View Abstract

A3823 - Retinoic Acid Upregulates Endothelial-Derived Angiocrine Factors to Promote Human Alveolar Epithelial Repair
Author Block: J. Alçada, D. Shao, M. J. Griffiths, C. Dean, M. Hind; National Heart and Lung Institute, Imperial College London, London, United Kingdom.
RATIONALE
Reactivation of developmental pathways holds promise as regenerative therapy for chronic lung diseases. Retinoic acid (RA) is essential for correct alveologenesis during lung development and can induce lung regeneration in animal models of alveolar insufficiency.
However, encouraging preclinical data translated poorly in clinical trials of RA and RAR gamma agonists in patients with COPD, in part due to a lack of models to explore retinoids biology in human lung. To investigate this, we studied the role of RA in human alveolar repair using isolated primary human pulmonary microvascular endothelial cells (HPMEC) and developed a novel human alveolar model using Precision-Cut Lung Slices (PCLS).
METHODS
All-trans RA (ATRA) effects on angiogenesis and migration of human pulmonary microvascular endothelial cells (HPMEC) and of the alveolar cell line A549 were assessed using a Matrigel-based tube formation and wound-healing assays (scratch) of confluent cell monolayers. PCLS (450 µm) were generated from agarose-inflated normal human lung surgical resection specimens, cultured in serum-free or ATRA-supplemented medium and analysed with confocal microscopy, Western-Blot and proteome profiler arrays at days 1 to 6. Inhibition of VEGF receptor-2 (VEGF-R2) was investigated with the selective inhibitor Ki8751 (Tocris®) or siRNA knockdown. HB-EGF and MCP-1 secretion was quantified by ELISA.
RESULTS
In HPMEC, ATRA induced tube formation in a dose dependent manner (p≤0.01, n=5). Ki8751 (Tocris®) abrogated this effect (p≤0.05, n=3), suggesting ATRA drives angiogenesis via VEGF-R2. In wound-healing assays, ATRA had no direct effect on the alveolar epithelial A549 cell line but significantly increased migration of HPMEC (p≤0.01, n=3). ATRA-induced HPMEC migration was inhibited with siRNA knock-down of VEGF-R2. A549 cell migration increased when exposed to conditioned medium of HPMEC treated with RA.
In PCLS generated from normal human lung, ATRA supplementation increased both endothelial (PECAM-1) and alveolar type 2 (Pro-SPC) cell markers (n=3). A proteome profiler array of PCLS, identified increased levels of several pro-angiogenic proteins in the ATRA-treated slices, in particular CXCL16, IGFBP-3, PIGF, VEGF-A, HB-EGF and MCP-1. When treated with ATRA, HPMEC exhibited increased secretion of HB-EGF and MCP-1. HB-EGF treatment significantly promoted A459 wound healing.
CONCLUSIONS
My work demonstrates that ATRA has biological activity in human lung with direct effects on human lung microvasculature including cell migration, VEGF-R2 dependent angiogenesis, and regulation of proteins that may have a paracrine effect on epithelial cell behaviours likely to be important in alveolar repair.