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The presence of immune stimulatory cells in fresh and cryopreserved donor aortic and pulmonary valve allografts.

BACKGROUND AND AIM OF THE STUDY
Heart valve allografts (HVA) used for valve replacement or ventricular outflow tract reconstruction may suffer from structural deterioration due to donor-specific immune responses. The presence of immune stimulatory cells, including dendritic cells and activated endothelial cells, has not been studied thoroughly in aortic or pulmonary HVA. The presence and distribution of these cells in both aortic and pulmonary HVA, before and after cryopreservation, was analyzed immunohistochemically.


METHODS
Aortic (n = 16) and pulmonary (n = 13) HVA, discarded for implantation due to morphological or technical reasons, were obtained from 12 heart-beating and nine non-heart-beating tissue donors. Aortic and pulmonary HVA were dissected longitudinally into two symmetric sections by splicing of the non-coronary aortic and non-facing pulmonary cusps. Each symmetric half contained one-and-a-half valve cusps attached to the vascular wall. Fresh halves were directly fixed in formaldehyde, and analyzed immunohistochemically. The corresponding halves of the valves were decontaminated, cryopreserved, stored for at least three weeks and thereafter thawed according to the Heart Valve Bank protocol before analysis.


RESULTS
Activated endothelial cells, expressing PECAM-1 (CD31), VCAM-1 and HLA class II molecules covered at least 50% of fresh valvular surfaces. A comprehensive vascular network was found in the myocardial rim and adventitial layer, which was covered entirely by activated endothelial cells. HLA class II-positive macrophages (CD68) and T lymphocytes (CD3) were found scattered in the stroma and subendothelial layer of the valve leaflets. Mononuclear cell clusters were found predominantly in relation to native degenerative foci, and more often in aortic valves. No difference in cellular distribution was observed between the two donor types. Dendritic cells positive for both S100 and CD45 were not found in immuno-double-stained sections. Cryopreservation resulted in minor structural alterations in the vascular wall, and an increase of cells with pycnotic nuclei and reduction of adhesion molecule expression on endothelial cells. All fresh and cryopreserved aortic and pulmonary HVA contained abundant HLA class II-positive endothelial cells and sparse distribution of mononuclear cells in the luminal and adventitial layers.


CONCLUSION
Cryopreservation minimally affected the extracellular matrix of HVA and diminished the cellularity of the valve graft, while the HLA class II expression of cells was not abrogated. Aortic valve allografts harbor more mononuclear cells than their pulmonary counterparts. The absence of dendritic cells (professional antigen-presenting cells) is compensated by the preservation of other cells expressing HLA class II molecules predominantly in the endothelium; this may be responsible for the initiation of a specific immune response against HVA.