MEDIAL SMOOTH-MUSCLE CELL LOSS IN ARTERIAL ALLOGRAFTS OCCURS BY CYTOLYTIC CELL-INDUCED APOPTOSIS

Objective: Experimental arterial allografts, used as models of chronic rejection, undergo marked loss of smooth muscle cells (SMC) from thei r media prior to the development of occlusive, intimal proliferative l esions. Medial SMC loss has been described in human heart transplants, and may be related to the development of occlusive coronary lesions w hich are the hallmark of chronic rejection. This SMC loss does not exh ibit the characteristics of necrotic cell death. We sought to determin e whether medial SMC loss in arterial allografts occurs by apoptosis. We further investigated these allografts for cytolytic cell-derived in ducers of apoptosis, Finally, we compared two different strain combina tions to assess the impact of varying histoincampatability on medial S MC loss. Methods: Evidence for intermucleosomal DNA degradation, which is characteristic of apoptosis, was sought by the in situ terminal de oxynucleotidyl transferase nick end labelling (TUNEL) method carried o ut on Lewis to Fisher rat femoral artery transplants (disparate at min or loci only) and Brown Norway to Lewis aortic transplants (fully disp arate at major and minor loci). Isografts (Lewis to Lewis) served as c ontrols. In a separate series of experiments graft mRNA was extracted and analysed by reverse transcription-polymerase chain reaction (RT-PC R) with primers for molecular inducers of apoptosis (TNF-alpha, Fas li gand, perforin, and granzyme-B) which are derived from cytolytic cells known to be present in allografts, Results: Allograft media contained large numbers of TUNEL stained nuclei in both strain combinations. Ne ither isografts nor ungrafted femoral artery segments stained positive for apoptosis. RT-PCR on whole allografts in both strain combinations revealed sustained upregulation of perforin, granzyme-B, Fas-ligand a nd TNF-alpha mRNA concomitant with medial SMC loss. Autografts demonst rated sustained up regulation of TNF-alpha, and perforin, but only bri ef upregulation of granzyme-B, and no upregulation of Fas-ligand. Conc lusions: These data strongly suggest that medial SMC loss in allograft arteriopathy occurs by apoptosis. Further, RT-PCR data indicate that cytolytic cell-derived inducers of apoptosis are upregulated in these grafts and may be accountable for medial SMC apoptotic cell death. Fin ally, fully-disparate (Brown Norway to Lewis) and minor-only incompati ble (Lewis to Fisher) strain combinations both resulted in marked inti mal proliferation, medial SMC loss by apoptosis, and similar patterns of expression of cytolytic cell derived inducers of apoptosis. Insofar as intimal proliferative lesion-formation may be dependent on medial damage (as in arterial-injury models), understanding the mechanism of medial SMC loss may provide a novel therapeutic approach to human card iac transplant arteriopathy. (C) 1998 Elsevier Science B.V. All rights reserved.