Adenovirus-mediated HIF-1α gene transfer promotes repair of mouse airway allograft microvasculature and attenuates chronic rejection

Abstract

Chronic rejection, manifested as small airway fibrosis (obliterative bronchiolitis [OB]), is the main obstacle to long-term survival in lung transplantation. Recent studies demonstrate that the airways involved in a lung transplant are relatively hypoxic at baseline and that OB pathogenesis may be linked to ischemia induced by a transient loss of airway microvasculature. Here, we show that HIF-1α mediates airway microvascular repair in a model of orthotopic tracheal transplantation. Grafts with a conditional knockout of Hif1a demonstrated diminished recruitment of recipient-derived Tie2⁺ angiogenic cells to the allograft, impaired repair of damaged microvasculature, accelerated loss of microvascular perfusion, and hastened denudation of epithelial cells. In contrast, graft HIF-1α overexpression induced via an adenoviral vector prolonged airway microvascular perfusion, preserved epithelial integrity, extended the time window for the graft to be rescued from chronic rejection, and attenuated airway fibrotic remodeling. HIF-1α overexpression induced the expression of proangiogenic factors such as Sdf1, Plgf, and Vegf, and promoted the recruitment of vasoreparative Tie2⁺ cells. This study demonstrates that a therapy that enhances vascular integrity during acute rejection may promote graft health and prevent chronic rejection.

Figure 1. Remodeling of tracheal microvasculature in chronic rejection following transplantation.

(A) Normal trachea. Arterioles and venules are located between cartilage rings and in the noncartilaginous (anatomically posterior) membranous portion. Capillaries are located overlying the cartilaginous portions. (B) Day-56 chronically rejected allograft. Only capillaries are evident in the different portions of the trachea. Fewer vessels are seen in the cartilaginous portion, and the microvasculature becomes tortuous and disorganized in the intercartilaginous and the membranous portions. Both panels are composited of separate images. Scale bar: 100 μm.

Figure 2. Recipient-derived Tie2⁺ cells contribute to microvascular remodeling in chronic rejection and to repair of donor vessels rescued from acute rejection by immunosuppression.

(A) Immunofluorescence (IF) staining of a cross section (with subepithelium marked by the black solid line on the right side of the images here and in the following figures) of day-56 allografts (Balb/c to FVB [actin-EGFP]) shows that the majority of CD31⁺ cells (red) are GFP⁺ (green) and of recipient origin. (B) The percentage of GFP⁺CD31⁺ cells among CD31⁺ cells was calculated as the percentage of the recipient contribution to the remodeled microvasculature (n = 6). (C) Chronically rejecting day-28 allografts (Balb/c to FVB[Tie2-LacZ]) shows that β-gal⁺ (arrows; green) cells form tube-like structures and become part of CD31⁺ (arrows; red) microvasculature (top panel). Perfusion coupled with IF staining analysis shows that β-gal⁺ (arrow; red) cells are perfused by FITC-conjugated lectin (arrow; green) (lower panel). (D) Recipient mice bearing acutely rejecting day-6 allografts (Balb/c to FVB[Tie2-LacZ]) were perfused with red India ink, and then whole-mount trachea allografts were prepared following X-gal staining. Recipient-derived Tie2⁺ cells (blue) are scattered in the allograft and mainly outside of the vessels (red). (E) Recipient-derived GFP⁺ cells of day-6 allografts (Balb/c to FVB[Tie2-EGFP]) were analyzed for CD45 and CD31 expression by flow cytometry. (F) Balb/c tracheas were first transplanted into B6 (with double transgene: Tie2-Cre, ROSA26EYFP) mice, and then the day-6 grafts were retransplanted into WT B6 mice subjected to immunosuppression; retransplanted grafts were harvested 28 days later. Grafts are histologically normal. IF staining shows that some YFP⁺ cells (arrow; green) become part of CD31⁺ (arrow; red) vasculature. Scale bars: 100 μm (A and D); 20 μm (C and F). Data are shown as mean ± SEM.

Figure 3. Hif1a is essential for early microvascular repair of trachea allografts.

(A) Western blotting of Hif1a and Sdf1 in day-2 and day-4 trachea allografts. Actin was used as an internal control. The lanes were run on the same gel but were noncontiguous. (B) IF staining of day-3 allografts (Balb/c to B6) shows that Hif1a⁺ cells (arrows; green) express CD31 (arrows; red). (C) IF staining of day-6 allografts (Balb/C to FVB[actin-EGFP]) shows that only a small percentage of Hif1a⁺ cells (arrow; red) are of recipient origin (arrow; green). (D) Microvascular perfusion of WT trachea allografts is completely lost at day 10, followed by partial revascularization at day 21. However, complete loss of vascular perfusion of Hif1a CKO trachea starts 2 days earlier (day 8), followed by minimal revascularization by day 21. (E) Percentage of perfusion area of trachea allograft was calculated (n = 4–6; *P < 0.05). (F) Real-time RT-PCR analysis of angiogenic factors of day-6 WT and Hif1a CKO allografts (n = 4; *P < 0.05). (G) FITC-conjugated lectin perfusion coupled with IF staining of day-8 WT allografts shows normal microvascular perfusion (green) and pseudostratified columnar epithelium (E-cadherin staining; red) (top panel). However, in the acutely rejecting Hif1a CKO allograft, there is a loss of perfusion in the subepithelial area, and the epithelium is flat with some basement membrane exposed (lower panel). (H) Day-6 Hif1a CKO or WT allografts demonstrated that fewer Tie2⁺ cells (blue) were recruited to Hif1a CKO allograft. (I) Tie2⁺ cells per high power field were calculated to confirm the result shown in H (n = 5; *P < 0.05). Scale bars: 20 μm (B, C, and F); 100 μm (D and G). Data are shown as mean ± SEM.

Figure 4. HIF-1α gene transfer prolongs microvascular perfusion of airway allograft and alleviates tissue hypoxia.

(A) Vascular perfusion of AdLacZ control adenovirus vector–treated allografts is lost at day 10, day 12, and day 14, followed by partial revascularization at day 21. However, perfusion of AdCA5-treated allografts is maintained at both day 10 and day 12, followed by partial loss at day 14 and near complete revascularization at day 21. (B) Percentage of perfusion area of AdLacZ- or AdCA5-treated allografts was calculated. (n = 4–6; *P < 0.05). (C) AdCA5-treated allografts have higher tissue pO₂ levels compared with AdLacZ-treated grafts. (n = 4–6; *P < 0.05 at individual time points). (D) FITC-conjugated lectin perfusion and laminin staining show that AdCA5-treated chronically rejected day-21 allografts (lower panel) have better laminin-invested vessels (arrows) than AdLacZ-treated grafts (top panel). (E) FITC-conjugated lectin perfusion and α-SMA staining show that AdCA5-treated chronically rejected day-21 allografts (lower panel) have more pericyte-covered vessels (arrows) than AdLacZ-treated grafts (top panel). Scale bars: 100 μm (A); 20 μm (D and E). Data are shown as mean ± SEM.

Figure 5. HIF-1α gene transfer increases expression of angiogenic factors and accelerates endothelial cell replacement by recipient-derived cells.

(A and B) Flow cytometry analysis of day-6 allografts shows that infiltrated CD4⁺ and CD8⁺ T lymphocytes are comparable in AdLacZ-treated (A) or AdCA5-treated (B) samples. The percentages indicate CD4⁺ or CD8⁺ cells among CD3⁺ mononuclear cells. (C and D) Real-time RT-PCR analysis of day-6 allografts demonstrated that the expression of proinflammatory cytokines is not significantly different between AdLacZ and AdCA5 treatment (C), but expression of proangiogenic factors Plgf, Sdf1, and Vegf is significantly different (D) (n = 6; *P < 0.05 of each proangiogenic factor). (E) AdLacZ- or AdCA5-treated Balb/c tracheas were transplanted into FVB (Tie2-EGFP) mice, and day-6 allografts were analyzed by flow cytometry. AdCA5-treated allografts recruit higher levels of GFP⁺ cells in comparison with AdLacZ-treated grafts (n = 6, *P < 0.05). (F) AdLacZ- or AdCA5-treated Balb/C tracheas were transplanted into FVB (Tie2-LacZ), mice and day-12 allografts were analyzed by X-gal staining. AdCA5-treated allografts have more recipient-derived Tie2⁺ vasculature (blue). Scale bar: 20 μm. Data are shown as mean ± SEM.

Figure 6. HIF-1α gene overexpression extends the time window for acutely rejecting allografts to be rescued from chronic rejection.

(A) Electron micrograph demonstrating abnormal appearance of endothelial cells with disrupted basement membrane and thrombus within the damaged vessel in day-10 allografts treated with AdLacZ. Original magnification, ×4,500. (B) Electron micrograph demonstrating normal appearance of vascular endothelial cells with relatively intact basement membrane and the existence of pericytes around the endothelial cells in day-10 allografts treated with AdCA5. (C) FITC-conjugated lectin perfusion coupled with IF staining of E-cadherin shows a flattened epithelial layer (red) and absence of subepithelial microvascular perfusion (green) in AdLacZ-treated samples. AdCA5-treated samples show a columnar epithelium with subepithelial vessel perfusion. (D) Day-12 allografts treated with AdLacZ or AdCA5 were retransplanted into naive B6 mice treated with immunosuppression, and the allografts were harvested 28 days following retransplantation. H&E staining results reveal that AdLacZ-treated retransplants developed chronic rejection (top panel), and acute rejection was successfully reversed in AdCA5-treated retransplants (lower panel). Scale bars: 20 μm.

Figure 7. HIF-1α gene overexpression attenuates fibrosis of airway allografts.

(A–C) RT-PCR analysis of Col1a1, Col3a1, and Col5a1 mRNA expression shows AdCA5-treated allografts express diminished levels of Col1a1 (A), Col3a1 (B), and Col5a1 (C) at day 14 and day 21 (n = 6; *P < 0.05). (D and E) Picrosirius red staining was used to estimate the levels of collagen protein in trachea allografts. Diminished collagen deposition is seen in AdCA5-treated day-14 (D, right panel) and day-21 (E, right panel) allografts. Scale bars: 20 μm. Data are shown as mean ± SEM.

Figure 8. Model of how HIF-1α promotes airway microvascular repair and prevents fibrosis.

(A) Scheme of recipient-derived Tie2⁺ cells contributing to microvascular repair in allograft rejection. Increased allograft hypoxia during rejection leads to increased expression of HIF-1α in endothelial cells, which further induces expression of proangiogenic factors that promote the recruitment and retention of recipient-derived angiogenic cells, including Tie2⁺ cells (green) and other non-Tie2⁺ cells (yellow). Enhanced expression of HIF-1α accelerates vascular repair. (B) Scheme of the effects of HIF-1α gene overexpression on airway perfusion and fibrosis. Perfusion is reestablished at day 4 following transplantation in both AdLacZ- and AdCA5-treated OTT after microvascular reconnection between the donor and the recipient. Progressive microvascular damage leads to a complete loss of perfusion at around day 10, followed by a partial microvascular reestablishment at day 21 in AdLacZ-treated allografts. Reestablished microvessels are phenotypically distinct from normal ones. In contrast, microvascular perfusion is prolonged to day 12 in AdCA5-treated allografts. This anatomic finding identifies day-12 allografts as “rescuable” to normal architecture by immunosuppression (versus day 8 for control airways; blue line). The onset and the intensity of the collagen deposition are significantly delayed and diminished respectively in AdCA5-treated airways (red line). Alleviation of fibrosis in HIF-1α–overexpressed airways is likely due to the decreased burden of tissue hypoxia (ischemia × time duration), which is represented by the yellow area.