Proc. Nati. Acad. Sci. USA Vol. 91, pp. 6463-6467, July 1994 Medical Sciences Chronic cardiac rejection: Identification of five upregulated genes in transplanted hearts by differential mRNA display (gene expreson/traplant arterlosclerosl/cardlac tnsplatatlon/polymerase chain reaction) ULRIKE UTANS*, PENG LIANG*, LAURI R. WYNERt, MoRRis J. KARNOVSKYt, AND MARY E. RUSSELL*tt§ *Cardiovascular Biology Laboratory, Harvard School of Public Health, tHarvard Medical School, and tCardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115 Communicated by Arthur B. Pardee, March 25, 1994 (receivedfor review November 18, 1993) ABSTRACT Tran t arteriosclerosis, the major man- planted heart that limits transplant survival (3, 4). Studies of festation of chronic rejection, develops after alogeneic (Lewis the process in humans have been restricted by the limited to F344) but not syngeneic (Lewis to Lewis) rat cardiac availability of tissue for analysis. Clinical specimens are transplantation. To identify transcriptionaly regulated medi- heterogeneous in their degree of chronic rejection, their ators asiated with chronic cardiac rejection, we adapted the extent of superimposed disease processes, and the period differential mRNA display technique for in vvo a nt between the time they are obtained and the time of trans- specimens. Gene tanscript patterns In four allogenec hearts plantation. Also, transplanted hearts obtained at autopsy are showing early signs of chronic rejection were compared with not suitable for analysis (which requires viable tissue), and those in two syngeneic hearts exposed to the same surgical the utility of endomyocardial biopsy specimens is limited by procedure but histologically normal. Twelve differentially ex- their small size. Moreover, the restricted extent of arterio- pressed cDNA bands were Identied. We improved the prob- sclerotic lesions that follow transplantation suggests that the ability of Ioating one or more allograft-specific cDNAs from process is locally regulated; thus, studies measuring systemic a single display band by first using recovered and reampled levels of factors implicated in chronic rejection may not PCR products as probes in RNA blot analysis. CDNA faents accurately reflect levels within the graft (5). cloned from individual bands were then used in a second RNA A heterotopic rat cardiac transplantation model (which blot analysis, which allowed for the correlation of specific results in long-term graft survival) has provided insight into mRNA scripts with cDNA clones. Five cDNA clones pro- the cellular development of chronic cardiac rejection (6, 7). duced time-dependent, allograft-specific hybridization. Se- Monocytes/macrophages accumulate in the first stage (days quence analysis demonstrated that two of these cDNAs corre- 7-30) ofarteriosclerotic thickening. In the intermediate stage sponded to unknown genes, whereas the other three repre- (days 30-75) both macrophages and smooth muscle cells sented known genes not previously associated with chronic accumulate, and in the later, more obliterative stage (after 75 rejection. The latter group included the macrophage lectin days) smooth muscle cells predominate. In previous studies specific for galactoe/N-acetylgalactomine (a cell-surface we used reverse transcription PCR to measure gene tran- receptor), the nuclear P1 gene (a homologue of a yeast repli- script levels for selected cytokine factors, and we showed cation protein), and a ubiquti n-lke gene. Our application of that interferon 'y and monocyte chemoattractant protein 1 the differential display tehnique allowed the direct identifica- transcript levels were upregulated in cardiac allografts in a tion of potential mediatos under in vivo conditions that specific and local manner (ref. 8 and M.E.R., A. F. Wallace, preserve the environment of the disease process-ncluding N. E. S. Sibinga, L.R.W., and M.J.K., unpublished data). infiltrating cell populations critical to the inflammatory re- However, chronic rejection is most likely mediated by a sponse. variety of known as well as unknown factors. Therefore we performed the present study to determine whether differen- For most genes, expression is regulated at the level of tial mRNA display could be used to identify unknown me- transcription; therefore, the evaluation of gene transcripts diators or known mediators not previously implicated in has become an important tool in the study of normal and chronic cardiac rejection. We adapted the technique to the pathologic processes. Conventional measurements ofmRNA evaluation of gene expression patterns directly in diseased transcript levels are usually confined to selected genes of tissues by comparing mRNA profiles in four arteriosclerotic interest and often require information about the gene se- rat hearts after allogeneic transplantation with those in two quence. In contrast, PCR-based differential display tech- control hearts subjected to syngeneic transplantation. Using niques circumvent this constraint by allowing comparison of 27 primer combinations, we identified two unknown and gene expression patterns between two cell populations (1) or three known genes whose expression is upregulated with time between various murine organs (2). One of the principal in hearts undergoing chronic rejection. advantages of differential display is that it permits the simul- taneous identification ofgenes that are upregulated as well as genes that are downregulated. Thus differential display has MATERIALS AND METHODS the potential to identify a spectrum of molecular factors- RNA Islation and RNA Blot Analysis. Heterotopic abdom- known and unknown-that are differentially regulated in inal cardiac transplantations were performed and samples cells under various conditions. were collected as described (8, 9). For allogeneic transplan- Little is known about molecular factors involved in the tations we used Lewis rats as graft donors and F344 rats as pathogenesis of chronic cardiac rejection. In contrast with recipients. This strain combination generates grafts that acute rejection, this multicellular process results in an oblit- survive beyond 7 days, the point after which rejection would erative neointimal thickening in the vessels of the trans- be considered chronic. The early signs ofchronic rejection- macrophage infiltration and adhesion to luminal surfaces- The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" §To whom reprint requests should be addressed at: Harvard School in accordance with 18 U.S.C. §1734 solely to indicate this fact. of Public Health, 677 Huntington Avenue, Boston, MA 02115. 6463 Downloaded by guest on September 26, 2021 6464 Medical Sciences: Utans et al. Proc. Natl. Acad. Sci. USA 91 (1994) were confirmed by histologic analysis as described (7, 9). RESULTS Syngeneic control procedures (Lewis to Lewis) were per- formed to assess the contribution of surgical manipulation to Differential mRNA Display. To identify transcriptionally the inflammatory response. Hearts were harvested at various regulated genes potentially involved in the early stages of points after transplantation and quick-frozen in liquid nitro- chronic rejection, we compared differential mRNA display gen. Total cellular RNA was extracted from heart tissue with patterns for hearts from syngeneic transplantations with RNAzol B (Tel-Test, Friendswood, TX) according to the those for hearts from allogeneic transplantations. Syngeneic manufacturer's instructions. Samples of total RNA (20 ug) hearts were normal on histologic examination, whereas 7- were fractionated in 2.2 M formaldehyde/1% agarose gels and 14-day allogeneic hearts showed luminal monocyte ad- and transferred onto nylon-supported nitrocellulose (Micron hesion and infiltration without intimal thickening (as de- Separations, Westboro, MA) by standard capillary blotting scribed previously; ref. 9). We performed PCR amplifications techniques. Specific probes were generated by labeling ream- with 27 primer combinations on all six samples and identified plified or cloned cDNA fragments with [a-32P]dCTP (random 12 PCR products, designated bands 1-12, that were differ- prime DNA labeling kit, Boehringer Mannheim). After hy- entially expressed between allogeneic and syngeneic tissue. bridization at 42TC and high-stringency washes at 600C in 0.3 Fig. 1 shows PCR amplifications obtained with three separate M NaCl/0.03 M trisodium citrate, pH 7/0.1% SDS, the blots primer combinations. Indicated are four representative PCR were exposed to Kodak X-omat AR film for 3-7 days with products (bands 1, 2, 11, and 12) that were reproducibly intensifying screens. Hybridization with a partial cDNA present in the allogeneic samples (lanes 3-6) but not in the fragment of the rat homologue of 36B4 (acidic ribosomal syngeneic samples (lanes 1 and 2) in each of the three phosphoprotein P0) was used as a loading control (10). analyses. Differential mRNA Display. Differential mRNA display RNA Blot Analysis with PCR-Ampld Fragments. To analysis was carried out as described (1, 11), except that in confirm the gene regulation patterns observed in the differ- vivo rather than in vitro samples were used and six rather than ential display study, we recovered the 12 selected bands, two samples were compared simultaneously. We studied reamplified them, and used them to probe RNA blots pre- total RNAs obtained from four allografts (two each harvested pared with RNAs from syn- and allogeneic