)-5 (6)-carboxyfluorescein-AM (BCECF; Molecular Probes) (1 mg/ml) in serum-free RPMI medium for 45 min at 37 °C. Following exposure to shear stress or BMP treatments in the presence or absence of noggin or vehicle, the endothelial cells were washed in RPMI medium before adding BCECF-loaded THP-1 cells (1:1 ratio). After a 30-min incubation at 37 °C under no-flow conditions, unbound monocytes were removed by washing the endothelial dishes five times with Hank's phosphate-buffered saline. Bound monocytes were quantified by either counting the cells under a fluorescent microscope or by measuring the fluorescent intensity of cell lysates by fluorescence spectrophotometry using a plate reader. Both assays showed similar results. Some studies were performed with MAEC pretreated with 5 μg/ml mouse-ICAM1 antibody (YN1; Southern Biotechnology) (22Kevil C.G. Patel R.P. Bullard D.C. Am. J. Physiol. Cell Physiol. 2001; 281: 8955-8960Google Scholar). NFκB Assay—NFκB activity was determined by using a NFκB reporter construct, NFκB-SEAP vector (1 μg; Clontech) expressing a secreted form of placental alkaline phosphatase driven by 4-κB sequences in tandem. This construct was co-transfected with 0.5 μg of either pAdTrack BMP4 or empty vector control using LipofectAMINE 2000. Six hours post-transfection, conditioned media were centrifuged and heat treated at 65 °C (to inactivate endogenous alkaline phosphatase) for 30 min, followed by chemiluminescence alkaline phosphatase assay according to the manufacturer's instructions. Statistical Analysis—Statistical significance was assessed by Student's t test using the Microcal Origin statistical package. Differential Regulation of the BMP4 Gene by LS and OS in Endothelial Cells—To identify the genes that may be responsible for the athero-protective and pro-atherogenic effects of LS and OS, respectively, we performed DNA microarray studies using cultured MAEC. Exposing MAEC to LS, but not OS, for 1 day using the modified "cone-and-plate" device (12Go Y.M. Boo Y.C. Park H. Maland M.C. Patel R. Pritchard Jr., K.A. Fujio Y. Walsh K. Darley-Usmar V. Jo H. J. Appl. Physiol. 2001; 91: 1574-1581Crossref PubMed Scopus (88) Google Scholar) induced a cell shape alignment to the direction of the flow from a typical polygonal "cobblestone shape" found in static cultured cells (Fig. 1). The total RNAs prepared from these cells were used to determine mRNA expression profiles by using Affymetrix and/or Motorola DNA chips according to the manufacturers' protocols. The analyses of these studies showed that LS exposure significantly and consistently inhibited BMP4 mRNA level in MAEC by more than 60–80% of static control levels (Fig. 2A). Unlike LS, however, exposure of endothelial cells to OS did not inhibit BMP4 mRNA expression (Fig. 2A). We also found that LS exposure up-regulated a well known mechanosensitive gene, endothelial nitric oxide synthase (eNOS), mRNA level by more than 5-fold (5.6 ± 1.2, n = 3) above static controls. In addition, eNOS protein level was also increased by ∼2-fold above controls as determined by Western blot analysis using a monoclonal antibody (data not shown), providing further confidence in our results. BMPs play an important role in bone formation, embryonic development, and differentiation (23Massague J. Nat. Rev. Mol. Cell Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar, 24Hogan B.L. Curr. Opin. Gen. Dev. 1996; 6: 432-438Crossref PubMed Scopus (662) Google Scholar). Although BMP4 protein has been found previously in calcified atherosclerotic plaques (25Dhore C.R. Cleutjens J.P. Lutgens E. Cleutjens K.B. Geusens P.P. Kitslaar P.J. Tordoir J.H. Spronk H.M. Vermeer C. Daemen M.J. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1998-2003Crossref PubMed Scopus (605) Google Scholar), its expression and functional importance in endothelial cells have not been determined. Therefore, we decided to verify the microarray results by independent methods at the levels of mRNA and protein as well as the functional roles of BMP4 in endothelial biology and pathobiology. First, we verified the BMP4 mRNA data by using a quantitative real-time PCR method. Exposure of endothelial cells to LS almost eliminated the BMP4 mRNA level (n = 6, p < 0.001) (Fig. 2B). In contrast, OS marginally, statistically not significant, increased BMP4 mRNA level compared with that of static control (n = 3). These results confirmed the DNA microarray results. Next, BMP4 protein expression was determined by immunoblot studies. BMP4 protein is synthesized as an inactive precursor (48–55 kDa) that is proteolytically cleaved by proprotein convertases, and the active ∼23-kDa protein is secreted (23Massague J. Nat. Rev. Mol. Cell Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar, 24Hogan B.L. Curr. Opin. Gen. Dev. 1996; 6: 432-438Crossref PubMed Scopus (662) Google Scholar). In endothelial cell lysates, the BMP4 precursor was detected as a 54-kDa protein, and the mature form (p23) was detected in the conditioned media collected from static or shear-exposed cells (Fig. 2C). Exposure of cells to LS significantly down-regulated expression of BMP4 precursor in a time-dependent manner (Fig. 2C). After 16–24 h of LS exposure, BMP4 precursor expression was virtually undetectable (Fig. 2C, left panel, p < 0.05). In contrast, exposure of MAEC to OS significantly increased BMP4 precursor protein level by 2-fold above control (Fig. 2C, middle panel, p < 0.05). Consistent with the cell lysate result, the conditioned media of MAEC exposed to LS (15 dyn/cm2) showed a barely detectable amount of secreted form of BMP4 (p23) (Fig 2C, right panel). In contrast, OS exposure did not significantly change the p23 BMP4 level in the conditioned medium (Fig. 2C, right panel, p < 0.05). Because the cells were exposed to LS (15 dyn/cm2) and OS (±5 dyn/cm2), we next determined whether it was the shear magnitude difference that accounted for our results observed so far. To address this question, we compared the effects of LS and OS using the same magnitudes (5 dyn/cm2 LS versus ±5 dyn/cm2 OS). As shown as Fig. 2C, right panel, at the same shear magnitude, OS-exposed cells had more than 3-fold BMP4 protein than that of LS. However, the higher LS magnitude (15 dyn/cm2) showed a much lower amount of BMP4 than that of lower LS (5 dyn/cm2). These results show that LS exposure inhibits BMP4 expression in a force-dependent manner, whereas OS maintains high BMP4 expression. BMP4 Expression in the Selective Patches of Endothelial Cells over Foam Cell Lesions in Human Coronary Arteries— Next, using the human coronary arteries we determined whether BMP4 protein is expressed in endothelial cells of human atherosclerotic lesions. The coronary arteries exhibiting a spectrum of atherosclerotic lesion complexity were obtained from patients undergoing heart transplants and examined by immunohistochemical staining (17Sorescu D. Weiss D. Lassegue B. Clempus R.E. Szocs K. Sorescu G.P. Valppu L. Quinn M.T. Lambeth J.D. Vega J.D. Taylor W.R. Griendling K.K. Circulation. 2002; 105: 1429-1435Crossref PubMed Scopus (776) Google Scholar). BMP4 protein expression was not apparent in the intimal endothelial cells in relatively normal, "minimally diseased" human coronary arteries (Fig. 3A) or in advanced lesions (data not shown). As shown in Fig. 3D, one exception was found in the endothelial cells (arrows) overlying foam cell lesions that were stained strongly against the BMP4 antibody. As shown in Fig. 3C, isotype-matched nonspecific mouse IgG used as a negative control further supported the specificity of BMP4 staining. In contrast, the medial smooth muscle cells and macrophages (Fig. 3, A and D) were most intensely stained against a monoclonal BMP4 antibody (smooth muscle cells and macrophages identified by α-actin and CD-68 staining, respectively; data not shown). To verify the identity of endothelial cells, the serial sections were stained with a von Willebrand factor antibody (endothelial marker, Fig. 3, B and E), demonstrating the location of BMP4 staining in select areas of endothelium. Furthermore, immunostaining with an ICAM1 staining showed that the expression of this pro-inflammatory adhesion molecule was selectively increased in the similar endothelial areas expressing BMP4 (Fig. 3, D and F, arrowheads). On the other hand, we failed to detect VCAM-1 in the adjacent serial sections (data not shown). This result is consistent with the finding reported by Endress et al. (7Garcia-Cardena G. Comander J. Anderson K.R. Blackman B.R. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4478-4485Crossref PubMed Scopus (458) Google Scholar). BMP4 Produced in Endothelial Cells by OS Stimulates Monocyte Adhesion—The selective expression of BMP4 protein in endothelial cells above foam cell lesions (an early form of atherosclerotic lesions) prompted a speculation that BMP4 may be involved in the inflammatory responses observed in lesion-prone areas (3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar, 4Libby P. Ridker P.M. Maseri A. Circulation. 2002; 105: 1135-1143Crossref PubMed Scopus (5922) Google Scholar). To begin to test the hypothesis, MAEC were treated with increasing amounts of BMP4 for 24 h and then monocyte adhesion to endothelium was determined. As a positive control, some cells were treated with a well known inflammatory cytokine, TNFα (100 units/ml). BMP4 stimulated monocyte binding in a concentration-dependent manner with a maximum activation of 4–7-fold over control (Fig. 4A, p < 0.05). As low as 0.1 ng/ml BMP4 induced a statistically significant increase, whereas 50 ng/ml BMP4 induced a maximum effect. A similar effect of BMP4 on monocyte adhesion was also observed by transfecting MAEC or HAEC with a vector expressing mouse BMP4 (data not shown). OS has been shown to induce monocyte adhesion both in vivo and in cultured endothelial cells by increasing surface expression of adhesion molecules (21Chappell D.C. Varner S.E. Nerem R.M. Medford R.M. Alexander R.W. Circ. Res. 1998; 82: 532-539Crossref PubMed Scopus (471) Google Scholar). Therefore, we used a BMP4 inhibitor, noggin (18Zimmerman L.B. De Jesus-Escobar J.M. Harland R.M. Cell. 1996; 86: 599-606Abstract Full Text Full Text PDF PubMed Scopus (1335) Google Scholar, 20Dale L. Jones C.M. Bioessays. 1999; 21: 751-760Crossref PubMed Scopus (150) Google Scholar), to examine whether OS induces monocyte adhesion in endothelial cells in a BMP4-dependent manner. Exposure of endothelial cells to OS for 24 h significantly increased monocyte adhesion (Fig. 4B, p < 0.05). Treatment of MAEC with noggin (50 ng/ml) inhibited OS-induced monocyte adhesion (Fig. 4B). In contrast, exposure of MAEC to LS for 24 h inhibited monocyte adhesion by ∼50% of static control level (Fig. 4C, p < 0.05) as expected (26Tsao P.S. Lewis N.P. Alpert S. Cooke J.P. Circulation. 1995; 92: 3513-3519Crossref PubMed Scopus (153) Google Scholar). Because LS exposure significantly inhibited BMP4 expression in endothelial cells (Fig. 2), we next examined whether the inhibitory effect of LS on monocyte adhesion could be reversed by BMP4 addition. For this study, we exposed MAEC in the presence of BMP4 during shear or static control for 24 h, followed by monocyte adhesion assay. The inhibitory effect of LS on monocyte adhesion was lost when MAEC were sheared in medium supplemented with BMP4 (Fig. 4C, p < 0.05). Taken together, these results suggest that BMP4 produced from endothelial cells by OS exposure leads to monocyte adhesion. BMP4 Stimulates Monocyte Adhesion by Inducing ICAM-1 Expression in an NFκB-dependent Manner—Next, we examined the mechanism by which BMP4 increases monocyte adhesion to endothelial cells. Adhesion of monocytes to endothelial cells is mediated by sequential coordinated molecular interactions between the integrins expressed on monocyte surface and several adhesion molecules expressed on the endothelial surface, including ICAM-1, VCAM-1, and E-selectin (3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar). Moreover, it has been shown previously that expression of ICAM-1, VCAM-1, and E-selectin on endothelial cell surface is increased in atherosclerosis-prone areas (3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19278) Google Scholar). Therefore, we first determined whether the endothelial expression of ICAM-1, VCAM-1, and E-selectin was modified in response to OS by FACS analysis. Exposure of HAEC to OS (1 day) increased ICAM-1 expression by 2.8-fold above control (Fig. 5A, p < 0.05). For comparison, TNFα stimulated ICAM-1 expression 4–5-fold above control cells (Fig. 5A). To determine whether ICAM-1 expression induced by OS was mediated by a BMP4-dependent mechanism, HAEC were either exposed to OS or static co
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