Eotaxin and capping protein in experimental vasculopathy

Abstract

Ischemia-induced tissue activation may contribute to the pathogenesis of graft vasculopathy, but the mediators implicated have only partially been characterized. To gain further insight into the molecular mechanisms involved, syngeneic rat aortic transplants with cold-storage-induced vasculopathy were studied for differentially expressed mRNA transcripts. Vessel segments were exposed to either 1 or 18 hours of cold ischemia, followed by transplantation into syngeneic recipients. After 3 days or 4 weeks, the grafts were removed and total mRNA was isolated and used for differential display to identify modulation of transcript expression related to prolonged storage. Using 15 sets of random primers, 17 polymerase chain reaction products were up-regulated and 2 were downregulated in grafts exposed to 18 hours of ischemia. Sequencing of these amplicons showed that 6 had a high degree of homology to known sequences whereas 13 had no homology to any of the genes in the database. Two of the differentially displayed amplicons (capping protein and eotaxin) were cloned, re-amplified, and used as probes for Northern blot analysis to confirm their differential expression. Immunohistochemistry using monoclonal antibodies against capping protein-alpha and eotaxin confirmed that both proteins are expressed in the media of normal aortas and that there was an increased expression in vessels exposed to prolonged ischemia albeit that the increase at the protein level seemed less compared with changes in transcript expression. Northern blots with RNA from aortic allografts exposed to prolonged ischemic storage also showed increased levels of capping protein and eotaxin mRNA whereas there was a decrease in the relative amount of these transcripts in vessels exposed to balloon denudation, suggesting that the increase after prolonged ischemic exposure is not the result of a nonspecific response to injury. Based on the biological characteristics of capping protein and eotaxin it is conceivable that they play a pathogenetic role in ischemia-induced vessel wall remodeling. It remains to be established whether these genes or their products serve as target molecules for therapeutic interventions to prevent or treat cold-storage-induced graft vasculopathy.

Figure 1.

Examples of differentially expressed genes in syngeneic aortic grafts transplanted after 1 or 18 hours of cold storage and removed on day 3 or week 4 after transplantation. Arrows indicate amplicons that were up-regulated in grafts exposed to 18 hours of ischemia but not in grafts exposed to 1 hour of ischemia; the up-regulated amplicon in A shows sequence homology with rat eotaxin; the up-regulated amplicon in B shows sequence homology with capping protein.

Figure 2.

Northern blots of [³²P]dCTP-labeled eotaxin (A), [³²P]dCTP-labeled capping protein (B), and [³²P[dCTP-labeled GAPDH (C) probes with RNA prepared from normal aortas or syngeneic aortic grafts exposed to 1 or 18 hours of cold storage and removed 3 days after transplantation. There was a 1.7- and a 9-fold increase in the eotaxin/GAPDH ratio between normal aortas and syngeneic aortic grafts exposed to 1 hour or 18 hours of storage, respectively (A). There was a 1.1- and 3.4-fold increase in the capping protein/GAPDH ratio between normal aortas and syngeneic aortic grafts exposed to 1 or 18 hours of cold storage, respectively (B).

Figure 3.

Northern blots using [³²P[dCTP-labeled eotaxin (A), [³²P[dCTP-labeled capping protein (B), and [³²P[dCTP-labeled GAPDH (C) probes with RNA prepared from carotid arteries exposed to balloon injury and removed on days 0, 3, 7, or 14 after injury. The mean eotaxin/GAPDH ratios were 2.3, 1.1, 1.2, and 1.94 on days 0, 3, 7, and 14, respectively; the mean capping protein/GAPDH ratios were 5.2, 2.6, 5.1, and 4.2 on days 0, 3, 7, and 14, respectively.

Figure 4.

Sequence homology of the cloned eotaxin amplicon showing a high degree of homology with Rattus norvegicus eotaxin cDNA.

Figure 5.

Sequence homology of the cloned capping protein amplicon showing a high degree of homology with Mus musculus capping protein α-1 subunit.

Figure 6.

Immunoperoxidase staining for eotaxin of a syngeneic aortic graft exposed to 18 hours of cold storage and 2 hours of residence in a syngeneic host. There is eotaxin expression in the medial smooth muscle cells.

Figure 7.

Semiquantitative evaluation of eotaxin expression in the intima, media, and adventitia of syngeneic aortic grafts exposed to either 1 or 18 hours of cold storage and studied 2 hours, 3 days, or 4 weeks after transplantation. The changes in eotaxin expression assessed by immunoperoxidase are not significant.

Figure 8.

Immunoperoxidase staining for capping protein α-1 subunit expression in a native aorta (A) and in a syngeneic aortic grafts after 1 hour (B) or 18 hours (C) of cold storage and α-smooth muscle actin (D) and monocyte (ED-1) staining (E) in syngeneic grafts exposed to 18 hours of cold storage. All grafts were removed 4 weeks after transplantation. A shows the constitutive expression of capping protein in the media of a normal aorta. B shows a few capping-protein-positive cells in the intima and media in grafts exposed to 1 hour of cold ischemia. C shows diffuse and pronounced expression of capping protein α-1 subunit in the neointima after 18 hours of ischemia. Staining of a serial section with anti-smooth muscle actin antibodies (D) or the anti-monocyte antibody ED-1 (E) shows that capping protein α-1 subunit expression is mostly in smooth muscle cells whereas there are only very few ED-1-positive cells in the intima. The arrow shows internal elastic lamina. Immunoperoxidase staining counterstained with Mayer’s hematoxylin; magnification, ×400.

Figure 9.

Semiquantitative evaluation of capping protein α-1 subunit expression in the intima, media, and adventitia of syngeneic aortic grafts exposed to either 1 or 18 hours of cold storage and studied 4 weeks after transplantation. The 0 time denotes nontransplanted normal aortas. The increase in capping-protein-positive cells in the intima and adventitia is significant in the group exposed to 18 hours of cold ischemia.

Figure 10.

Accumulation of capping protein α-1 subunit-positive cells around surgical material in the adventitia of a syngeneic graft 8 weeks after transplantation, suggesting that some macrophages may also express capping protein α-1 subunit. Magnification, ×400.