Trends in Cardiovascular Medicine
Volume 15, Issue 1 , Pages 1-8, January 2005

Role of Progenitor Cells in Transplant Arteriosclerosis

  • Jan-Luuk Hillebrands

      Affiliations

    • Corresponding Author InformationAddress correspondence to: J.L. Hillebrands, PhD, Dept. of Cell Biology, Section Immunology & Histology, University Medical Center Groningen (UMCG), A. Deusinglaan 1, NL-9713 AV Groningen, The Netherlands. Tel.: (+31) 050-3638995; fax: (+31) 05f0-3632512.
    • ,
  • Geanina Onuta
    • ,
  • Jan Rozing

Jan-Luuk Hillebrands, Geanina Onuta, and Jan Rozing are at the Department of Cell Biology, Section Immunology & Histology, University Medical Center Groningen, The Netherlands.

Article Outline

To date, chronic transplant dysfunction (CTD) is recognized as the major cause of transplant loss long term after transplantation. CTD has the remarkable histologic feature that the luminal areas of the intragraft arteries become obliterated as a result of occlusive neointima formation. Neointimal lesions contain predominantly vascular smooth muscle cells (VSMCs) and extracellular matrix admixed with inflammatory cells. At the luminal side, neointimal lesions are covered with a monolayer of endothelial cells (ECs). The etiology of transplant arteriosclerosis (TA) is largely unknown, and adequate prevention and treatment protocols are not available. In contrast to the largely accepted “response-to-injury” hypothesis for the development of TA that attributes an important role to graft-derived ECs and VSMCs, recent data indicate that host-derived vascular progenitor cells play a major role in the development of TA. The process leading to TA appears to be heterogeneous, and neointimal ECs and VSMCs can be recruited from different sources, possibly depending on the severity and duration of vascular damage. These data suggest a significant role of host-derived circulating EC/VSMC progenitor cells, which may be partly bone marrow derived. Circulating vascular progenitor cells are potential targets for therapeutic intervention to ameliorate TA development. Therefore, identification of mediators and cellular mechanisms that promote recruitment of vascular progenitors to sites of injury is warranted to dissect their detrimental and possible beneficial effects in the development of TA.

 

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Transplant Arteriosclerosis 

Although solid organ transplantation has almost become a routine procedure in clinical practice, it has not achieved its goals as a long-term treatment for patients with end-stage organ failure. Whereas the short-term results of organ transplantation have significantly improved over recent decades due to improved immunosuppressive regimens, long-term success has remained at the same level, the lone exception being renal transplantation (Hariharan et al. 2000). To date, chronic transplant dysfunction (CTD) is considered to be the major cause of long-term (>1 year) allograft loss and can be defined clinically as the progressive irreversible loss of graft function (Orosz and Pelletier 1997). In addition to functional deterioration, CTD is associated with organ-specific histopathology and also features common histopathologic changes in the intragraft arteries. This vasculopathy is referred to as transplant arteriosclerosis (TA) and is preferred to transplant atherosclerosis, because TA is usually—in contrast to atherosclerosis—general and concentric, often lacking a lipid-rich core typical of atheroma (Libby and Pober 2001). TA is characterized by infiltration of smooth muscle α actin (SMA)-positive vascular smooth muscle cells (VSMCs), culminating in the formation of an occlusive neointima. Although a causal relation between deteriorated function and the histologic presence of TA still has to be proven, progressive vessel occlusion leading to downstream ischemic tissue damage and disruptive fibrosis has generally been accepted as the main cause of CTD.

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Pathogenesis of TA: The Response-to-Injury Hypothesis 

The response-to-injury paradigm (Ross 1993) has been accepted widely for the development of TA, holding that graft endothelial cells (ECs) become damaged by transplant-related trauma and rejection. Consequently, a remodeling process is initiated that is coordinated by cytokines and growth factors produced by ECs as well as parenchymal cells and infiltrating leukocytes. Eventually, this cascade results in medial VSMC migration and proliferation during which they change their phenotype from “contractile” to “synthetic.” According to this concept, the neointimal ECs and VSMCs in TA are considered to originate from the grafted tissue and therefore should be donor derived. However, several phenomena argue against this working hypothesis for the development of TA. First, migration of medial VSMCs into the subendothelial space is hardly documented, whereas several studies have shown in experimental models in mice that blood-borne cells attach to the luminal side of the artery after mechanical injury prior to intimal hyperplasia (Sata et al. 2000). We had similar findings in the aortic transplant model in rats and showed that the first SMA-positive cells in the developing neointima are found in a scattered pattern on the luminal side of the vessel wall, suggesting direct entry from the lumen (Hillebrands et al. 2002a). Moreover, it is commonly observed in these experimental models that intimal ECs and medial VSMCs are universally killed (due to severe and generalized vascular wall damage) and disappear before a neointima starts to develop. This observation also suggests that the vascular cells constituting the neointima are not derived from the injured vascular wall. Finally, we previously showed that implantation of artificial biodegradable vascular grafts in rats resulted in the development of completely new vascular wall structures in time, and the cells constituting these newly developed vascular wall structures are derived from the recipient by definition. These observations led to our hypothesis that the neointimal cells in TA are of recipient and not donor origin, and are derived from a pool of circulating (possibly BM-derived) progenitor cells that are recruited to sites of vascular damage and locally differentiate into mature ECs and VSMCs (Nieuwenhuis et al. 2000). This article recaps the experiments we have performed to test this hypothesis.

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Origin of Neointimal VSMCs in TA 

The concept of the existence of blood-borne ECs and VSMCs is not from recent years; in 1963, Stump et al. demonstrated the existence of blood-borne vascular precursor cells by showing that Dacron hubs, implanted in the aorta of pigs, became covered with ECs and VSMC-like cells originating from the peripheral blood. The question of whether such cells are also involved in the development of TA in transplanted allografts, however, has only recently raised considerable interest. The first data suggesting involvement of host VSMCs in TA development were reported by Plissonnier et al. (1995), who performed flow cytometric analysis on isolated rat aortic neointimal cells. We used a different strategy in order to exclude the risk of sample contamination with recipient-derived inflammatory cells as might have occurred in the previous study. We performed origin analyses in two different experimental transplant models in rats (aortic and cardiac transplantation) in which severe TA develops after allografting (Hillebrands et al., 2001a, Hillebrands et al., 2001b). To determine the contribution of host VSMCs in the development of TA, a single-cell, polymerase chain reaction (PCR)-based method was developed (sry-gene served as target) and this assay was sufficiently sensitive to detect male-derived cells at the single-cell level. This PCR was then performed on microdissected single nuclei of neointimal SMA-positive VSMCs from aortic and cardiac allografts that were transplanted in the female to male combination. This method excluded the risk of sample contamination with recipient-derived inflammatory cells. Virtually all neointimal VSMCs in both aortic and cardiac allografts were of recipient origin and not donor origin (Hillebrands et al., 2000, Hillebrands et al., 2001b). These findings have now independently been confirmed in a variety of rat (Religa et al. 2002) and mouse (Li et al., 2001, Sata et al., 2002, Shimizu et al., 2001) studies. The use of such experimental animal models obviously also makes it possible to address questions on the anatomic origin of the neointimal VSMCs, and one of the potential sources are stem cells (SCs) that reside in the bone marrow (BM).

Although previously BM SCs were considered to be predominantly hematopoietic precursor cells, recent data have shown that adult BM still holds the potential to differentiate into many different tissues (i.e., adult SC plasticity). This plasticity implies that adult SCs can switch—once relocated and appropriately stimulated—to a whole series of other progeny (Poulsom et al. 2002). Using BM chimeric rats, allowing for discrimination between BM- and non-BM-derived cells, we analyzed by confocal laserscanning microscopy (CLSM) using major histocompatibility (MHC)-class I haplotype-specific antibodies for the presence of BM-derived neointimal VSMCs in aortic allografts. These experiments revealed that the host-derived VSMCs were predominantly, if not all, derived from a non-BM source (Hillebrands et al. 2003). This observation is in line with recent reports by others (Li et al., 2001, Religa et al., 2002, Shimizu et al., 2001) also showing a minor contribution of BM-derived VSMCs in the development of TA after experimental aortic allografting in mice. One study (Sata et al. 2002) showed a major contribution (∼82%) of BM-derived VSMCs in the development of TA after cardiac allografting in mice. The authors did not, however, perform double staining for transgene expression (BM origin) and SMA, and therefore it is not possible to deduce what proportion of those cells were actually SMA-positive VSMCs. Thus, one can conclude that although the BM can provide VSMCs found in TA, other non-BM sources also provide precursor cells. Such host non-BM-derived VSMC precursors must be radio resistant, because all studies described above used irradiation to create BM-chimeric animals. Based on our results, we cannot exclude the possibility that host-derived neointimal VSMCs result from ingrowth of adjacent medial VSMCs. However, Shimizu et al. (2001) demonstrated in their aortic transplant model in mice that neointimal VSMCs are not derived from contiguous extension from the host media, and this is in line within the hypothesis that the contribution of circulating VSMC precursors in the development of TA.

From experimental studies, there is now compelling evidence that VSMCs in TA are frequently and in majority derived from recipient cells, which is in sharp contrast to the response-to-injury paradigm (Ross 1993). In contrast to experimental studies, all human studies on the origin of VSMCs in TA in cardiac allografts describe the presence of no or only low percentages of recipient-type VSMCs (Atkinson et al., 2004, Glaser et al., 2002, Hruban et al., 1993). In contrast to human cardiac allografts, Grimm et al. (2001), studying renal allograts, showed that 80% to 90% of neointimal VSMCs were of recipient origin. Analysis of the reciprocal combinations, however, clearly demonstrated the existence of a persistent population of donor-type cells. Whether organ-specific differences in this process exist—which might explain the observed differences between the cardiac and renal transplants—remains to be elucidated.

Several phenomena might contribute to the differences observed between the experimental and clinical studies. First, preserved medial VSMCs in the intragraft arteries of transplanted allografts may serve as a source for VSMCs that can populate the neointima. Because in most of the rodent models no immunosuppressive drugs are administered, medial cell destruction often leads to complete medial necrosis. In contrast, in human allografts, different immunosuppressive regimens are used to prevent rejection, most likely resulting in protection of medial VSMC destruction. One can envision that when viable medial VSMCs remain available, these cells can provide a source of neointimal cells, whereas in the case of complete medial destruction, other sources—by definition—are required. However, in our model of TA in cardiac allografts, a small rim of presumably donor-derived medial VSMCs was present, whereas virtually all neointimal VSMCs were found to be of recipient origin. Another difference between rodent models and humans is the presence of pre-existing intimal lesions in human allografts at the time of engraftment (Wong and Yeung 2001). These intimal lesions, containing donor-type VSMCs, may provide the basis for further outgrowth of VSMCs during the subsequent development of (posttransplant) TA (Atkinson et al. 2004). Such pre-existing intimal lesions are rarely found in animal tissues used for transplantation. Taken together, although strong experimental evidence exists favoring recipient involvement, the possibility of medial or intimal VSMC contribution in TA development should be taken into account.

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Origin of Neointimal ECs in TA 

According to the response-to-injury hypothesis, neointimal ECs are of graft origin. In case the EC monolayer becomes damaged and partly denuded (e.g., due to ischemia and the alloimmune response), neighboring graft ECs will re-endothelialize the injured areas, thereby restoring the functional EC monolayer. However, in 1965, Medawar generated the concept of EC chimerism, proposing that allografts are repopulated by host-derived ECs. EC chimerism may have important implications for maintaining graft integrity for two reasons. First, rapid recovery from EC loss (i.e., re-endothelialization of denuded areas) will inhibit thrombus formation and subsequent tissue damage. Secondly, re-endothelialization with host ECs can also be expected to result in graft adaptation due to gradually reduced immunogenicity of the graft vasculature (Medawar, 1965, Pober, 2001). Based on recent data, however, graft adaptation due to EC chimerism is questionable, as is discussed below.

Since the concept of graft adaptation was proposed, several groups have analyzed in clinical studies whether ECs covering the luminal surface of the graft vessels after transplantation are of donor origin or have been replaced by host-derived ECs. Both data supporting donor (Hruban et al., 1993, Yousem and Sonmez-Alpan, 1991) as well as recipient (Lagaaij et al., 2001, O'Connell et al., 1991, Quaini et al., 2002, Sedmak et al., 1988, Simper et al., 2003) origin have been reported, indicating heterogeneity of the underlying process. In most studies describing EC chimerism, EC replacement was only partial (variation from 0% to 40%), and therefore it is questionable whether this adaptive mechanism provides sufficient protection against alloimmunity and thereby ameliorates TA development. To test the concept of graft adaptation, we determined the origin of neointimal ECs in both aortic and cardiac allografts transplanted in rats. Aortic allografts were transplanted in the MHC mismatched Brown Norway (BN) to Lew and the Piebald Virol Glaxo (PVG) to Albino Oxford (AO) rat strain combinations. Four weeks (BN to Lew) and 12 weeks (PVG to AO) after transplantation, the origin of the neointimal ECs was determined by MHC class I haplotype-specific immunohistochemistry. Both strain combinations presented with severe TA at the time of analysis and revealed complete EC replacement with host-derived cells (Hillebrands et al. 2001b). Normal vascular architecture with graft ECs (and without inflammation and TA) was preserved after daily treatment with cyclosporin A. Recently, we confirmed our EC replacement data in another aortic transplant model using human placental alkaline phosphatase (hPAP) transgenic (Tg) rats as recipients. hPAP-Tg rats constitutively express hPAP in all nucleated cells (Kisseberth et al. 1999), thereby making this rat extremely useful for cell-tracking studies. Dark Agouti (DA) aortic allografts were transplanted into Tg recipients and after 8 weeks the origin of neointimal ECs was analyzed by four-color CLSM. The results are summarized in Figure 1 and show that the neointimal ECs were positive for both the EC marker and the transgene, indicating that these ECs are recipient derived. Also, the microvascular ECs of newly formed vascular structures in the adventitia (indicative of angiogenesis) were positive for both markers (not shown). Moreover, the majority of the SMA-positive cells within the neointima expressed hPAP, thereby confirming our previous single-cell PCR data showing that the neointimal VSMCs are derived from the host. In contrast to aortic transplants, neointimal ECs in cardiac allografts remain of donor type, whereas the neointimal VSMCs are of host origin (Hillebrands et al. 2001b). This difference between the two models might be explained by the fact that in the cardiac transplant model, recipients were immune modulated (thereby ameliorating the rejection response) (Hillebrands et al. 2001a), whereas aortic transplant recipients were not immune suppressed. These data suggest that EC replacement represents a reparative response to injury rather than a constant mechanism of tissue maintenance. This is supported by findings in clinical and experimental kidney transplantation showing that the percentage of EC replacement correlates with the severity of rejection (Lagaaij et al., 2001, Xu et al., 2002).

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  • Figure 1. 

    Neointimal endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in DA aortic allografts transplanted in human placental alkaline phosphatase (hPAP) transgenic F344 rats are host derived. (A) Schematic overview of the transplantation protocol. DA abdominal aortic grafts were orthotopically transplanted into transgenic F344 recipient rats that constitutively express the hPAP gene in all nucleated cells. The origin of neointimal ECs and VSMCs was analyzed with a confocal microscope after performing four-color immunofluorescent staining 2 months after engraftment. At this time point, severe transplant arteriosclerosis (TA) (inflammation, media necrosis, and neointima development) was present, as illustrated in the schematic cross-section. The black square indicates the region at which the photomicrographs of Figure 1B were taken. (B) Immunofluorescent staining was performed with the use of the following monoclonal antibodies: asm-1 (smooth muscle α actin [SMA], green), rat endothelial cell antigen (RECA)-1 (EsCs, blue), and α-hPAP (transgene, red). Nuclear counterstaining was performed using 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (cyan). Figures a through e show the negative control (only incubation with conjugated isotype-specific second-step antibodies and DAPI). Only the elastin fibers in the media are visible, due to autofluorescence (c and e) (original magnification ×945). Figures f through j show the same region after incubation with the primary monoclonal antibodies: RECA-1 (g), asm-1 (h), and α-hPAP (i). Transgene expression was detected throughout the neointima in both the VSMCs and ECs, indicating host origin of these cells (original magnification ×945). The insets show high-power magnifications of neointimal VSMCs expressing both α-actin and hPAP (h—j). Figures k through o show high-power magnifications of the neointimal ECs demonstrating co-localized expression of both RECA-1 and hPAP (l, n, and o). Arrows indicate double-positive neointimal ECs (original magnification ×9000). a, adventitia; lu, lumen; m, media; ni, neointima. (These data are based on results that were previously published by Hillebrands et al. 2001b.).

To evaluate the potential source of the host-derived neointimal ECs in rat aortic allografts, BN recipients were reconstituted with Lew BM cells and subsequently transplanted with a DA aortic allograft. In addition, the reciprocal combination (BNBM → Lew) was included in this analysis. Discrimination between BM- and non-BM-derived ECs was again determined by CLSM using MHC class I haplotype-specific antibodies and an EC marker. These experiments revealed that the host-derived ECs originated predominantly from a non-BM source, whereas only few BM-derived neointimal ECs were detected (<5%) (Hillebrands et al. 2002b). Figure 2 summarizes the findings of these experiments performed in BM chimeric hosts. A striking observation was the scattered distribution of host-derived ECs in the graft in early TA, supporting blood-borne origin of these cells rather than ingrowth from the anastomosis. Similar data were recently reported by others using an aortic transplant model in transgenic mice, although their percentage of BM-derived ECs (∼35%) was higher (Hu et al. 2003). Taken together, these data indicate that both BM- and non-BM-derived ECs, presumable derived from a pool of circulating (progenitor) cells, contribute to graft re-endothelialization after transplantation. Two important questions remain to be answered: (a) What is the nature of the cells that give rise to ECs after vascular injury? and (b) What is the relevance of graft re-endothelialization with circulating cells in the context of neointima development? These questions are addressed below.

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  • Figure 2. 

    Neointimal endothelial cells (ECs) in aortic allografts are predominantly non-bone-marrow (BM) derived. Allogeneic BM transplantation was performed in the LewBM → BN and BNBM → Lew strain combinations (using lethally irradiated recipient rats), resulting in the establishment of complete and stable BM chimerism as determined by Fluorescence Activated Cell Sorter analysis (not shown). DA aortic allografts were transplanted in chimeric animals 6 to 8 weeks after BM transplantation. Grafts were analyzed 3 months after aorta engraftment. Cross-sections were taken from the center of the graft and used for histologic analysis. Confocal laser scanning microscopy was performed with the use of the following panel of monoclonal antibodies: asm-1 (smooth muscle α actin [SMA],), RECA-1 (ECs), OX18 (pan major histocompatibility complex [MHC] class I), MN4-91-6 (DA MHC class I), and OX27 (BN MHC class I). Nuclear counterstaining was performed using DAPI (blue). Figures a through d: LewBM → BN combination and Figures e through i: BNBM → Lew combination. (a) Staining for α actin (green) and ECs (red). Arrowheads indicate RECA-1-positive ECs and the asterisk indicates SMA-positive neointimal vascular smooth muscle cells (VSMCs) (original magnification ×2520). (b) Staining for SMA (green) and aorta donor (DA) MHC class I (red). Neointimal ECs are MN4-91-6 negative, suggesting host origin (original magnification ×1260). (c) Staining for SMA (green) and pan MHC class I (red). Arrowheads indicate MHC class I-positive ECs (original magnification ×2520). (d) Staining for host (BN) MHC class I (green) and ECs (red). Arrowheads indicate RECA-1 and OX27 double-positive ECs, indicating host, non-BM, origin (original magnification ×2520). (e) Staining for SMA (green) and aorta donor (DA) MHC class I (red). Arrowhead indicates a donor MHC class I-positive EC, indicating graft origin. Arrow indicates an MN4-91-6 negative cell, suggesting host origin. Asterisk indicates the nucleus of SMA-positive medial VSM cell (original magnification ×5670). (f) Staining for SMA (green) and ECs (red) on a serial section of e. Arrowheads indicate RECA-1-positive staining, thereby confirming that the cells depicted in e are ECs (original magnification ×5670). (g) Staining for SMA (asm-1, green) and pan MHC class I (red). Arrowheads indicate MHC class I-positive ECs (original magnification ×2520). (h) Staining for BM-donor (BN) MHC class I (green) and ECs (red). Arrowheads indicate RECA-1 and OX27 double-positive ECs, indicating that these ECs are BM derived (original magnification ×5040). (i) Staining for SMA (green) and ECs (red). Arrow indicates a cluster of RECA-1-positive ECs and the asterisk indicates SMA-positive medial VSMCs (original magnification ×2520). iel, internal elastic lamina; m, media; ni, neointima. (Reproduced with permission from Hillebrands et al. 2002b p. 194–195. Copyright 2002, Nature Publishing Company.)

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Endothelial Progenitor Cells 

Accumulating evidence supports the existence of three types of circulating ECs in the peripheral blood. The major proportion (approximately 95%) of the circulating ECs is derived from non-BM tissues and are presumably sloughed, mature, circulating ECs (CECs) that randomly enter the circulation as a result of blunt vascular injury. CECs do not express the SC/progenitor cell markers CD133 and CD34, and this cell population has limited growth capability compared with BM-derived endothelial progenitor cells (EPCs) (Lin et al., 2000, Rafii and Lyden, 2003). CECs can be cultured early (<1 week) from buffy-coat mononuclear cells (MNCs), whereas EPCs appear as colonies late (∼1 month) after initial cell culture. EPCs are thus responsible for approximately 5% of the circulating cells capable of endothelial-like phenotype and these cells are believed to originate from hematopoietic SCs (HSCs) (Hristov et al., 2003, Rafii and Lyden, 2003). Residing in the BM, EPCs express CD133, CD34, and Flk1 (vascular endothelial growth factor receptor-2 [VEGFR-2]) resembling an angioblastic phenotype. After entry into the circulation, EPCs mature and start to express EC-specific markers such as CD31 (platelet-endothelial cell-adhesion molecule [PECAM-1]), CD146 (vascular endothelial cadherin [VE-cadherin]), von Willebrand factor, and endothelial nitric oxide synthase, whereas they gradually lose expression of CD133 and CD34 (except for microvascular ECs that retain CD34 expression). Once EPCs enter the circulation, these cells are referred to as circulating endothelial progenitor cells (CEPs) that are capable of generating outgrowth endothelial cells. In addition to the EPCs/CEPs that originate from HSCs, Rehman et al. (2003) recently reported on a population of monocyte-derived endothelial-like cells that are also referred to as EPCs (Rehman et al. 2003). In contrast to HSC-derived EPCs, monocyte-derived EPCs express the common leukocyte marker CD45 as well as monocyte/macrophage-specific markers CD11c, CD11b (Mac-1), and CD14 and the EC marker CD31 (PECAM-1) (Rehman et al. 2003). The various EPC populations identified thus far have been used in different therapeutic studies and are discussed below.

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Endothelial Progenitor Cells in TA 

To date, only one study has been reported that links the frequency of circulating ECs with the development of TA. Simper et al. (2003) determined the numbers of CECs and EPCs in the peripheral blood of cardiac transplant subjects with or without established TA in the coronary arteries. In subjects with TA, the frequency of EPCs was significantly reduced compared with subjects without established TA, whereas the frequency of CECs was similar between both patient groups. Moreover, the authors demonstrated seeding of host ECs at sites displaying TA in a separate series of experiments. These data provide circumstantial evidence, but no proof, for the depletion of EPCs from the circulation due to recruitment to areas of vascular injury. From this study (Simper et al. 2003), it cannot be concluded whether the EPCs were truly derived from the BM, and it is not unlikely that other SC/progenitor cell niches exist outside of the BM that can give outgrowth of ECs that contribute to re-endothelialization at sites of vascular injury. Indeed, Hu et al. (2004) recently showed that the adventitia of the aortic root contains a population of non-BM-derived Sca-1+ cells that can differentiate into ECs in vitro upon stimulation with VEGF and are characterized by CD34, CD31, and VE-cadherin expression (Hu et al. 2004).

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Reendothelialization and Neointima Formation 

From the data currently available, it is not clear whether re-endothelialization with host-derived ECs (EPCs/CEPs or CECs) has any ameliorating effect on the development of TA after allogeneic transplantation. Our experimental data (Hillebrands et al. 2001b) suggest that independent of whether EC replacement with host-derived ECs takes place, TA develops, and this observation favors against a beneficial role of re-endothelialization in the process of TA development. However, re-endothelialization has been shown to ameliorate neointima formation and improve vascular wall function in various recent reports studying restenosis (Walter et al., 2002, Fujiyama et al., 2003, Werner et al., 2003, Gulati et al., 2004). The histopathologic finding of restenosis shows many similarities with TA (formation of an occlusive neointima), although the underlying mechanism may be different. Restenosis develops at high frequency after percutaneous transluminal coronary angioplasty (with or without additional stenting), a treatment that results in severe vascular damage including EC denudation.

BM-derived ECs and VSMCs have been shown to contribute in various degrees to neointima formation after mechanical injury, depending on the model used (Tanaka et al. 2003). Several reports describe experimental studies in which re-endothelialization of mechanically injured vessels is enhanced by either pharmacologic treatment with statins (Walter et al. 2002) or by cell transfer of EPCs that are obtained from different sources (Gulati et al., 2003, Fujiyama et al., 2003, Werner et al., 2003). The general observation described in these reports is that enhanced re-endothelialization of the injured vascular wall results in reduced neointima formation and improved EC function (Gulati et al. 2004). These studies thus clearly indicate that in restenosis, therapeutically enhanced re-endothelialization is beneficial. Whether the same holds true for the development of TA remains to be elucidated in future studies. One should, therefore, take into account that there is at least one major difference between the development of TA and restenosis—that is, the duration of the damage inducing insults. In restenosis, there is only one insult that induces damage that needs to be repaired (balloon inflation and the stenting procedure), whereas after allografting, the intragraft vasculature is continuously exposed to damage-inducing factors due to ongoing subclinical rejection. It is therefore not unreasonable to speculate that enhanced re-endothelialization after allografting does not result in reduced neointima formation. In addition, recent data suggest that EPCs may even act as promotors of TA rather than having a favorable effect on neointima development. Hu et al. (2003) observed after allogeneic aorta transplantation in BM-chimeric mice not only neointimal EC replacement (with non-BM derived cells), but also neovascularization of the neointimal lesions with microvessels that only consisted of BM-derived ECs (Hu et al. 2003). These microvessels appeared in allografts earlier than neointimal formation, suggesting that vasculogenesis within the intima may be a crucial event for the development of TA. If this dual role of circulating endothelial cells in the development of TA holds true, future strategies should on the one hand aim at enhancing re-endothelialization with non-BM derived CECs, and on the other hand, impair microvessel formation within the intima due to EPC directed vasculogenesis.

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Plasticity of the Process: Different Sources for ECs and VSMCs 

Because different anatomic origins of neointimal ECs and VSMCs in TA have been described to exist, we hypothesize that both EC and VSMC precursors are not a single entity, but can be recruited from a variety of niches depending on the severity and duration of vessel injury (Hillebrands et al. 2003). During limited superficial damage of the vessel wall, with a remaining vascular structure, intact ECs neighboring the injured endothelial cell layer and medial VSMCs themselves will probably provide sufficient repair potential. In this case, the neointimal vascular cells will be donor derived. More severe vascular damage—including more extensive intimal EC and medial VSMC injury—over a limited period of time may signal ingrowth of ECs and VSMCs from adjacent (host) vessels. In this respect, it should be mentioned that mature ECs can also give rise to smooth muscle-like cells via endothelial—mesenchymal transdifferentiation (Frid et al. 2002). Severe but time-restricted vessel damage—including full disrupture of medial VSMC layers—will lead to recruitment from non-BM sources, whereas similar damage over a prolonged period of time will probably need additional VSMC precursor recruitment from the BM.

Our hypothesis that the BM can contribute to neointima formation, but only after induction of severe damage, is supported by several findings. First, Wagers et al. (2002) demonstrated that hematopoietic SCs—which have been shown to harbor developmental plasticity and can differentiate into vascular wall cells—do not contribute to nonhematopoietic tissues under physiologic conditions without induced vascular damage (Wagers et al. 2002). These data thus indicate that transdifferentiation of hematopoietic SCs and their progeny is an extremely rare event under normal conditions. Second, Tanaka et al. (2003) studied different restenosis models and demonstrated that BM-derived VSMC and EC precursors only contribute to neointima formation after wire injury, causing complete EC denudation and medial VSMC loss. Third, in regeneration of infarcted myocardium, tissue damage appears to be the major determinant required for the transdifferentiation of primitive BM cells into ECs and VSMCs (Orlic et al. 2001).

One possible source of non-BM-derived neointimal VSMCs is adventitial fibroblast that can differentiate into SMA-positive myofibroblasts and migrate toward the lumen and contribute to neointimal formation after vascular injury (Sartore et al. 2001). In addition to mature fibroblasts, however, the tunica adventitia also contains a local niche of non-BM-derived Sca-1+c-kit+CD34+ SCs that can give rise to both VSMCs and ECs (Hu et al. 2004). This cell population has been shown to contribute to the development of atherosclerosis, and these findings support the hypothesis that mesenchymal progenitor cells that reside in the adventitia contribute to neointimal lesion formation by either recruitment through the vascular wall toward the subendothelial space or by release into the blood to form circulating progenitor cells. Recently, it has been shown that the human peripheral blood indeed contains a population of circulating VSMC progenitor cells. Simper et al. (2002) demonstrated that in vitro culture of human peripheral blood mononuclear cells in the presence of platelet-derived growth-factor-enriched medium resulted in the generation of so-called smooth muscle outgrowth cells expressing SMA, myosin heavy chain, and calponin. The precise molecular identity of these circulating VSMC progenitors remains to be determined. Moreover, it is unclear whether circulating ECs and VSMC precursors arise from a common progenitor in blood or BM. It has been shown that Flk-1-expressing embryonic SCs can give rise to VSMCs both in vitro and in vivo, suggesting that adult EPCs may also serve as VSMC progenitors (Yamashita et al. 2000). Thus, the data described above indicate that ECs and VSMCs can develop from different precursors derived from different anatomic locations. In clinical transplantation, it is likely that the entire spectrum of EC and VSMC precursor derivation occurs, perhaps even in one and the same patient, depending on the severity of damage throughout the tissue.

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Conclusions 

Our data indicate that host-derived ECs and VSMCs contribute to the development of TA. Different anatomic locations appear to contain niches of vascular progenitor cells. It is likely that, depending on the duration and severity of the vascular damage, progenitors will be recruited from different sources to the site of damage. Based on the results presented, future strategies should aim at targeting recipient VSMC precursor cells rather than medial VSMCs. Moreover, based on restenosis data, one should strive for rapid re-endothelialization by enhanced recruitment of EC progenitors to ameliorate neointima formation, although it remains to be elucidated whether this also holds true for the development of TA. Identification of mediators and cellular mechanisms that promote recruitment of vascular progenitors to sites of injury is warranted, to dissect their detrimental and beneficial effects in the development of TA. These studies will reveal new therapeutic approaches for today's major transplantation-related clinical problem.

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Acknowledgments 

This study was supported in part by grants from The Netherlands Organisation for Health Research and Development (ZonMw grant 916.46.104), the Dutch Kidney Foundation (grant C03.6015), and the Ubbo Emmius Foundation. The authors would like to thank Dr. M.C. Harmsen and Dr. E.R. Popa from the Division Medical Biology, Dept. Pathology & Laboratory Medicine, Groningen University Medical Center, for providing hPAP transgenic F344 rats.

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PII: S1050-1738(04)00153-7

doi:10.1016/j.tcm.2004.10.002

Trends in Cardiovascular Medicine
Volume 15, Issue 1 , Pages 1-8, January 2005

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