Development of intimal hyperplasia in transplant arteriosclerosis

Vascular disease is the main cause of disability and mortality in the western world and the major limiting factor for long- term survival of transplanted organs. Occlusive vascular lesions lead to ischemia and structural changes in organs and in transplant arteriosclerosis and restenosis after endovascular procedures, narrowing of the vessel lumen is partly due to intimal hyperplasia caused by smooth muscle cells (SMCs). In this thesis, I have examined the mechanisms involved in intimal hyperplasia in a rat aortic allograft model of transplant arterioscleroris with an emphasis on the biology of SMCs in this process.

First, the phenotypic properties of the SMCs involved in the formation of intimal hyperplasia were studied by electron microscopy and immunohistochemistry. A segment of the abdominal aorta was transplanted orthotopically from Fischer to Lewis rats and the transplanted vessels examined after 1-12 weeks. After I week, loss of endothelial cells, adhesion of platelets and leukocytes to the luminal surface, and a phenotypic modification of SMCs in the media were observed. Subsequently, modified SMCs appeared in the intima and lymphocytes and macrophages were found to infiltrate the intima as well as the adventitia. This occurred together with detachment of endothelial cells and activation of SMCs in the media as determined by the induction of cellular retinol-binding protein-1. Later, SMalpha-actin positive SMCs were observed to migrate into the intima and proliferate as judged by staining for cyclin D I and proliferating cell nuclear antigen. TUNEL- and Fas/CD95-positive SMCs, indicating apoptosis, appeared in the media which was followed by a reduction in SMalpha-actins taining in this layer.

The continued development of the neointima was associated with a decrease in SMalpha-actin-positive SMCs, an increased staining for the extracellular matrix components fibronectin and osteopontin, and a further accumulation of inflammatory cells. The maximum growth in size of the neointima with an increase both in the number of SMCs and the content of extracellular matrix occurred 4-8 weeks after transplantation. SMCs and monocytes/macrophages in the neointima and in the media were also noted to accumulate lipid, turn into foam cells, and eventually show signs of necrosis and apoptosis. Within the lipid-rich cell remnants, calcification also occurred. Finally, after 12 weeks, the growth in mass of the intimal lesions ceased and reformation of an endothelial lining was detected.

In order to examine if SMCs derived from the host animal can participate in intimal hyperplasia, we transplanted aorta of F344 female rats to Lewis male rats with or without cyclosporin A treatment. As a control, one group of animals was transplanted with aortic isografts exposed to prolonged cold ischemia. Infiltration of SMCs and inflammatory cells into the intimal lesions, cell proliferation, and apotosis were analyzed by immunostaining and laser micro-dissection followed by real-time PCR for the SRY gene to determine cell origin.

Early after transplantation, proliferating and apoptotic graft SMCs were observed in the neointima and leukocytes and immunoglobulins appeared in the grafts. At this time apoptosis of medial SMCs occurred and proliferating, SRY-positive, host-derived SMCs started to accumulate in the neointima. After 8 weeks, the neointima was mainly composed of host-derived SMCs. Inummosupression with cyclosporin A significantly decreased the number of host SMCs in the neointima and only a small number of host SMCs were observed in isografts exposed to prolonged ischemia.

To explore the possible bone marrow origin of allograft cells, female LEW rats were irradiated and substituted with bone marrow from male LEW rats by transplantation of vascularized bone marrow or by infusion of bone marrow cells, followed by transplantation of aorta from female F344 rats. Immunostaining for cell-specific markers and real-time PCR for the SRY gene showed that the number of leukocytes was lower than the number of bone marrow-derived cells in intimal lesions. Primed in situ labeling for the SRY gene combined with immunostaining confirmed the presence of SM-like cells of male origin in the vessel wall in the intima. Similar observations were made after balloon injury of the carotid artery.

The findings add further support for the prior assumption that early loss of endothelial cells contribute to the initial response and activation of SMCs, whereas the inflammatory process may be the dominating factor that influence vessel structure later after transplantation. The observations also suggest that progenitors of bone marrow origin give rise to cells with SM-like properties during the formation of intimal hyperplasia after allotransplantation as well as after balloon injury. In addition, the results provide the basis for a novel theory of transplant arteriosclerosis which suggests that the development of intimal hyperplasia in this vasculopathy is a dynamic two-stage process that involves apoptosis of resident graft SMCs triggered by an allogenic immune response which also promotes recruitment of host-derived SMCs.

List of scientific papers

I. Bojakowski K, Religa P, Bojakowska M, Hedin U, Gaciong Z, Thyberg J (2000). Arteriosclerosis in rat aortic allografts: early changes in endothelial integrity and smooth muscle phenotype. Transplantation. 70(1): 65-72.
https://pubmed.ncbi.nlm.nih.gov/10919577

II. Religa P, Bojakowski K, Gaciong Z, Thyberg J, Hedin U (2003). Arteriosclerosis in rat aortic allografts: Dynamics of cell growth, apoptosis and expression of extracellular matrix proteins. Molecular and Cellular Biochemistry.

III. Religa P, Bojakowski K, Maksymowicz M, Bojakowska M, Sirsjo A, Gaciong Z, Olszewski W, Hedin U, Thyberg J (2002). Smooth-muscle progenitor cells of bone marrow origin contribute to the development of neointimal thickenings in rat aortic allografts and injured rat carotid arteries. Transplantation. 74(9): 1310-5.
https://pubmed.ncbi.nlm.nih.gov/12451271

IV. Religa P, Bojakowski K, Gaciong Z, Thyberg J, Hedin U (2002). Intimal hyperplasia in rat aortic allografts: Role of allogenic immune response in the recruitment of host derived smooth muscle cells. [Submitted]