The human fetal lung xenograft: validation as model of microvascular remodeling in the postglandular lung

Abstract

Background: Coordinated remodeling of epithelium and vasculature is essential for normal postglandular lung development. The value of the human-to-rodent lung xenograft as model of fetal microvascular development remains poorly defined.

Aim: The aim of this study was to determine the fate of the endogenous (human-derived) microvasculature in fetal lung xenografts.

Methods: Lung tissues were obtained from spontaneous pregnancy losses (14-22 weeks' gestation) and implanted in the renal subcapsular or dorsal subcutaneous space of SCID-beige mice (T, B, and NK-cell-deficient) and/or nude rats (T-cell-deficient). Informed parental consent was obtained. Lung morphogenesis, microvascular angiogenesis, and epithelial differentiation were assessed at 2 and 4 weeks post-transplantation by light microscopy, immunohistochemical, and gene expression studies. Archival age-matched postmortem lungs served as control.

Results: The vascular morphology, density, and proliferation of renal subcapsular grafts in SCID-beige mice were similar to age-matched control lungs, with preservation of the physiologic association between epithelium and vasculature. The microvasculature of subcutaneous grafts in SCID-beige mice was underdeveloped and dysmorphic, associated with significantly lower VEGF, endoglin, and angiopoietin-2 mRNA expression than renal grafts. Grafts at both sites displayed mild airspace dysplasia. Renal subcapsular grafts in nude rats showed frequent infiltration by host lymphocytes and obliterating bronchiolitis-like changes, associated with markedly decreased endogenous angiogenesis.

Conclusion: This study demonstrates the critical importance of host and site selection to ensure optimal xenograft development. When transplanted to severely immune suppressed, NK-cell-deficient hosts and engrafted in the renal subcapsular site, the human-to-rodent fetal lung xenograft provides a valid model of postglandular microvascular lung remodeling.

Figure 1. Morphology of preimplantation lung, control lung and xenografts

A. Representative preimplantation lung at 17 weeks’ gestation (pseudoglandular - early canalicular stage of development). B. Representative control lung at 21 weeks’ gestation (canalicular stage). C–D. Representative photomicrographs of renal subcapsular (C) and subcutaneous (D) xenografts, obtained at 17 weeks’ gestation and studied at post-transplantation week 4. E–F Scanned images of representative renal subcapsular (E) and subcutaneous (F) xenografts, obtained at 17 weeks’ gestation and studied at post-transplantation week 4. A–F: Hematoxylin-eosin stain. A–D: original magnification X100; E and F: original magnification X5.

Figure 2. Analysis of surfactant protein-C gene expression in grafts

Values represent mean ± SD of 4–6 animals per group, normalized to GAPDH expression. Open bars: kidney grafts; closed bars: subcutaneous grafts. SP-C: surfactant protein-C *: P < 0.001 versus kidney graft; º: P < 0.05; ºº: P < 0.01 versus day 14.

Figure 3. Analysis of graft vascularization and endothelial cell proliferation

A and D: Representative CD31-immunohistochemical analysis of renal subcapsular xenografts (obtained at 17 weeks’ gestation, studied at post-transplant week 4). A dense capillary network is noted within the septa, forming a near-continuous single or double capillary pattern in subepithelial position. Graft-derived capillaries extend deep into adjacent murine renal cortex (Fig. 3A, right). B and E: Representative CD31-immunohistochemical analysis of subcutaneous xenografts (obtained at 17 weeks’ gestation, studied at post-transplant week 4). Graft capillaries are seen as short, interrupted profiles within the septa. Some areas appear devoid of vessels (Fig. 3B, top). Rare vascular extension from graft into surrounding fibrous capsule is noted (Fig. 3B, asterisk). Airspaces appear dilated. C and F: Representative control lung at 21 weeks’ gestation showing an intensely CD31-immunoreactive capillary network, organized as a single or double network below the epithelium. G: Representative renal subcapsular graft at post-transplant week 4 showing a well developed capillary network with numerous proliferating endothelial cells, exhibiting double immunoreactivity for Ki-67 (green, nuclear) and CD31 (red, cytoplasmic). H. Representative subcutaneous graft at post-transplant week 4 showing focal presence of septal capillaries. Only rare endothelial proliferative activity is seen. I. Representative control lung at 21 weeks’ gestation showing proliferating endothelial cells in a dense capillary network. A–F: CD-31 (PECAM-1) immunohistochemical analysis (DAB-peroxidase staining with hematoxylin counterstain). A–C: original magnification: X100; D–F: original magnification: X200. G–I: Ki-67 (Alexafluor-green) and CD31 (Cy3-red) double immunofluorescence; original magnification: X400).

Figure 4. Vascular density

Analysis of areal fraction of CD31-immunoreactive enodothelial cells relative to air-exchanging (septal) parenchyma, expressed as a percentage. Values represent mean ± SD of at least 4 animals per group. Open bars: kidney grafts; closed bars: subcutaneous grafts; grey bar: control lung (21 weeks’ gestation). *: P < 0.05 versus kidney graft.

Figure 5. Analysis of angiogenesis-related gene expression

Values represent mean ± SD of 5–6 animals per group, normalized to GAPDH expression. Open bars: kidney grafts; closed bars: subcutaneous grafts. PECAM-1: platelet endothelial cell adhesion molecule (CD31); VEGFA: vascular endothelial growth cell factor-A; ANGPT2: angiopoietin-2. *: P < 0.05; **: P < 0.01; ***: P < 0.001 versus kidney graft; º: P < 0.01 versus day 14.

Figure 6. Morphology and vascularization of renal xenograft in the nude rat

A. Representative photomicrograph of renal subcapsular xenograft at post-transplant week 4 showing a band-like inflammatory infiltrate along the graft-kidney border. B–C. Renal graft at post-transplant week 4 showing prominent inflammation along the graft-kidney interface on the left side of the graft (Fig. 6B), corresponding with decreased vascularization on that side of the graft (Fig. 6C). D–F. Renal graft at post-transplant week 4 showing dense inflammatory infiltrates deep within the graft. Fibroproliferative plugging of bronchioles is noted, replicating the histopathologic findings of obliterative bronchiolitis in humans (Fig. 6E, arrows). Vascularization is markedly diminished in this severely inflamed graft (Fig. 6F). A, B, D and E: Hematoxylin-eosin staining; C and F: CD-31 (PECAM-1) immunohistochemical analysis (DAB-peroxidase staining with hematoxylin counterstain). E: original magnification X200; others: original magnification X100.