Lung transplantation may be the only viable option for patients suffering from otherwise incurable end-stage pulmonary diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. that lymphatic vessel formation after lung transplantation mediates HA drainage and suggest that treatments to stimulate lymphangiogenesis have promise for improving graft outcomes. Introduction Lung transplantation remains the optimal treatment to prolong survival and improve quality of life for patients with end-stage lung disease (1). While outcomes after lung transplantation continue to improve, the 5-year survival rate is still far below the rates achieved following other solid organ transplantations (2). Despite aggressive immunosuppressive treatments, acute rejection happens in up to 55% of lung allograft recipients and is among the leading factors behind morbidity through the 1st yr after transplantation (3, 4). Furthermore, recurrent severe rejection represents an initial risk element for the introduction of bronchiolitis obliterans symptoms (BOS), or chronic allograft rejection, Mitoxantrone IC50 which really is a main impediment to long-term success (5C8). Because the effectiveness of immunosuppression can be significantly less than ideal, continuing exploration of book therapeutic options can be imperative. Lung transplantation involves airway, arterial, and venous connections at the time of surgery (9). Notably, anastomosis of severed donor lymphatic vessels to those of the recipient is not performed due to technical challenges, resulting in complete interruption of lymphatic drainage. Reestablishment of a lymphatic continuum after transplantation relies on formation of new lymphatic vessels (lymphangiogenesis), the exact roles of which remain enigmatic and somewhat controversial in transplant pathophysiology (10, 11). While some studies have shown a beneficial role of lymphatic vessels in transplantation (12C15), the prevailing view holds that they contribute to alloimmune responses that will result in the exacerbation of allograft rejection (16C19). Therefore, it has been suggested that inhibiting lymphangiogenesis could be critical for graft tolerance and survival (20, 21). However, lung allografts are acutely rejected within 7 days after transplantation in animal transplant models (22C25), whereas spontaneous Mitoxantrone IC50 restoration of lymphatic drainage from the transplanted lung to the lymph nodes occurs no earlier than day 7 after transplantation (26, 27). Since the onset of lung rejection precedes the reestablishment of lymphatic continuity, it is possible that insufficient lymphatic drainage could be responsible, at least in part, for acute lung allograft rejection. Hyaluronan (HA) is a ubiquitously distributed extracellular matrix glycosaminoglycan that exists physiologically as a high-MW (HMW) polymer but undergoes extensive fragmentation in response to tissue injury (28C31). HA has been previously associated with lung injury and repair through multiple pathways driven by HA receptors such as CD44 and TLR2 and TLR4 (29, 31, 32). More Mitoxantrone IC50 recently, low-MW Rabbit Polyclonal to PLA2G4C HA was shown to play important roles in the development of BOS through TLR2/4-dependent pathways, leading to increased numbers of neutrophils and alloantigen-specific T lymphocytes, while HMW HA decreased graft inflammation (33). Increased HA content and fragmentation contribute to transplant rejection (32C34), although it is unclear whether the abundant presence of HA also reflects a lack of its effective drainage in rejected allografts. Since the turnover of HA (several grams/day in humans) occurs primarily through lymphatics (approximately 85%) (35, 36), where uptake is mediated by the lymphatic vessel endothelial HA receptor LYVE-1, we hypothesized that HA clearance impairment due to severely compromised lymphatic drainage might lead to its aberrant accumulation in lungs undergoing rejection. To test this hypothesis, we performed gain-of-function (therapeutically inducing lymphangiogenesis to promote HA drainage) as well as loss-of-function (blocking HA uptake by lymphatic endothelial cells [LECs]) experiments in a mouse model of orthotopic lung transplantation. In addition, we analyzed sequential transbronchial biopsy (TBB) specimens from human lung transplant recipients to determine whether the observations made in the mouse model are consistent with clinical events. Results Acute lung allograft rejection leads to loss of lymphatic vessels. To assess the fate of lymphatic vessels in lung transplants, we first performed mouse orthotopic left lung transplantation using the 3-cuff technique for vascular and airway anastomoses (Figure Mitoxantrone IC50 1, A and B), as previously described (37, 38). We sacrificed the animals 30 days after transplantation and visualized the lymphatic vessels using antibodies against LYVE-1, a widely used marker of LECs (39C41). Consistent with previous observations (22, 23),.