Stretch-induced upregulation of VEGF gene expression in murine pulmonary culture: a role for angiogenesis in lung development.
OBJECTIVE
The authors' have shown pulmonary alveolarization (capillary and alveolar growth) both after fetal tracheal occlusion and postnatal pulmonary distension. The trophic and developmental mechanisms responsible for this growth remain largely unknown; however, experimental systems have defined an enhanced expression of angiogenic proteins in response to tissue stretch. The authors hypothesize that the stimulation of pulmonary alveolarization after stretch is secondary to upregulation of the potent endothelial cell mitogen vascular endothelial growth factor (VEGF) and that the endothelial cell represents the central stimulus of parenchymal growth.


METHODS
A mixed primary pulmonary cell culture obtained by enzymatic digestion of fetal, neonatal, and adult mouse lung was plated on Bioflex elastomer bottom plates, grown to confluence, rendered quiescent, and subjected to continuous cycles of stretch-relaxation with nonstretched cells as controls. Cells were harvested at time-points 0, 30 minutes, 2 hours, 4 hours, 8 hours, and 24 hours. RNA was extracted and VEGF gene expression analyzed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Similar cell groups were harvested, processed, and analyzed utilizing Western Blot techniques. VEGF PCR of mRNA isolated from fetal sheep subjected to surgical creation of diaphragmatic hernia both with (DH-TL) and without (DH) tracheal ligation also was analyzed.


RESULTS
VEGF mRNA isoforms 120, 164, and 188 showed increased expression in all stretched groups, which was noted by 30 minutes with maximal expression seen at 2 to 4 hours and a return to baseline expression by 24 hours. VEGF protein was similarly elevated in all stretched cell groups. In preliminary studies, DH/TL sheep showed upregulation of VEGF compared with DH sheep alone.


CONCLUSIONS
These data show in an in vitro system that "pulmonary stretch" upregulates VEGF mRNA and protein expression supporting the role of angiogenesis in the stretch-induced pulmonary alveolarization. The authors speculate that such angiogenic activity is a rate-limiting factor in stimulating alveolar epithelial development, and as a treatment modality, therapeutic angiogenesis may provide a noninvasive method with which to treat pulmonary hypoplasia.
Muratore
C S
CS
Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Nguyen
H T
HT
Ziegler
M M
MM
Wilson
J M
JM
D000375:Aging; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000235:genetics* / Q000378:metabolism; D005333:Fetus; D015870:Gene Expression; D006548:Hernia, Diaphragmatic / Q000235:genetics / Q000503:physiopathology; D065630:Hernias, Diaphragmatic, Congenital; D008026:Ligation; D008168:Lung / Q000098:blood supply / Q000254:growth & development* / Q000378:metabolism; D008222:Lymphokines / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D018919:Neovascularization, Physiologic; D010812:Physical Stimulation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D014132:Trachea / Q000502:physiology; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D020033:Protein Isoforms; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors