Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation Noel M. Caplice*†‡, T. Jared Bunch§, Paul G. Stalboerger*, Shaohua Wang*, David Simper*, Dylan V. Miller¶, Stephen J. Russell†, Mark R. Litzowʈ, and William D. Edwards¶ Divisions of *Cardiovascular Diseases, ʈHematology, and ¶Anatomic Pathology, †Molecular Medicine Program, and §Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905 Communicated by Ernest Beutler, The Scripps Research Institute, La Jolla, CA, February 6, 2003 (received for review December 26, 2002) Atherosclerosis is the major cause of adult mortality in the devel- arteries. Control same-gender bone marrow transplant subjects oped world, and a significant contributor to atherosclerotic plaque were used to assess FISH sex chromosome discrimination and to progression involves smooth muscle cell recruitment to the intima detect vascular tissue evidence of background fetal and maternal of the vessel wall. Controversy currently exists on the exact origin microchimerism (16). of these recruited cells. Here we use sex-mismatched bone marrow transplant subjects to show that smooth muscle cells throughout Methods the atherosclerotic vessel wall can derive from donor bone mar- Patients and Autopsy Tissue. Coronary artery specimens from 13 row. We demonstrate extensive recruitment of these cells in subjects (eight gender-mismatched subjects and five gender- diseased compared with undiseased segments and exclude cell– matched control subjects) who received BMT were studied. All cell fusion events as a cause for this enrichment. These data have patients had survived 1 month or more after transplant with broad implications for our understanding of the cellular compo- clinical report of engraftment. The institutional review board at nents of human atherosclerotic plaque and provide a potentially Mayo Clinic approved the use of all autopsy specimens and all novel target for future diagnostic and therapeutic strategies. patients gave consent before transplant for their tissue to be used in research studies. precursor ͉ bone marrow ͉ plaque ͉ intima ͉ chimerism Combined Immunohistochemical and FISH Analysis. Formalin-fixed, therosclerosis is a complex disease, and our current under- paraffin-embedded blocks were cut into 4-␮m sections and Astanding represents a synthesis of numerous hypotheses placed on microscope slides. Multiple sections through diseased developed over the past century and a half (1–4). The hypotheses and undiseased coronary artery segments were obtained from have been continually modified to take into account new exper- each subject. The sections were deparaffinized by using CitriSolv imental and clinical data (5–9). Currently, the pathogenesis of (Fisher Scientific) and rehydrated in an ethanol series. Immu- atherosclerosis is thought to involve a sequence of biologic nohistochemical analysis was performed by using monoclonal events within the intima of the vessel wall which includes vascular antibodies against smooth-muscle ␣-actin, myosin heavy chain, injury, monocyte recruitment and macrophage formation, lipid and calponin as described (15, 17). The secondary antibodies deposition, platelet degranulation and thrombosis, and vascular used were antimouse antibodies conjugated to Cy-3 (Molecular smooth muscle cell migration, proliferation and extracellular Probes) (red stain) or AlexaFluor488 (Molecular Probes) (green matrix synthesis (9). stain). Until recently, the smooth muscle cell component of primary After immunostaining, FISH was immediately performed. atherosclerotic plaque was thought to solely derive from the The tissue was dehydrated twice in 100% ethanol for 1–2 min and surrounding local vessel wall with migration of phenotypically then heated in a steamer in preheated 1 mM EDTA (pH 8.0) for distinct smooth muscle cells from the media through fenestra- 20 min, followed by 0.05 ␮g͞␮l proteinase K (Sigma) or pepsin ͞ ⅐ ͞ tions in the internal elastic lamina to the intima where prolif- A (2,100 units mg) in buffer (0.05M Tris HCl 2 mM CaCl2,pH eration of these altered cells occurs (2, 7, 10). These latter 7.8͞0.01 M EDTA͞0.01 M NaCl) at 37°C for 15 min. The tissue assumptions have been questioned in the light of animal studies was then rinsed in an ethanol series. Subsequently, the hybrid- suggesting that bone marrow progenitor cells can infiltrate the ization probe mixture (Vysis, 30-804824) was applied to the atherosclerotic intima and differentiate in vivo to form smooth sections. The DNA probes used were specific for the ␣ satellite muscle cells within the plaque (11–14). We have also recently regions of the X and Y chromosomes, and were fluorescently shown that phenotypically distinct smooth muscle cells can be labeled. The X chromosome probe (CEP X, Vysis, B-7322) was grown in culture from human peripheral blood, suggesting the labeled with cyanine-3 (red), and the Y chromosome probe existence of a circulating smooth muscle progenitor (15). How- (CEP Y, Vysis, B-6927) was labeled with fluorescein isothio- ever, no in vivo data currently exist to support a bone marrow cyanate (green). For combined immunostaining and FISH, Cy3 origin for smooth muscle cells within primary atherosclerotic actin staining with Y chromosome, or Alexa Fluor actin staining plaque of human subjects. with X chromosome combinations were used. In separate ex- To investigate whether smooth muscle cells participating in periments a probe to the centromere of human chromosome 18 human atherosclerotic plaque derive from the bone marrow, we (CEP 18 SpectrumAqua-light blue-Vysis) was also combined studied diseased and undiseased coronary artery segments of with X and Y chromosome analysis to evaluate cell ploidy and deceased subjects who had previously undergone gender- exclude cell fusion. Ploidy analysis was performed on all gender- mismatched bone marrow transplantation (BMT) and were mismatched subjects. found to have coexistent coronary atherosclerosis at autopsy. Immunohistochemistry for smooth muscle specific markers was combined with flourescence in situ hybridization (FISH) for the Abbreviations: BMT, bone marrow transplantation; FISH, fluorescence in situ hybridiza- X and Y chromosomes to discriminate male and female smooth tion; DAPI, 4Ј,6-diamidino-2-phenylindole. muscle cells of donor and recipient origin in diseased coronary ‡To whom correspondence should be addressed. E-mail: [email protected]. 4754–4759 ͉ PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0730743100 Downloaded by guest on September 24, 2021 Table 1. Clinical and pathologic characteristics of gender-mismatched and gender-matched (control) BMT subjects Graft Patient Gender Gender Age, duration, no. [D] [R] years Underlying disease Conditioning regimen days Coronary pathology Gender-mismatched bone marrow transplant subjects 1 Male Female 42 Chronic lymphocytic Cyclophosphamide TBI 964 Grade III fibrocellular leukemia plaque 2 Male Female 46 Chronic granulocytic Cyclophosphamide 501 Grade II fibrocellular leukemia busulphan plaque 3 Male Female 33 Chronic granulocytic Cyclophosphamide TBI 41 Grade III fibrocellular leukemia plaque 4 Male Female 30 Myelodysplastic syndrome Cyclophosphamide TBI 90 Diffuse intimal thickening 5 Female Male 39 Myeloma Cyclophosphamide 1235 Grade III fibrocellular busulphan plaque 6 Female Male 29 Myelodysplastic syndrome Cyclophosphamide TBI 78 Grade III fibrocellular plaque 7 Female Male 45 Acute myelogenous Cyclophosphamide TBI 131 Grade III fibrocalcific leukemia plaque 8 Female Male 58 Myelofibrosis Cyclophosphamide 91 Diffuse intimal busulphan thickening Gender-matched bone marrow transplant subjects 9 Male Male 55 Myelodysplastic syndrome Cyclophosphamide TBI 655 Grade II fibrocellular plaque 10 Male Male 46 Acute lymphocytic Cyclophosphamide TBI 527 Grade III fibrocellular leukemia plaque 11 Male Male 45 Myelodysplastic syndrome Cyclophosphamide TBI 153 Diffuse intimal thickening 12 Female Female 46 Chronic granulocytic Cyclophosphamide 108 Diffuse intimal leukemia busulphan thickening 13 Female Female 52 Chronic granulocytic Cyclophosphamide TBI 901 Grade II fibrolipid leukemia plaque [D] and [R] indicate donor and recipient, respectively. TBI, total body irradiation. Coverslips were affixed and sealed with rubber cement, and ploidy (1,000 nuclei per diseased and undiseased segment per the slides were incubated at 80°C for 3 min to denature all DNA gender-mismatched patient). A pathologist who was unaware of and then were incubated at 37°C overnight. After hybridization, the age or sex of the patient independently assessed the histology the slides were washed in 2ϫ SSC͞0.1% Nonidet P-40 solution of the coronary arteries to classify the presence or absence of at room temperature, counterstained with 0.03 ␮g͞ml 4Ј,6- arterial disease. Comparisons between diseased and undiseased diamidino-2-phenylindole (DAPI), and mounted with Vectash- groups were made by ANOVA, and P Ͻ 0.05 was considered ield (Vector Laboratories, H-1200). FISH signals were enumer- significant. ated by using a Zeiss Axioplan microscope equipped with a triple-pass filter (Vysis). Results Characteristics of Study Subjects. Thirteen autopsy subjects were Data and Statistical Analysis. Sections were reviewed on the same studied, eight subjects (four male, four female) had undergone microscope to ensure the images were consistent and reproduc- gender-mismatched and five control subjects (three male, two ible. Only sections that contained clear morphology