Aging and Transplant Arteriosclerosis in Absence of... : Transplantation (original) (raw)
Current immunosuppressive protocols have led to better control of acute rejection, yet almost half of all transplanted vascularized organ grafts will be lost at a certain stage posttransplantation due mainly to transplant arteriosclerosis (1). Chronic rejection of vascularized organs translates into arteriosclerosis, which is characterized by the progressive occlusive disease forming in the vessel’s lumen, giving rise to a thick neointima mainly made of smooth muscle cells. Conversely, the growing need for organ donors has led to donor-pool expansion to include old donors (2, 3), even though the response to vascular injury or remodeling has long been known to be severe in old vessels and results in a thicker neointima (4–6). Impact of age on organ transplantation has raised a great interest within the transplantation community, but evaluating the effects of donor and recipient ages in clinical settings is often complicated by the use of many immunosuppressive drugs that may influence the graft outcome (7, 8) through modulation of the immune system or by direct toxicity to the graft. Furthermore, inclusion criteria differ from a clinical trial or survey to another, and the length of the follow-ups is also variable. Of a total 43,172 renal transplants reported to the United Network for Organ Sharing, Terazaki et al. (2) have reported a 5-year graft survival falling from 81% when donors were aged 21 to 30 to 39% when the donors’ age was over 60. In another analysis, a 13% difference in 3-year graft survival rates was reported when recipients of kidneys from donors over or under age 55 were considered separately, and the graft half-life was 11 years for younger donors and 6 years when the donor was older (9). Others have reported a similar trend in 10-year survival of cardiac transplants between two groups of patients in which organs were from young donors or 50-year and older donors (3).
Whether the recipient’s age affects graft survival and function is still a matter of controversy. Recipient age has been recognized as being most important in allograft rejection outcome (10, 11). Indeed, Liu et al. (10) have reported that advanced recipient age was associated with more severe allograft injury, and that the best outcomes were observed for young recipients, irrespective of whether allografts were derived from young, adult, or old donors. A more recent study has found that the recipient’s age is a determining factor of patient and graft survival, where the latter was poorer in patients older than 60, even though they have the lowest risk for acute rejection (11). This observation could not be explained by any combination of patient age with donor age, delayed graft function, or immunosuppression.
Collectively, the current available data suggest that both donor and recipient ages affect survival outcomes after organ transplantation. However, in clinical settings, it is difficult to dissociate between the effects of donor and recipient ages on the graft’s outcome, which will be ultimately determined by many variables, such as donor-recipient degree of major histocompatibility complex disparity, immunocompetence of the recipient, drug-related toxicity to the graft, and other factors, which altogether may eclipse the contribution of age on the development of transplant arteriosclerosis. Therefore, we were prompted to reexamine the influence of age on the formation of neointimal formation in vascular grafts in the absence of alloimmune responses and immunosuppressive drugs. A syngeneic transplant model of the aorta was used to eliminate variables other than age, such as alloreactivity and the use of immunosuppressive drugs, and where neointima formation was initiated by vascular injury. The purpose of this study was to determine whether the age of the graft or the age of the recipient matters the most in transplant arteriosclerosis.
MATERIALS AND METHODS
Animals
Female Fisher (F344) rats of 2 (young) and 22 months (old) of age were purchased from the National Institute of Aging. All animals received humane care in compliance with the Principles of Laboratory Animal Care, formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences, and published by the National Institutes of Health (NIH Publication No. 86–23, revised 1985).
Animal Models and Experimental Design
All operative procedures were carried out under anesthesia with Isoflurane (Baxter Pharmaceuticals, Deerfield, IL). Injury to the thoracic aorta was inflicted with a balloon catheter (Fogarty, size 2F; Baxter Healthcare Corp., Irvine, CA), adapted to a custom angiographic kit (Scimed; Boston Scientific, Natick, MA), using a modified, previously described technique (12, 13). Because aortas of old donors are larger than younger ones, the balloon catheter was inflated accordingly to ensure an equal pressure (between 1.5 and 1.6 atmospheres) regardless of the aorta luminal diameter. Proper and uniform endothelium denudation has been verified in sample denudation by in vivo Evans blue staining. Briefly, 3 ml of 2 mg/ml Evans blue (Sigma, St. Louis, MO) were injected intravenously 30 minutes prior to sacrifice and tissue harvesting, where the de-endothelialized area incorporates the dye and stains blue as pictured “en face” on a flat-open aorta. To confirm that aging exaggerated the response to injury, thoracic aortas of young and old rats (no transplant involved) were de-endothelialized on day 0 and were harvested 30 days later to quantify the neointimal thickening. To determine the influence of donor and recipient age on neointimal development in the absence of alloimmune factors, the thoracic aortas from young or old donor rats were balloon injured in vivo under anesthesia before being transplanted into syngeneic sex-matched old or young syngeneic recipients. Control groups consisted of young aortas transplanted into young recipients and aortas from old donors transplanted into old recipients. To control for a possible ischemia-induced injury, a group of animals received age-matched and age-mismatched donor aortas that did not undergo balloon injury before transplantation. The injured graft was harvested and flushed with 5 ml cold Ringer’s lactate solution and kept in the same solution on ice until transplanted to the abdominal aorta of the recipient in an end-to-end fashion for both proximal and distal anastomoses using continuous 8–0/10–0 Surgilene sutures (Sherwood, Davis & Geck; St. Louis, MO) as previously described (13). Total ischemia time, defined as the time interval between transection of the donor descending thoracic aorta and release of both proximal and distal clamps on the recipient aortas, ranged from 55 to 69 min. There was no significant difference in the mean ischemic times among groups. Aortic grafts were explanted for morphometric analyses on days 14, 30, and 60 after transplantation.
Quantitative Morphometry
Three different segments from each graft were fixed with 10% buffered formalin and were embedded in paraffin. From each segment, 5-μm sections were cut, mounted, and stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), Sirius red (SR), and elastin van Gieson (EVG). Images from H&E-, PAS-, and EVG-stained slides were recorded using a digital charge-coupled device video camera on an Olympus BH-2 light microscope (Melville, NY). A polarized light filter system (Olympus) was used for the Sirius-based colorimetric assay according to a previously described method (14). Images were processed using PhotoShop version 5.0 software (Adobe Systems, San Jose, CA) color recognition, picture element dissection, and histogram determination properties. The degree of neointimal formation was calculated as total neointimal area/total medial area ratio (N/M) on full circular rings of EVG-stained cross-section pictures as we described earlier (15).
Neointimal collagen and elastin contents (collagen- or elastin-positive neointimal area/ total neointimal area), and cell density (total number nuclei/neointimal area) were calculated from SR, EVG, and PAS stained slides, respectively. Three images (magnification ×100) covering at least 60% of the neointimal area from each section were recorded. Three sections from each segment (proximal, middle, and distal thirds) of each graft were analyzed. For each animal (graft) and each determination, results are presented as the mean of nine acquired readings from three sections.
Immunohistochemical Staining and α-Actin-Positive Cells Quantification
α-Actin-containing cells were identified with monoclonal mouse antibody (1:25; Dako, Carpinteria, CA; M0851). The bound primary antibody was detected with a peroxidase-coupled streptavidin-biotin kit (Dako; k0690). The reaction was visualized with a peroxidase substrate kit (Vector Laboratories, Burlingame, CA; sk-4100). Quantification of immunostained slides was carried out as follows: three microscopic images were dissected using the property of color recognition of Photoshop v5.0 software. The α-actin positive (brown) area of the neointima was expressed as a fraction of the total area calculated as (α-actin positive area/total neointimal area) ×100.
5-Bromo-2-Deoxyuridine Incorporation Assay
5-Bromo-2-Deoxyuridine (BrdU; Sigma) was delivered in drinking water (0.8 mg/ml in water, changed daily) from day 2 to day 7 after aortic transplantation, as previously described (16). It is likely that water consumption will be variable between rats. This is not expected to affect the number of BrdU-positive cells but is likely to affect only the intensity of the staining; the animals drinking more BrdU in water are expected to have a higher number of BrdU molecules incorporated per each newly duplicated nucleus, which then will stain stronger. Injured aortas were harvested on day 14 posttransplantation, and BrdU-labeling assay was performed on double-stained sections (α-actin and BrdU), as we and others have described elsewhere (15, 17). Five-micron serial paraffin sections were used to estimate cellular proliferation. Permeabilization and BrdU staining of tissue sections were carried out by overnight incubation with sheep anti-BrdU monoclonal antibody (Amersham Biosciences Corp., Piscataway, NJ), diluted in a 0.8% Triton X-100, 5% rabbit serum, and further incubated with a biotinylated rabbit anti-sheep IgG (1:150 in phosphate-buffered saline, 0.4% Triton X-100, 2% rabbit serum) for 1.5 hr at room temperature after rinsing excess first antibody. BrdU-labeling index (the number of BrdU+ nuclei/unit length of internal elastic lamina corresponding to 0.1 mm) was calculated from five random areas in the neointimal layer of each section. The number of BrdU-positive nuclei per high power field was counted on 60 juxtaposed squares of an Adobe Photoshop grid per acquired picture (three magnification ×40 pictures/section). A minimum of five sections for each group was analyzed.
Statistical Analysis
Unless otherwise stated, data are represented as mean ± SD. Comparisons of more than two groups were carried out using analysis of variance followed by Newman-Keuls test. A P value equal or less than 0.05 was considered statistically significant.
RESULTS
Age-Related Exaggerated Response to Vascular Injury
For a same degree of vascular insult, older animals develop more neointima than the young ones. First, we sought to confirm the exaggerated neointima formation with advance in age reported elsewhere (4–6), as it is the prerequisite model for the following experiments reported herein. Young (2-month-old) and old (22-month-old) rats underwent balloon injury of the thoracic aortas. Proper endothelium denudation has been verified by in vivo Evans blue staining, where de-endothelialized area incorporates the blue dye stain uniformly in blue (Fig. 1A). Thirty days later, the injured vessels were harvested for morphometric analysis. On day 30 postinjury, old vessels had a significantly (P = 0.0009) higher degree of neointima formation as compared with young vessels (0.23 ± 0.06, n = 10; and 0.10 ± 0.02, n = 9, respectively) (Fig. 1). Because the size of the media is also larger in older vessels, the difference in hyperplasia between old and young rats would have been superior even if neointima total area was provided instead of a neointimal area/medial area ratio. This data confirm the exaggerated intimal hyperplasia witnessed with advanced age.
Age-related exacerbated neointima formation. Neointima formation was initiated in thoracic aorta of old (22-month-old) and young (2-month-old) rats by endothelium denudation with balloon catheter. (A) Proper and uniform endothelium denudation has been verified by in vivo Evans blue staining. Briefly, 3 ml of 2 mg/ml Evans blue (Sigma) were injected intravenously. 30 minutes before sacrifice and harvesting, where the de-endothelialized area incorporates the blue dye as pictured “en face” on a flat-open aorta. (B) Neointima/media ratios were estimated 30 days posttransplantation by computer-assisted morphometry on EVG-stained whole circular histologic cross-sections. Ratios are the averages from three cross-sections from three different aortic segments of an injured aorta of each of n animals. (C) Severity of the neointima in young and old aortas is depicted (in between arrowheads) in representative micrographs of cross-sections of EVG-stained slide (magnification ×20).
The Degree of Intimal Hyperplasia in the Vascular Graft is a Function of Recipient Age, Not Donor Age
Having confirmed that advanced age is associated with a more severe neointima formation as initiated by vascular injury (Fig. 1), we then sought to determine whether vessels from old donors would develop the same kind of exaggerated intimal hyperplasia when transplanted into young recipients.
First, to exclude age-related and transplant-related ischemia in neointima formation in our model, we transplanted intact (without balloon injury) young or old aortas in age-matched and age-mismatched recipients. At 30 days after transplantation, we found that graft ischemia alone with no injury did not lead to any neointimal development in any of the grafts, irrespective of donor-recipient age combinations (n = 3; Fig. 2B).
Recipient age dictates the severity of age-related neointima formation in aortic grafts. Old (22-month-old) aortas were de-endothelialized and transplanted into old (22-month-old) and young (2-month-old) syngeneic sex-matched F344 rat recipients. (A) Neointima/media ratios on days 14, 30, and 60 after injury and transplantation were estimated by computer-aided morphometric analysis in three circular cross-sections of each of three different rings of paraffin-embedded aortas explanted from n animals of each combination group. The sections were stained with EVG and analyzed blindly at magnification ×20 using a conventional light microscope. (B) To ensure that no spontaneous neointima forms due to ischemia or age, age-mismatched aortic transplants were performed without endothelium denudation. Photomicrographs are representative of aortic grafts harvested 30 days after transplantation.
To assess the role of the recipient in the age-related exaggerated neointima formation, injured aortas from young or old donors were transplanted into age-mismatched recipients, and neointimal formation was assessed at 14, 30, and 60 days posttransplantation. Donor and recipient rats of the same sex and strain (Fisher rats) were used to avoid the anticipated H-Y-mediated (18) and alloimmune-mediated hyperplasia that we reported (15), which may eclipse the contribution of age on neointimal development.
Figure 2 summarizes the neointima formation in response to injury for each of the three time points. Injured aortas from old donors that were transplanted into old recipients (old donor → old recipient) developed a more pronounced neointimal thickening as compared with those placed in young ones (old donor → young recipient; P = 0.011). This was a consistent observation for aortas at all time points (day 14, 30, and 60) posttransplantation (Fig. 2A, B). On the other hand, balloon-injured aortas from young donors developed more neointimal thickening when transplanted into old recipients (young donor → old recipient) than when transplanted into young ones (young donor → young recipient; P = 0.005). This response was observed as early as 14 days and was consistent at all time points (days 30 and 60) after transplantation (Fig. 2A, B). Importantly, in young recipients, neointimal growth seems to stabilize as early as 2 weeks posttransplantation, while it still persists in old recipients even when assessed at 60 days. Together, these results clearly show that same donor age yields different severity of neointima formation depending on the age of the recipient. Regardless of the donor’s age, young recipients always develop less neointima than the old ones. Similarly, the older the recipient, the more severe the neointima formation regardless of the donor’s age.
Cellular Proliferation Accounts, at Least in Part, for the Exacerbated Neointimal Formation in Older Recipients
Mechanistically, we assessed whether in vivo proliferation is higher in old recipients as compared with the young. BrdU incorporation in proliferating cells was assessed in separate animal groups where BrdU was administered in vivo from day 7 posttransplantation to the day of harvest on day 14. The number of neointimal cells that stained positive for anti-BrdU monoclonal antibody was counted on different areas representative of the bulk neointima. After transplantation, regardless of the age of the donor, the BrdU labeling index and the absolute number of BrdU-positive cells was higher in the neointima of the aortas transplanted into old recipients as compared with those transplanted into the young recipients (Fig. 3). Because all BrdU-positive cells also stained positive (brown) for α-smooth actin in a peroxidase-based immunostaining (Fig. 3), it is assumed that most of the proliferating cells in the neointima display the vascular smooth muscle cell phenotype. While the variability between rats in drinking more or less water is likely, this is likely to affect only the intensity of the staining and is not expected to affect the number of BrdU-positive cells. The animals drinking more BrdU in water will have a higher number of BrdU molecules incorporated in the newly duplicated nucleus, which therefore will stain stronger.
Cell proliferation accounts, at least in part, for exacerbated neointima formation in older recipients. Influence of the recipient age on accumulation of proliferating BrdU-positive cells in the neointima. BrdU labeling was performed on histologic cross-sections of aorta retrieved 14 days postinjury and transplantation. BrdU was supplied in drinking water (0.8 mg/ml) from day 7 to 14 postinjury and transplantation. (A) Density of BrdU-positive cells per high power field and the number of BrdU-positive nuclei/0.1 mm of internal elastic lamina. (B) Photomicrograph of a representative aortic section double stained with peroxidase-coupled streptavidin mouse anti-α actin monoclonal antibody (brown) and biotinylated sheep anti-BrdU polyclonal antibodies, in violet (similar to nuclei pointed by arrowheads).
Cellularity and Content of Collagen and Elastin in the Neointima Unaffected by Donor or Recipient Age
No statistically significant differences were seen between the transplanted groups with regard to neointimal content of elastin and collagen. At day 30 posttransplantation, the neointima consisted mainly of α-actin-positive vascular smooth muscle (VSMC)-like cells. There is a trend to lower cellularity in the neointima of aortas originally from old donors (Table 1). The results reflect a combined average of data acquired from the proximal, middle, and distal segments of the aorta, which may account for the large variations among the same group.
Morphometric analysis of the neointima in aortic grafts of old and young donors transplanted into age-matched or age-mismatched F344 rat recipients
DISCUSSION
In clinical transplantation, the use of organs to cure end-stage diseases has been the treatment of choice. However, the increasing number of patients on the transplantation waiting list has led to the enlargement of the donor pool to include old cadaveric donors (2, 3). The impact of age on organ transplantation has raised a great interest among the transplantation community in recent years. The incidence of arteriosclerosis is known to increase with age where remodeling in the vessels is severe in old vessels and results in a thicker neointima (4–6). Migration/attachment and proliferation of vascular smooth muscle cells are fundamental steps in the development of arteriosclerosis and neointimal formation after endothelial injury (19, 20). Accumulation of VSMCs, their proliferation, and production of extracellular matrix are critical events in the development of a neointima (21). However, the mechanism by which aging promotes exaggerated neointimal formation after vascular injury is poorly understood. Some studies demonstrated that VSMC proliferation increased with age (4, 6, 22), while others showed the opposite (5, 23, 24). These conflicting data may reflect the inherent limitations associated with in vitro study systems, as VSMCs are known to alter their phenotypes in cultures (25).
To discern between neointimal hyperplasia promoted by aging from that promoted by the alloimmune response, our strategy consisted of using vascular endothelium denudation as the initiator of intimal thickening in donor aortas transplanted into age-mismatched, syngeneic, sex-matched recipients. Our results showed that regardless of the donor’s age, all aortas transplanted into old recipients exhibited a more pronounced neointimal hyperplasia than when transplanted into young recipients. These findings reported herein lend a greater role to the age of the recipient than was previously thought (2, 3, 6, 9). In our controls for ischemia and in the absence of injury induced by endothelium denudation, we did not witness any neointimal thickening even in old donor grafts transplanted into old recipients. A minimal neointima formation in isogenic femoral artery transplants retrieved from adult rat recipients 40 days posttransplant has been reported elsewhere (26). The absence of any neointima in our experiments may be attributed to the type of vascular graft, where we used thoracic aorta instead of femoral artery, or else to cold ischemia time.
The relevance of the widely used balloon catheter model to transplant injury and chronic rejection is a legitimate concern, which could be addressed by the lack of models to initiate neointima formation in organs without resorting to immune-mediated chronic rejection or some other type of injury. Balloon catheter is widely used as an experimental extreme mimetic of organ injury that yields a uniform endothelium denudation with subsequent development of a consistent neointimal hyperplasia within a reasonable time. Ischemic models often yield variable degrees of neointimal thickening, from no thickening to a high degree of thickening, and may not be suitable for experiments where the readout is intimal hyperplasia. In clinical situations, it is difficult to assess age-related arteriosclerosis in transplanted organs because the alloimmune response due to histoincompatibility and subsequently implemented drugs regimens may overshadow the neointimal formation promoted by aging. The other reason we chose balloon injury is to ensure that old and young aortas received the same degree of injury under strict pressure control even though they had different diameters/calibers. This would have been much more difficult to achieve using a wire that does not accommodate to the varying vessel diameter. Our results indicate that neointimal formation and proliferation of VSMCs within the neointima (BrdU uptake) were greater in old recipients, regardless of the donor’s age (Fig. 3). These findings suggest that one possible mechanism whereby the aging biological milieu affects more neointimal formation is by increasing the proliferation of smooth muscle cells. In an earlier separate study in mice, we reported that aging results in a significant resistance to apoptosis and enhanced proliferation in neointimal VSMCs (27). This is in agreement with other findings, which showed that glucose-induced apoptosis was greater in VSMCs from young rats compared with that of VSMCs derived from old rats (28). Aging VSMCs also share the characteristic of higher proliferation and increased resistance to apoptosis with aging fibroblasts as described elsewhere (29). Advance in age is associated with higher levels of inflammatory cytokines (30), higher tumor necrosis factor (TNF)-α in the coronary arteries (31), and high levels of lipopolysaccharide-induced TNF-α in the arterial wall (32). Recently, Csiszar et al. (31) demonstrated that the increased susceptibility to apoptosis in the endothelial cell layer in aging coronaries is likely mediated by the proinflammatory phenotype, which may directly contribute to endothelium denudation and further neointimal accumulation of cells in the elderly. In an in vitro study focusing on mitogen-activated protein kinases (ERK, JNK, P38), Li et al. (33) found that age differentially influenced activation of signaling pathways in VSMCs exposed to high glucose or TNF-α, which may thus contribute to the increased risk for vascular disease associated with aging and diabetes mellitus.
Morphologically, modifications were shown to accumulate with time in the aortic intima of old rats. These age-related modifications include disruption of the internal elastic lamina, intimal thickening, and increases in growth factors and collagenase activity. Metalloproteinases-2, transforming growth factor-β, and intercellular adhesion molecule-1 levels were elevated and localized to the thickened intima of old rats (34, 35). Increases in collagen content and collagen/elastin ratio and decreases in elastin density and the number of medial VMSCs have been reported in intact aortas of old rats (34). However, in transplanted aortas, we could not find significant differences in cellularity (number of nuclei/area) or collagen and elastin ratios; values were similar in the neointima of injured aortas, regardless of the recipient’s age. Hypertension may be regarded as another factor in old recipients, but the F344 rat model we used differs from humans in that it is not complicated by hypertension (36, 37), although it presents the same aortic stiffening (36).
The previous paradigm on intimal hyperplasia assumes that neointima formation is the result of migration and proliferation of local smooth muscle cells from the media (19, 20). However, this concept has been challenged recently as more evidence suggested that not all neointimal cells come from the medial layer (38–40). It has been demonstrated that over 50% of neointimal smooth muscle cells derive from circulating precursors of bone marrow origin (39). Bone marrow-derived neointimal VSMCs have been demonstrated in both mechanical vascular injury (39) and allograft models (40). These observations are in line with our findings that the recipient’s age plays an important role in de novo or transplant arteriosclerosis.
In summary, this study addresses the contribution of age to transplant arteriosclerosis in a rat aortic transplant model in absence of variables such as alloimmune-mediated chronic rejection and the chronic use of immunosuppressive drugs. We demonstrated that neointima formation increases with age and that its severity in a transplantation context is primarily determined by the recipient’s age rather than the age of the aortic graft. Factors in the old recipients that may affect the development and the severity of transplant arteriosclerosis may include, but are not limited to, cellular and soluble elements within the blood such as circulating stem cells and progenitor cells, growth factors, hormones, advanced-glycation end products, and reactive oxygen species. The inference from our data is that in addition to targeting the alloimmune response, strategies aiming at increasing the graft’s lifespan have to also take into account the recipient’s aging milieu.
ACKNOWLEDGMENT
The authors are indebted to Mireya Hernandez for her expert technical help with morphometric analyses.
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Keywords:
Arteriosclerosis; Organ procurement; Aorta/artery; Recipient/donor age; Old/aging/young; Graft/transplant
© 2005 Lippincott Williams & Wilkins, Inc.