Two Hsp70 family members expressed in atherosclerotic lesions Zhihua Han, Quynh A. Truong, Shirley Park, and Jan L. Breslow* Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Contributed by Jan L. Breslow, December 16, 2002 Gene expression profiling was carried out comparing Con A elicited chromosomes 10, 14, and 19 (10). The responsible genes at these peritoneal macrophages from C57BL͞6 and FVB͞N wild-type and loci are not currently known. apolipoprotein (apo)E knockout mice. An EST, W20829, was ex- Because macrophages play such a fundamental role in the pressed at higher levels in C57BL͞6 compared with FVB͞N mice. initiation and progression of atherosclerotic lesions, differen- W20829 mapped to an atherosclerosis susceptibility locus on chro- tially expressed genes were sought between elicited peritoneal mosome 19 revealed in an intercross between atherosclerosis- macrophages from C57BL͞6 and FVB͞N wild-type and apoE susceptible C57BL͞6 and atherosclerosis-resistant FVB͞N apoE knockout mice. In the course of this analysis, one of the genes knockout mice. A combination of database search and Northern overexpressed in C57BL͞6 macrophages corresponded to an -analysis confirmed that W20829 corresponded to 3؅-UTR of a EST that maps to the chromosome 19 atherosclerosis suscepti hitherto predicted gene, named HspA12A. Blasting the National bility locus. This gene, HspA12A, and another closely related Center for Biotechnology Information database revealed a closely mammalian homologue that maps to a different region of the related homologue, HspA12B. HspA12A and -B have very close genome, HspA12B, were found to be distant members of the human homologues. TaqMan analysis confirmed the increased Hsp70 family. Both genes were shown to be expressed in HspA12A expression (2.6-fold) in elicited peritoneal macrophages atherosclerotic lesions but not in nonlesional regions of the from C57BL͞6 compared with FVB͞N mice. TaqMan analysis also aorta. These genes now become candidates for playing a role in revealed increased HspA12A and HspA12B expression (87- and the atherosclerotic process, and HspA12A is also a candidate for 6-fold, respectively) in lesional versus nonlesional portions of the involvement in the chromosome 19 susceptibility locus. thoracic aorta from C57BL͞6 apoE knockout mice on a chow diet. In situ hybridization confirmed that both genes were expressed Materials and Methods within lesions but not within nonlesional aortic tissue. Blasting of Animals. Mice were housed in a specific pathogen-free humidity- HspA12A and HspA12B against the National Center for Biotech- and temperature-controlled room with a 12-h light͞dark cycle nology Information database (NR) revealed a hit with the Con- and fed normal chow diet. Eight-week-old C57BL͞6, FVB͞N, served Domain database for Hsp70 (pfam00012.5, Hsp70). Both and C57BL͞6 apoE knockout mice were from The Jackson genes appear to contain an atypical Hsp70 ATPase domain. The Laboratory. FVB͞N apoE knockout mice were created by BLAST search also revealed that both genes were more similar to ͞ ϫ backcrossing apoE knockout heterozygous F1 (C57BL 6 primitive eukaryote and prokaryote than mammalian Hsp70s, 129͞Sv) mice with wild-type FVB͞N mice for 10 generations and making these two genes distant members of the mammalian Hsp70 then performing brother͞sister mating to derive homozygous family. In summary, we describe two genes that code for a apoE knockouts. subfamily of Hsp70 proteins that may be involved in atheroscle- rosis susceptibility. RNA Isolation from Elicited Peritoneal Macrophages. Male mice of each genotype were used to isolate peritoneal macrophages. n the last decade, induced mutant mice have become useful Each mouse was injected in the peritoneum with 0.5 ml of 80 Imodels of atherosclerosis. The first of these models, the ␮g͞ml Con A (Sigma) in PBS. After 72 h, mice were euthanized apolipoprotein (apo)E knockout mouse, has been widely used to by asphyxiation in CO2 and the peritoneal cavity immediately study lesion formation, test pharmacological therapies, and lavaged with 9 ml of PBS at 4°C. The lavagates from 5–15 mice evaluate the influence of candidate genes on the atherosclerotic were then combined and spun at 500 ϫ g and pelleted cells process (1, 2). In the apoE knockout mouse on a chow diet, resuspended in RPMI 1640 medium (American Type Culture monocyte͞macrophage attachment occurs at 6–8 weeks of age Collection). The cells were then plated in P100 plastic tissue followed by the appearance of subintimal lipid-laden macro- culture dishes (Corning), incubated at 37°C for 2 h, and washed phages, foam cells, at 8–10 weeks of age. Between 15 and 25 three times with cold PBS to remove unattached cells. By using weeks of age, macrophage necrosis occurs, and the fibrous cap the RNeasy Mini kit (Qiagen, Chatsworth, CA), attached cells forms. Candidate genes that decrease macrophage number, were lysed and total RNA isolated. monocyte͞macrophage attachment, and homing to lesion-prone regions dramatically decrease atherosclerotic lesion formation cDNA Microarray Expression Analysis. Two micrograms of total and progression in mouse models (3–8). The morphology of the RNA was used to synthesize fluorescently labeled cDNA probes atherosclerotic plaque and the results from experiments with with a 3DNA submicro kit (Genisphere, Montvale, NJ). Glass candidate genes all suggest a major role for macrophages in slides printed with Ϸ9,000 cDNAs or ESTs were a generous gift atherosclerosis. from Raju Kucherlapati at Albert Einstein College of Medi- Mouse strains have been described with differing susceptibil- cine (11). Arrays were hybridized according to their recom- ities to atherosclerosis based initially on responsiveness to diet mended protocol (http:͞͞sequence.aecom.yu.edu͞bioinf͞ (9). With the advent of induced mutant mice that develop microarray͞protocol4.html). Hybridized arrays were scanned atherosclerotic lesions on chow or more physiologically relevant Western-type diets (1, 2), these models are now being used in genetic crosses to reveal atherosclerosis modifier loci. In an Abbreviation: apo, apolipoprotein. intercross between an atherosclerosis-susceptible strain, Data deposition: The sequences reported in this paper have been deposited in the GenBank C57BL͞6 apoE knockout, and an atherosclerosis-resistant database (accession nos. AY196789 and AY196790). strain, FVB͞N apoE knockout, three loci were revealed on *To whom correspondence should be addressed. E-mail: [email protected]. 1256–1261 ͉ PNAS ͉ February 4, 2003 ͉ vol. 100 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.252764399 Downloaded by guest on September 26, 2021 with a ScanArray4000 (General Scanning, Watertown, MA) RNA probes (ribo-probes) were generated by in vitro transcrip- and analyzed by SCANALYZE software (http:͞͞rana.stanford. tion with T7 RNA polymerase (Roche Diagnostics). Sense edu͞software). ribo-probes were generated by in vitro transcription with SP6 RNA polymerase from the same plasmids. After perfusion as Creation of cDNA Probes and Northern Blot Analysis. cDNAs corre- above, the thoracic aorta was removed from 6-mo-old chow-fed sponding to the EST W20829, the midpoint, and the beginning C57BL͞6 apoE knockout mice, fixed in 4% paraformaldehyde at of the predicted exon 12 of HspA12A were made by PCR. The 4°C for 72 h, immersed in 25% sucrose at 4°C for 96 h, immersed primers used corresponded to the sequences indicated in Table in OCT, and frozen at Ϫ80°C until sectioning. Sections at 8-␮m 3, which is published as supporting information on the PNAS intervals were made with a cryostat at Ϫ23°C, collected onto web site, www.pnas.org. The PCR reaction used High Fidelity silanized glass slides (PGC Scientific, Gaithersburg, MD), and Taq (Invitrogen) and the template was mouse brain͞heart cDNA stored at –80°C until hybridized. Before hybridization, sections (Ambion, Austin, TX). Twenty-five nanograms of each cDNA were fixed in 4% paraformaldehyde, permeabilized with 0.01 was radiolabeled by nick-translation ECA prime II (Ambion) mg͞ml proteinase K, treated with 0.2 M HCl, and acetylated in and 32P-dATP (Perkin–Elmer). Nitrocellulose blots containing 1.3% triethanolamine and 0.25% acetic anhydride (Sigma). RNA from multiple mouse tissues of BALB͞c strain were After each of these steps, the slides were washed once with 0.1 purchased from CLONTECH and hybridized with Express Hyb M phosphate buffer (pH 7.4). Sections were then hybridized with Rapid Hybridization Buffer (CLONTECH) according to the antisense or sense ribo-probes in Hybriwell chambers (PGC manufacturer’s directions. After each hybridization, the blot was Scientific) at 55°C for 24 h. Sections were then washed in 5ϫ SSC stripped by incubation in 1 liter of 0.5% SDS solution at 100°C at 60°C for 20 min, 2ϫ SSC, and 50% formamide at 60°C for 30 for 15 min and exposed to x-ray film for 24 h to ensure that there min, treated with 1 mg͞ml RNaseA at 37°C for 30 min, and were no residues from previous hybridizations. Northern blot washed with 2ϫ SSC and 50% formamide at 60°C for 30 min. analysis of human HspA12A and HspA12B was carried out by a The sections were then treated with anti-DIG antibody–alkaline similar method, and the primer sequences used are specified in phosphatase conjugate (Roche) at room temperature for 18 h. Table 3. After washing with 0.1 M phosphate buffer (pH 7.4), nitroblue tetrazolium͞5-bromo-4-chloro-3-indolyl phosphate substrates TaqMan (Real-Time Quantitative RT-PCR) Analysis. Total RNA from (Roche Diagnostics) were added and blue color developed in the peritoneal macrophages was isolated as above. Total RNA was dark for 3 h. Sense-strand ribo-probes were used as negative isolated from aortic tissue as follows: four 10-mo-old C57BL͞6 controls in adjacent tissue sections. apoE knockout mice fed a chow diet were anesthetized, the chest MEDICAL SCIENCES was opened, the right atrium nicked, and perfusion carried out Results through the left ventricle with 10 ml of PBS.