Advance Publication by J-STAGE Circulation Journal Official Journal of the Japanese Circulation Society http://www.j-circ.or.jp

Localization of Latent Transforming Growth Factor-β Binding -1 in Human Coronary Atherosclerotic Plaques Rahmi Öklü, MD, PhD; Robin Hesketh, PhD; Stephan Wicky, MD; James C. Metcalfe, PhD

Background: Transforming growth factor-β (TGFβ) and its receptors have been detected by immunohisto- chemistry in the normal vessel wall and in atherosclerotic lesions of human coronary arteries. However, TGFβ is normally secreted as an inactive complex associated with a latent TGFβ-binding protein (LTBP). Therefore, detection of TGFβ antigen only in the arterial wall does not imply the activated form of the growth factor.

Methods and Results: In situ hybridization and immunohistochemistry demonstrated LTBP1 mRNA and pro- tein expression throughout the media and intima of early coronary artery lesions, with the highest levels of protein at the luminal surface. In advanced lesions, LTBP1 mRNA and protein were detected mainly in regions of high cell density, such as the fibrous cap.

Conclusions: Assays of the TGFβ signalling pathway will be required to determine the activity associated with TGFβ antigen in the vessel wall.

Key Words: Atherosclerosis; Coronary heart disease; ; Immunohistochemistry; Molecular biology

ransforming growth factor-β (TGFβ) is a multifunc- latent TGFβ-binding protein (LTBP)13,14 of which there are tional cytokine that regulates a variety of cellular 4 types, LTBP1–4. Association of the 25-kDa active TGFβ T processes, including proliferation, differentiation and homodimer with the LAPs is sufficient to render the com- migration in a wide range of cell types.1 For example, TGFβ plex biologically inactive in vitro15,16 and in vivo,15 whereas inhibits the in vitro proliferation and migration of vascu- LTBP1 has been implicated in the normal assembly and lar smooth muscle cells, and maintains their differentiated secretion of TGFβ1.17,18 LTBP1 may also target the latent phenotype in vitro and in vivo.2–5 Control of TGFβ activity complex to the cell surface and extracellular matrix,19–21 and is therefore potentially important for homeostasis of the arte- therefore regulate access to activating enzymes that release rial vessel wall6–8 and abnormalities in TGFβ signaling may the active 25-kDa homodimer. The addition of excess free be critical in the pathogenesis of vascular disease. Both LTBP1, or a peptide fragment of LTBP1, inhibits activation TGFβ antigen and the TGFβ type I and type II receptors of TGFβ in the LLC.19 However, the presence of LTBP1 in (TGFBRI and TGFBRII) have been detected in normal the human arterial wall and in atherosclerotic lesions has not human coronary and carotid arteries, the aorta and in athero- been reported. We have therefore examined the expression sclerotic plaques.9–12 However, atherosclerotic plaques have of LTBP1 mRNA by in situ hybridization (ISH) and of been examined in 2 studies for an inactivating mutation in the LTBP1 protein by immunohistochemistry in human coro- TGFBRII that inhibits TGFβ signaling through heterodimer nary atherosclerotic plaques. Other isoforms, LTBP2–4, in formation of TGFBRI and TGFBRII complexes. The muta- atherosclerosis were not investigated, primarily because their tion was detected in 1 study in a high proportion of plaques,11 relationship with TGFβ assembly, secretion and activation but not in the other.9 Consequently the issue of whether have not been as extensively demonstrated. TGFβ antigen in the vessel wall is biologically active and whether it affects lesion development remains unresolved. Methods TGFβ is generally secreted in its inactive form as a large latent complex (LLC) consisting of the active form of the Study Population and Sample Preparation cytokine non-covalently associated with latency-associ- The study population consisted of 11 patients referred for ated peptides (LAPs), 1 of which is covalently attached to the cardiac transplantation between June 1997 and February

Received April 20, 2010; revised manuscript received August 12, 2010; accepted August 13, 2010; released online November 7, 2010 Time for primary review: 21 days Harvard Medical School, Massachusetts General Hospital, Vascular Imaging and Interventions, Boston, MA (R.Ö., S.W.), USA; Section of Cardiovascular Biology, Department of Biochemistry, University of Cambridge, Cambridge (R.H., J.C.M.), UK Mailing address: Rahmi Öklü, MD, PhD, Harvard Medical School, Massachusetts General Hospital, Vascular Imaging and Interven- tions, 55 Fruit Street, Boston, MA 02155, USA. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-10-0334 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Advance Publication by J-STAGE ÖKLÜ R et al.

Figure 1. Detection of SM-α-actin, LTBP1 mRNA and protein in human coronary arteries. Sections show haematoxylin and eosin (H&E) staining (A,F,K), SM-α-actin immunofluorescence (green; B,G,L), dark field in situ LTBP1 mRNA expression (C,H,M) and LTBP1 immunofluorescence detected by the tyramide signal amplification system (red; D,I,N and E,J,O) in serial sections from the same stage II, Va and Vc lesions. C* denotes a control section using the sense LTBP1 probe. Nuclei were visual- ized with DAPI (blue). * in K denotes the same region in L,M and N in the stage Va lesion. Arrow indicates the internal elastic membrane of the arterial wall. Original magnification ×100 (A–H,K and M) and ×200 (I,J,L,N and O). DAPI, 4’,6-diamidino- 2-phenylindole; LTBP, latent TGFβ-binding protein; SM, smooth muscle; TGF, transforming growth factor.

1998. The study was approved by the Huntingdon Local (1:50; Sigma). The reactivity of the antibody mAb 388 with Research Ethics Committee and informed consent for use of LTBP1 was confirmed by western blotting under reducing tissue for experimentation was given by each patient. conditions using purified platelets as the source of LTBP1, Human coronary arteries (n=16) were dissected immedi- which yielded a single band of 160 kDa (not shown). ately following cardiectomy, mounted in embedding medium Endothelium was visualized using a rabbit polyclonal anti- (Cryo-M-Bed; Bright Instrument Company Ltd) and snap- body against von Willebrand factor (1:500, Dako; HRP-con- frozen in liquid nitrogen. Tissue sections (8.5μ m) were pre- jugated anti-rabbit IgG (1:500; Sigma) and the Cyanine-3 pared using a cryostat, thaw-mounted onto Superfrost Plus TSA kit). The presence of platelet specific microparticles was slides (BDH Laboratory Supplies), and stored at –85°C. investigated using a goat polyclonal antibody against CD42 Samples from 2 groups classified according to the established (1:100, Santa Cruz Biotechnology; HRP-conjugated anti-goat criteria of Stary et al22,23 using haematoxylin and eosin and IgG (1:500; Sigma) and the Cyanine-3 TSA kit). Smooth Oil-Red-O staining (early lesion, stage II and III, no atheroma- muscle cell (SMC) differentiation status was analyzed using tous plaques, n=6; advanced lesion, stages V and VI, n=10) a FITC-labelled SM-α-actin monoclonal antibody (1:500, were examined. Sigma). All incubations were carried out at 37°C for 60 min with Immunohistochemistry the primary antibody, 30 min with the secondary antibody and LTBP1 protein in human coronary artery sections was de- 15 min with streptavidin – HRP. Sections were counterstained tected using a monoclonal antibody (mAb) 388 (5μ g/ml; for 10 min with 300 nmol/L 4’,6-diamidino-2-phenylindole R&D Systems) and a rabbit polyclonal antibody (pAb 39; (DAPI, Molecular Probes) and mounted with Prolong-Anti- 1:50; gift of Dr Kohei Miyazono), followed by biotin-conju- fade (Molecular Probes). For controls, the primary antibody gated anti-mouse IgG (1:100; Sigma) or biotin-conjugated was omitted from the staining protocol and substituted with anti-rabbit IgG (1:20; Sigma), respectively. After incubation equal amounts of non-immune IgG (Sigma). with streptavidin-conjugated horseradish peroxidase (HRP, 1:500; Sigma), LTBP1 immunoreactivity was visualized ISH using a Cyanine-3 Tyramide Signal Amplification kit (TSA; Isolation of a LTBP1 fragment (681 bp) common to all re- NEN Life Sciences) according to the manufacturer’s instruc- ported LTBP1 spliced variants was obtained using reverse tions. Tyramide staining patterns were compared with conven- transcriptase-polymerase chain reaction (RT-PCR) and total tional fluorescein isothiocyanate (FITC)-labelled anti-mouse RNA isolated from adult human aortae.24 The forward primer IgG and FITC-labelled anti-rabbit IgG secondary antibodies (5’-CCATGGTGCCAGCGTGCCTAAACTTTATC-3’) and Advance Publication by J-STAGE Localization of LTBP1 in Coronary Atherosclerosis

Figure 2. Comparison of LTBP1 antigen detection methods in coronary artery sections of a stage I lesion. (A) LTBP1 antigen (red) was detected using the monoclonal antibody 388 and the Cyanine-3-TSA system. (B) LTBP1 antigen was detected using the monoclonal antibody 388 and a FITC-labeled anti-mouse IgG (green). (C) LTBP1 antigen was detected using the poly- clonal antibody 39 and a FITC-labeled anti-rabbit IgG (green). Arrows indicate the internal elastic membrane of the arterial wall. Original magnification ×200. FITC, fluorescein isothiocyanate; LTBP, latent TGFβ-binding protein; TGF, transforming growth factor.

the reverse primer (5’-GGTAGGATCCGGCCCAAATCCT- uniform levels throughout the neointima and media of early ATTTTG-3’) for PCR correspond to nucleotides 414–442 lesions (Figure 1C). In the advanced lesions the neointi- and 1068–1095, respectively, of the human LTBP1S cDNA mal expression of LTBP1 mRNA was very heterogeneous (Accession number M34057).25 The amplification product was (Figures 1H,M) compared with that seen in the neointima of gel-purified, sequenced to confirm identity and cloned into a early lesions (Figure 1C) with extensive areas of low LTBP1 pGEM-T Easy vector (Promega). Sense and antisense ribo- mRNA expression. However, in areas of high cell density in probes were synthesized from linearized pGEM-LTBP DNA the neointima of advanced lesions (Figure 1H), and particu- templates in the presence of [α35S]-UTP (~1,500 Ci/mmol, larly in regions of dense connective tissue at fibrous caps 20 mCi/ml; Amersham-Pharmacia Biotech) using an in vitro (Figure 1M), high levels of LTBP1 mRNA expression were transcription kit (Maxi-Script, Ambion). mRNA ISH was detected. Within these regions expression was restricted to performed as previously described.26 Sections were exposed a minor proportion (15–20%) of the cells (Figures 1H,M). to autoradiographic emulsion for 25 days at 4°C, developed The levels of LTBP1 mRNA in the media were similar in all and counterstained with haematoxylin and eosin. samples within each group of lesions (compare media in Figures 1C and H).

Results and Discussion Expression of LTBP1 Protein Differentiation Status of SMCs in the Coronary Artery In this study the experiments on LTBP1 protein expression Sections were performed with both mAb 388 and pAb 39. The pat- Sections stained with haematoxylin and eosin typical of an terns of LTBP1 antigen expression using both antibodies early lesion with a highly cellular neointima (stage II) and of detected by the tyramide signal amplification system and 2 more advanced lesions (stage Va and stage Vc) are shown FITC-labelled anti-IgG were very similar (Figure 2) and all in Figures 1A,F and K, respectively. Sections from the same data shown are for the mAb. lesions stained by immunofluorescence for the SMC differ- LTBP1 protein in early lesions consistently showed very entiation marker, SM-α-actin, are shown in Figures 1B,G high levels of expression in the endothelial layer overlying and L. The neointima of early lesions showed less SM-α-actin lower levels of uniform expression in the neointima and fur- staining than the media and very little staining in the layer of ther reduced levels in the media (Figure 1D). It is therefore cells towards the luminal surface. In the stage Vc lesions the clear that the LTBP1 protein levels near the luminal surface amount of SM-α-actin was also markedly reduced at the are substantially higher, relative to LTBP1 mRNA expres- neointimal surface, but was similar in the cellular interior of sion, than those observed in the media (compare Figures 1C the neointima and the media (Figure 1G). The neointima of and D). These results imply that factors other than the stage Va lesions showed only heterogeneous expression of amount of mRNA determine LTBP1 protein levels. For SM-α-actin near the surface, with very little detectable anti- example, it is possible that dedifferentiated SMCs in the neo- gen in the lipid core (Figure 1L). The most prominent reduc- intima drive translation of LTBP1 mRNA into protein more tion in SM-α-actin staining occurred mainly in areas of high efficiently than in the media. Alternatively, LTBP1 protein cell density in the fibrous connective tissue layer near the stability may be substantially higher in the neointima (eg, if luminal surface (Figure 1G) and in areas of very low cell the LLC is not degraded proteolytically to release TGFβ). density in the lipid core of advanced lesions (Figure 1L). The high levels of LTBP1 protein near the luminal surface These observations are consistent with previous studies on might also be explained by accumulation of LLC from the SMC differentiation in such lesions.27 The presence of an circulation, although there are no reports of LTBP1 protein intact endothelial layer in these same lesions was confirmed in platelet-poor plasma. Deposition in the lesion of cells by immunostaining for von Willebrand factor (not shown). from the circulation that express very high levels of the pro- tein might also account for the high levels of LTBP1 protein Expression of LTBP1 mRNA near the luminal surface. Platelets are a major source of LLC ISH showed that LTBP1 mRNA was expressed at similar, in the circulation28 and we therefore used immunofluorescent Advance Publication by J-STAGE ÖKLÜ R et al. staining with anti-CD42 antibody to assay for the presence of platelet microparticles carrying glycoprotein IIb/IIIa.29 No References specific staining was detected in early or late lesions, indicat- 1. Massague J. TGF-beta signal transduction. Annu Rev Biochem ing that platelet deposition and granule release are unlikely to 1998; 67: 753 – 791. account for the observed accumulation of LTBP1. 2. Bjorkerud S. Effects of transforming growth factor-beta 1 on human arterial smooth muscle cells in vitro. 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