| Abstrakt: |
Retinoic acid (RA) is essential for correct mammalian lung development and remarkably can induce alveolar regeneration in rodent models of COPD. However, clinical trials of RA and RA-Receptor gamma agonists in patients with COPD were disappointing. The reasons for the lack of effect in COPD patients are unknown but there is a clear need for a greater understanding of the underlying biology driving RA-induced lung regeneration. To investigate this, I studied the role of RA in human alveolar repair using isolated human pulmonary microvascular endothelial cells (HPMEC), alveolar epithelial cells (A549) and developed a novel human alveolar model using Precision-Cut Lung Slices (PCLS). In HPMECs, All-transRA (ATRA) induced angiogenesis in a dose dependent manner (p≤0.01, n=5). Pharmacological inhibition of VEGF receptor-2 with the selective inhibitor Ki8751 (Tocris) abolished this effect (p≤0.05, n=6) (figure 1). A proteome profiler array of PCLS, demonstrates an increase in pro-angiogenic proteins in the ATRA group, including CXCL16, IGFBP-3, PIGF, VEGF-A, HB-EGF and MCP-1. In addition, ATRA treatment of PCLS generated from histologically normal human lung lead to increased endothelial (PECAM-1) and alveolar type 2 (Pro-SPC) cell markers (n=3). Further investigation revealed that in wound-healing (scratch) assays of confluent cell monolayers, ATRA had no direct effect on the rate of wound healing in alveolar epithelial cells (A549) but significantly increased healing in HPMECs (p≤0.01, n=3). Moreover, siRNA knock-down of VEGF-R2 inhibited ATRA-induced wound-healing in HPMECs. Conditioned media from ATRA-treated HPMEC increased wound healing in A549 cells suggesting that the effects of RA on alveolar epithelial repair are mediated indirectly via the vascular network. HPMEC secreted HB-EGF with ATRA stimulation and HB-EGF treatment significantly increased A549 wound healing (n=2) suggesting it may act as a paracrine endothelial-epithelial regulator. My work demonstrates that RA has biological activity in human lung with direct effects on human lung microvasculature including cell migration, angiogenesis, and regulation of proteins likely to be important in alveolar repair. Together my data significantly advances our understanding of the mechanisms of RA induced repair in human lung tissue.[Figure] |
