CTD is the primary cause of allograft loss after the first perioperative year and is today’s most important problem in clinical organ transplantation. The hallmark of CTD is development of TA; i.e., progressive intimal thickening (neointima formation) consisting of α-actin–positive VSM cells intermingled with recipient-derived mononuclear cells (T cells and macrophages) (2
). Current thought on the process of TA holds that medial VSM cells of affected arteries migrate into the subendothelial space and start to proliferate, leading to vascular narrowing and occlusion (2
). In this paper, we analyzed whether neointimal VSM cells as well as ECs are truly donor-derived, using two transplant models (aorta and heart) in rats (28
). To address this question, donor and recipient MHC class I haplotype-specific immunohistochemistry and PCR analyses of DNA sequences allowing discrimination between donor and recipient origin were performed.
First we analyzed development of TA after transplantation of aortic grafts that were ex vivo 50 Gy γ-irradiated (to inhibit proliferation and migration of medial VSM cells) prior to transplantation. It has been shown that γ-irradiation of cultured VSM cells in vitro inhibits proliferation of these cells after stimulation (45
). Three months after transplantation, irradiated grafts showed severe TA, similar to nonirradiated grafts, suggesting recipient origin of neointimal cells. Aziz et al. performed similar experiments using liquid nitrogen to destroy medial VSM cells in aortic allografts prior to transplantation. These frozen allografts showed TA to the same extent as unmodified grafts, also suggesting recipient origin of neointimal cells (22
Subsequently, aorta recipients were treated with 5 mg/kg or 10 mg/kg CsA for 28 days to study the effect of immunosuppressive treatment on the development of TA. CsA treatment completely blocked neointima formation and preserved the vascular media, confirming that development of TA is primarily alloimmune-mediated. Neointima formation is dependent on, and proportional to, the extent of medial VSM cell damage (46
). By suppressing the alloimmune response by CsA treatment, no medial VSM cell (and possibly also no EC) damage occurs; thereby development of TA is prevented. However, Stoltenberg et al. showed that administration of the same amounts of CsA, resulting in similar 24-hour CsA trough levels, resulted in significant inhibition, but no prevention of neointima formation 3 and 6 months after aorta transplantation in the A×Cgg35Irish (ACI) to Lew strain combination (33
). Differences between the rat strains used and the follow-up period might explain the different results.
To confirm the origin (donor vs. recipient) of neointimal endothelial and VSM cells in rat aortic allografts displaying extensive TA, we first performed recipient MHC class I specific immunohistochemistry. Results indicate that ECs indeed are of recipient origin in nontreated and irradiated aortic allografts. CsA treatment prevented neointima formation, and MHC class I staining showed that ECs layering the internal elastic lamina are still of donor type and are not being replaced by recipient-derived ECs. In contrast to the aortic allografts, neointimal ECs are still of donor type in cardiac allografts showing severe TA 530 days after engraftment. Similar results were found by Hasegawa et al. (47
), who showed that after MHC-mismatched cardiac allografting in mice ECs in CAs with neointimal lesions are still of donor origin at 12 weeks after transplantation by using MHC class II–specific immunohistochemistry.
The origin of ECs (donor vs. recipient) after organ allografting has been debated since Woodruff (48
) and Medawar (49
) suggested that replacement of donor ECs with recipient ECs (graft adaptation) might be an important factor in establishing transplantation tolerance in long-term allograft survivors. Since then, several groups reported data supporting (11
) and discounting (17
) repopulation of graft vessels by recipient-derived ECs after solid organ transplantation (kidney, heart, and lung) (reviewed in ref. 50
). Taken together, results from literature and data presented in this paper suggest that EC replacement is not a general phenomenon after allogeneic organ transplantation. In the absence of immunosuppression, donor ECs become necrotic and disappear within 2 weeks after allogeneic aorta transplantation in rats (21
). This severe damage of donor ECs might explain the repopulation of the graft with recipient-derived endothelium. After cardiac transplantation following intrathymic immune modulation and short-term immunosuppression, remaining alloreactivity against graft endothelium as well as alloimmune-independent factors do not result in sufficient EC damage leading to EC replacement. Recently, we reported that after intrathymic immune modulation the intragraft cytokine profile in long-term allografts (>200 days) has shifted from a Th1 type (high IL-2 and IFN-γ) toward a Th3 or Tr1 type (high IFN-γ, TGF-β, and IL-10; no IL-2) profile. This altered immune response might preserve graft endothelium, but it does not prevent the development of TA (30
). Studying human kidney biopsies, Lagaaij et al. recently suggested a correlation between the degree of EC replacement by recipient-derived endothelium and the severity of vascular rejection (16
). Replacement of donor endothelium by recipient-derived ECs seems to be related to graft dysfunction, rather than to graft function as suggested by Woodruff and Medawar (48
), and seems to play a role in endothelial repair processes upon (severe) vascular injury.
The origin of the neointimal VSM cells in the arteriosclerotic lesions still remains questionable. The original paradigm concerning the development of TA implies intimal hyperplasia to be derived from the donor media (8
). This is supported by findings of Hruban et al., who showed donor origin of neointimal VSM cells in two patients using fluorescence in situ hybridization on tissue sections (20
). However, data have also been reported indicating recipient origin of neointimal VSM cells in experimental transplant models (21
). MHC class I haplotype-specific immunochemistry did not allow distinction between donor and recipient origin of neointimal VSM cells in our transplant models, since both neointimal and medial α-actin–positive VSM cells do not express sufficient amounts of MHC class I antigens for detection. A similar lack of expression has been described for MHC class II antigens on neointimal VSM cells (47
Therefore we addressed the question of whether neointimal VSM cells are truly graft-derived using two unrelated PCR-based analyses (Tap2 and HY) on aortic and cardiac allografts, enabling discrimination between donor and recipient-derived cells.
Using both types of PCR analysis on aortic allografts and microdissected CAs with severe TA, we found that at least a significant part of the cells in these tissues displays recipient-type DNA. Similar data were obtained for irradiated aortic allografts. However, neointimal lesions have been reported to contain an appreciable number of recipient-derived infiltrating leukocytes (43
). Also, the aortic allografts analyzed in this study contained neointima-infiltrating mononuclear cells, consisting predominantly of ED1+
macrophages and some T cells. Such contaminating recipient-derived leukocytes can account for the recipient-specific signal in our analysis of aortic allografts and microdissected CAs, especially for the male-specific analysis, since the HY PCR is much more sensitive than the Tap2 PCR. Similar problems may also have influenced the results of Plissonnier et al. (21
) and more recently Brazelton et al. (23
) on the origin of VSM cells in vascular allografts. These groups used allospecific immunofluorescent staining of cell suspensions (21
) and tissue sections (23
) of aortic and femoral arterial allografts. Although their conclusion, that neointimal VSM cells are of recipient origin, in itself is probably correct, their type of analysis cannot distinguish between the infiltrating (recipient-derived) leukocytes and neointimal VSM cells.
Since our male-specific nested HY PCR allows detection of male-derived cells at the single-cell level (32
), we analyzed microdissected single nuclei of neointimal VSM cells. Nuclei were isolated from neointimal lesions of both aortic allografts and CAs with severe TA by positive selection (α-actin staining), thereby eliminating the risk of contaminating our samples with infiltrating recipient-derived mononuclear cells. HY PCR analysis of single nuclei of α-actin–positive neointimal cells showed that at least 90% of these cells in neointimal lesions of both aortic allografts and CAs from cardiac allografts are of recipient origin.
Although Hasegawa et al. claim that VSM cells in neointimal lesions of CAs after cardiac transplantation in mice are probably still of donor origin at 12 weeks after transplantation, their type of analysis, MHC class II specific immunohistochemistry, does not allow determination of origin of neointimal VSM cells because of lack of MHC class II antigen expression on these neointimal VSM cells (47
Since our data show recipient origin of neointimal VSM cells (aortic and cardiac allografts) and endothelium (aortic allografts), the question arises: what is the anatomical origin of these neointimal cells? Kouchi et al. demonstrated bloodstream origin of endothelial and α-actin–positive smooth muscle cells present on Dacron grafts transplanted in dogs (51
). Recently, recirculating bone marrow–derived CD34+
EC progenitor cells (EPCs) have been identified in the peripheral blood of humans (52
). The frequency of circulating EPCs increases in response to regional ischemia in both mice and rabbits. This increased frequency of circulating EPCs contributes to neovascularization of ischemic regions (54
), indicating that EPCs are mobilized from the bone marrow into the circulation and subsequently “home” to sites needing neovascularization. Using bone marrow chimeric dogs, Shi et al. demonstrated donor (bone marrow) origin of ECs attached on implanted Dacron grafts, also indicating existence of circulating bone marrow–derived ECs (55
). In another study, Bhattacharya et al. recently showed enhanced re-endothelialization of Dacron grafts after seeding with bone marrow–derived CD34+
cells prior to implantation (56
). Taken together, these results suggest an important role for bone marrow–derived CD34+
progenitor cells as a source for ECs.
The anatomical source of the neointimal VSM cells is still unknown. One possibility might be ingrowth of medial VSM cells from the recipient side of anastomosis, as suggested by Aziz et al. (22
). VSM cell destruction by freezing the recipient aortic ends at both sides near the anastomosis resulted in less pronounced intimal thickening compared with untreated aortic allografts. However, the frozen recipient aortic ends remained devoid of α-actin–positive VSM cells during the observation period of 60 days. If the neointimal VSM cells originate from the recipient media, one should expect migration of α-actin–positive VSM cells along the frozen aortic ends toward the mid-graft region. This was, however, not clear from the study by Aziz et al (22
). A second possibility is transdifferentiation from ECs into α-actin–positive VSM cells. Studying quail embryos, de Ruiter et al. showed transdifferentiation of embryonic ECs into α-actin–expressing mesenchymal cells in vivo and in vitro (57
The third possibility for the anatomical origin of neointimal VSM cells is the existence of VSM progenitor cells that differentiate into α-actin–positive VSM cells. Circulating VSM progenitor cells, however, have not been identified so far. Bucala et al. reported the existence of a non–bone marrow–derived circulating cell population with fibroblast properties that specifically enters sites of tissue injury (58
). In addition, Kouchi et al. described the presence of α-actin–positive smooth muscle cells on Dacron grafts, which apparently originated from the bloodstream (51
). Using whole vessel wall organ cultures, Slomp et al. showed migration of adventitial myofibroblasts to the luminal surface, where they transdifferentiated into endothelial-like cells as well as into a VSM cell phenotype in intimal thickening (59
). These results suggest the existence of a myofibroblast cell lineage that can serve as an endothelial and smooth muscle stem cell population. In our aortic transplant model, sequential immunohistological analysis of developing TA at an early stage after transplantation indeed suggests a blood-borne influx of regenerating VSM cells (not shown).
In conclusion, in contrast to current thought about the process of TA, the α-actin–positive VSM cells in the hyperplastic neointima in aortic and cardiac allografts are of recipient and not of donor origin. Neointimal ECs in aortic allografts are replaced by recipient-derived endothelium, whereas in cardiac allografts neointimal ECs are not replaced and are still of donor origin. So, independent of EC replacement, donor VSM cells in the graft are replaced by VSM cells of recipient origin in these two transplant models.
We propose that the development of TA is an attempt to restore vascular function upon immunological injury (alloreactivity) and is essentially part of a normal healing process. Basically, as a result of the immunological injury, medial VSM cells disappear, leaving the elastin network as a scaffold allowing restoration of the vessel wall by recipient-derived cells. However, in TA this remodeling does not seem to stop, eventually leading to total occlusion of the vessels involved. According to this concept, at least two windows in time seem to exist that would allow therapeutic intervention to prevent the development of severe TA. The first is early after transplantation, when primary vascular graft damage through either ischemia or alloreactivity should be preventable. Perhaps current immunosuppressive therapies should even be intensified or adapted to suppress nonclinical rejection episodes causing vascular damage eventually leading to TA. The second window for intervention would be later, when TA starts to develop. Our data suggest that TA is the result of a repair process proceeding beyond the needs of functional repair. Future intervention strategies at this stage should aim at regaining proper control of this dysregulated process.