In this study, we have shown that deoxygenated pulmonary artery blood is shunted to transplanted lungs soon after transplantation. We show for the first time that the relative percentage of blood flow to the transplanted lung is relatively constant in the first year after transplantation. The values noted for relative perfusion are similar to values presented in a prior study demonstrating 83% of perfusion to the transplant in COPD lungs and 69% of perfusion to the transplant in IPF lungs (12
). Reconstructed CT-angiography studies suggest that transplant airway hypoxia may be due, in part, to the lack of a radiologically demonstrable BA circulation in the lung after transplantation. The differences in BA lengths between transplant and native lungs were highly significant; a finding illustrating that the BA was uniformly abbreviated in length on the transplant side within individual patients and that, collectively, transplant BAs were significantly shorter than native airway BAs. Interestingly, the one exception to this was observed in the latest patient to be evaluated (612 d after transplant). It is possible that given time, a systemic artery may regrow into the graft. Indeed, this form of collateralization has been observed in several cases of heart-lung transplant recipients (10
), wherein the coronary arteries serve as a source of vessels to the presumably hypoxic airways. However, to date, this kind of collateralization has not been observed in single- or double-lung transplants.
To our knowledge, the current study is the first to attempt to measure airway tissue O2
saturations in humans. We found that transplant airway tissue remains relatively hypoxic compared with airways in native and normal lungs. The fact that the 2°carina, which are just distal to the anastomosis, were not significantly hypoxic compared with the contralateral native airway, suggests the possibility that the systemic circulation may be growing slowly (possibly due to hypoxia-driven angiogenesis) from the recipient into the donor at the microvascular level. It should be noted that parenchymal tissue O2
saturations are always significantly lower than blood O2
saturations, with hemoglobin O2
saturation of blood in the microvascular tissue spaces typically running closer to venous saturation than to arterial saturation. Although reference ranges for tissue O2
saturations have not been established in health and disease, measured values of O2
saturation for many tissues are typically 71 ± 3%, or a 95% confidence interval of 65 to 77% (R. Kum; Spectros; personal communication). In the current study, the only clear and relevant difference between the higher O2
(64–65%) saturation groups (native and normal airways) and the lower O2
saturation (60%) group (transplant airways) was the presence of a BA in the higher O2
saturation group. The tissue oximeter used in this study is a Food and Drug Administration–approved device routinely deployed through a colonoscope to assess intestinal oxygenation during vascular surgery (14
). Future studies are anticipated to further validate this new approach by expanding its use in lung transplantation and other pulmonary diseases.
Although the results demonstrate that transplant airways are relatively hypoxic, a 5% O2
saturation difference is of unclear biological relevance. There are a number of factors to consider with the results of the current study. First, the T-Stat oximeter assesses tissue oxygenation rather than arterial O2
saturation. As such, these are more mixed venous oxygen (capillary) saturations, which likely underestimate the true local arteriolar O2
saturation difference and rather represent tissue oxygen deficit. Furthermore, we currently do not have the technology to directly assess exertional airway tissue oxygenation, which would likely reveal greater tissue hypoxia in transplanted airways with exercise as there would be no source, to our knowledge, of highly oxygenated blood to supply airways during increased metabolic demand. Additionally, our animal studies demonstrate that inflammation itself greatly exacerbates airway tissue hypoxia and could potentially unmask a larger difference during rejection than the differences currently noted (6
). In our group's animal model, which assesses the tissue Po2
(rather than O2
saturation) of functional tracheal transplants, the tissue Po2
can go as low as 7 mm Hg (from a baseline in the low 30s) with unmitigated rejection (data not shown). Finally, the tissue oxygen saturation values reported in the current study were obtained in healthy, noninfected, nonrejecting, and non-BOS patients, and the observed differences simply indicate that the baseline quiescent healthy lung transplant is relatively hypoxic. It is clearly possible that without other concomitant risks, such as infection and rejection, this relative hypoxia is, in itself, an insufficient cause for BOS development.
The mechanisms by which hypoxia and ischemia contribute to postinflammatory fibrosis are not established but the clustering of hypoxia, ischemia, inflammation, and fibrosis is routinely observed in several clinical situations, such as normal skin wound healing (16
) and chronic kidney diseases (17
). In pulmonary fibrosis in mice and in humans, microarray data sets reveal hypoxic signaling among the most statistically important dysregulated pathways (18
). In vitro
studies have demonstrated profibrotic phenotypic change of fibroblasts in response to hypoxia (21
). Epithelial and endothelial cells can undergo mesenchymal transition under ischemia to become another source of activated fibroblasts (23
). Hypoxia likely directly contributes to the progression of fibrosis by increasing the release of major extracellular matrix proteins (24
). Transforming growth factor-β2
–induced fibrosis is associated with intense vasoconstriction and tissue hypoxia (25
). Which of the above phenomena (i.e., activated fibroblasts, mesenchymal transition, release of matrix proteins) contributes the most to airway fibrosis is not currently known, but it is clear that inflamed tissue subject to low Po2
and ischemia is at considerable risk for fibrotic remodeling.
It has been estimated in a canine study that about 50% of blood flow to the main bronchi normally comes from the BAs and 50% from the poorly oxygenated pulmonary artery circulation (26
). Although there has been some debate about the existence of a functional bronchopulmonary vascular anastomosis in normal lungs, most evidence suggests that these connections likely exist in normal lungs with networks forming at the precapillary level (27
). The BA circulation is highly conserved through evolution (29
), and the ramifications of living without this circulation are not known. In the absence of a BA revascularization step at the time of transplantation, the bronchi are presumably 100% supplied by the pulmonary circulation, which would result, as illustrated by this study, in the airways being more hypoxic. Preclinical and preliminary clinical studies demonstrate that BA revascularization improves tissue perfusion with more highly oxygenated blood (30
), is durable (32
), is associated with less epithelial metaplasia (33
), and is protective of pulmonary endothelium and type II pneumocytes (34
). With modern surgical techniques, bronchial anastomoses healed well without BA reconnection, and for largely this reason, the highly oxygenated BA circulation is now sacrificed in all lung transplant recipients. Lung transplants now rely on the persistence of a microcirculation presumably arising from the deoxygenated pulmonary circulation to provide perfusion to the airways.
The Copenhagen group, led by Gosta Pettersson, demonstrated that BA revascularization is durable with vessels remaining fully patent for 2 years (32
). A follow-up study showed that BA revascularization (BAR) may also postpone the development of BOS and improve patient survival (35
) and that reanastomosed vessels remain patent for at least 2 years (32
). It is not clear if the BOS that was ultimately seen in some of the patients within this BAR cohort was attributable to a late failure (beyond 2 years) of the BA grafts; if so, such a late failure could have occurred on the basis of rejection of the vasculature alone. If restoring a functional BA circulation is protective against the development of BOS, simply performing BAR itself is no guarantee of long-term airway perfusion because the donor-derived vasculature will remain a target for alloimmune injury. At a minimum, the BAR experience in Copenhagen suggests that a large patent vascular system from the outset is protective. Just as prevention of early graft injury likely has long-term beneficial effects (36
), additional steps to preserving early airway microvascular integrity may similarly prove critical for BOS prevention (6
There are other sequelae, beyond hypoxia, that may occur after the loss of the BA circulation and could contribute to airway disease. These include the loss of airway nutrition, altered lymphatic flow, decreased airway lining fluid, attenuated innate immune defenses, diminished clearance of small particles, and reduced control of airway temperature and humidity (38
). The bronchial circulation is responsible for the formation of the epithelial lining fluid, which plays a role in the local defenses against inhaled irritants and foreign substances. In contrast to the pulmonary circulation, BA vascular transudates appear to contribute to lymphatic flow (40
). What happens to lymphatic flow in the absence of this BA contribution is not known. A functional BA circulation is required for the maintenance of normal mucociliary transport (41
). Interrupting the BA likely leads to interference with absorbing and clearing airway particles (40
). Finally, the airway mucosa responds to the cooling of airways after the inhalation of cold air by increasing bronchial blood flow, and by doing this improves heat and water transfer from the air. This same circulation is capable of conserving moisture in dry environments such that only one-tenth of the normal humidity is exhaled (41
). Therefore, the loss of a bronchial circulation could negatively impact the regulation of airway temperature and humidity. In summary, although exaggerated tissue hypoxia with inflammation (beyond what was measured here in quiescent transplants) could occur in the absence of BA circulation, there are several other functions normally performed by the BA circulation and not assessed in the current study that could also contribute to tissue fibrosis.
Although the Papworth Hospital autopsy studies suggested that microvascular dropout occurs before the development of BOS, perhaps through alloimmune-mediated damage of the donor vasculature, they also demonstrated a robust increase in the microvasculature in established BOS (1
). Based on experimental orthotopic tracheal transplant studies (6
), we have previously argued that the increased angiogenesis observed in airway fibrosis may simply be a response to airway tissue hypoxia and ischemia in rejection. Hypoxia-induced angiogenesis in airways may be primarily attributed to the actions of hypoxia-inducible factor-1α (HIF-1α). HIF-1α is a transcriptional regulator that is responsive to a reduction of Po2
in tissues and actively promotes hypoxia-associated angiogenesis and vasculogenesis (42
). HIF-1α–mediated vessel growth is mediated through the production of multiple angiogenic growth factors and by its differentiating effects on endothelial cells and bone marrow–derived endothelial progenitor cells. Accordingly, pharmacological or molecular approaches that engage hypoxic adaptation at the point of a major sensor like HIF-1α could conceivably lead to a profound sparing of hypoxic tissue and enhanced recovery of function after alloimmune inflammation. Therapy that protects vascular endothelial cells from injury or optimizes their recovery may limit chronic rejection.
As recently noted by Contreras and Briscoe (37
), “a robust vasculature appears to be the ‘silver lining’ that is necessary to sustain long-term allograft function.” The lung transplant may be an especially vulnerable allograft given the relatively hypoxic starting point of its airways. Table E1 in the online supplement summarizes preclinical and clinical studies germane to this issue and suggests how BAR may result in a healthier organ after transplantation beyond simply improving the healing of the airway anastomosis. Viewed through the new perspective of a lung transplant as an oxygen-poor organ threatened by frank ischemia with rejection of its vascular supply, new therapeutic approaches focusing on surgical BAR at the time of transplantation and on stabilization of microvascular integrity have strong potential for limiting the development of chronic rejection.