Abstract
Morbidity and mortality for neonates with congenital diaphragmatic hernia-associated pulmonary hypoplasia remains high. These patients may be deficient in vascular endothelial growth factor (VEGF). Our lab previously established that exogenous VEGF164 accelerates compensatory lung growth (CLG) after left pneumonectomy in a murine model. We aimed to further investigate VEGF-mediated CLG by examining the role of the heparin-binding domain (HBD). Eight-week-old, male, C57BL/6J mice underwent left pneumonectomy, followed by post-operative and daily intraperitoneal injections of equimolar VEGF164 or VEGF120, which lacks the HBD. Isovolumetric saline was used as a control. VEGF164 significantly increased lung volume, total lung capacity, and alveolarization, while VEGF120 did not. Treadmill exercise tolerance testing (TETT) demonstrated improved functional outcomes post-pneumonectomy with VEGF164 treatment. In lung protein analysis, VEGF treatment modulated downstream angiogenic signaling. Activation of epithelial growth factor receptor and pulmonary cell proliferation was also upregulated. Human microvascular lung endothelial cells (HMVEC-L) treated with VEGF demonstrated decreased potency of VEGFR2 activation with VEGF121 treatment compared to VEGF165 treatment. Taken together, these data indicate that the VEGF HBD contributes to angiogenic and proliferative signaling, is required for accelerated compensatory lung growth, and improves functional outcomes in a murine CLG model.
Highlights
Abbreviations congenital diaphragmatic hernia (CDH) Congenital diaphragmatic hernia compensatory lung growth (CLG) Compensatory lung growth extracorporeal membrane oxygenation (ECMO) Extracorporeal membrane oxygenation heparin-binding domain (HBD) Heparin-binding domain Heparin-binding epidermal growth factor-like factor (Hb-EGF) Heparin-binding epithelial growth factor HMVEC-L Human microvascular lung endothelial cells high-power fields (HPF) High power field H&E Hematoxylin and eosin IHC Immunohistochemistry phosphate-buffered saline (PBS) Phosphate-buffered saline PBST Phosphate-buffered saline with 0.05% Triton-X pulmonary function testing (PFT) Pulmonary function test(ing) post-operative day (POD) Post-operative day PVDF Polyvinyl difluoride pulmonary hypoplasia (PH) Pulmonary hypoplasia pEGFR Phosphorylated epithelial growth factor receptor pHTN Pulmonary hypertension
VEGF120 demonstrated a milder dose-dependent increase in lung volume, neither dose reached statistical significance compared to saline control
Inactive VEGF120 or VEGF164 did not produce a significant increase in lung volume or total lung capacity (TLC) at POD4 after pneumonectomy (Supplemental Fig. S1A,B)
Summary
Abbreviations CDH Congenital diaphragmatic hernia CLG Compensatory lung growth ECMO Extracorporeal membrane oxygenation HBD Heparin-binding domain Hb-EGF Heparin-binding epithelial growth factor HMVEC-L Human microvascular lung endothelial cells HPF High power field H&E Hematoxylin and eosin IHC Immunohistochemistry PBS Phosphate-buffered saline PBST Phosphate-buffered saline with 0.05% Triton-X PFT Pulmonary function test(ing) POD Post-operative day PVDF Polyvinyl difluoride PH Pulmonary hypoplasia pEGFR Phosphorylated epithelial growth factor receptor pHTN Pulmonary hypertension. Animal models and human patients with CDH-associated PH may be deficient in vascular endothelial growth factor (VEGF), especially in the alveolar stage of lung development[3,4]. Murine left pneumonectomy-induced compensatory lung growth (CLG) shares molecular patterning similar to developmental alveolarization and serves as a model to study pulmonary hypoplastic d iseases[11,12]. In accordance with the prior observation of decreased VEGF mRNA expression in patients with CDH in the alveolar stage of development, our group administered exogenous VEGF164, the most abundant murine isoform of the protein, in a murine CLG model, and demonstrated accelerated CLG to completion by POD413,14. In working towards the goal of translating systemic VEGF therapy into clinical practice for PH, we hypothesized that a VEGF protein lacking heparin-binding ability may potentially be effective in accelerating CLG without interacting with exogenous heparin given clinically. We aimed to further delineate the role of the HBD in VEGFmediated pulmonary growth, development, and function
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