Abstract 15599: Klf4 Regulates the Extent of Lung Microvessel Endothelial Cell Regeneration
Rationale: Transcription factor Krüppel like factor (Klf)-4 might regulate endothelial cell (EC) regeneration; however, the underlying mechanisms are poorly understood.
Objective: The goal of this study was to delete EC-Klf4 in a timed manner using the lox-P elements, Cdh5 (VE-cadherin) as a promoter to drive the Cre recombinase, and to address the role of Klf4 in adult lung microvessels.
Methods and Results: After 12 generations of backcrossing, we transferred Klf4fl to a C57BL background. Male Klf4fl/+ were crossed with female Klf4fl/+ mice to generate Klf4fl/fl (25%) offspring. In the next breeding scheme, Klf4fl/fl mice were crossed with male tg-Cdh5CreERT2 (tamoxifen inducible) mice to produce heterozygous Klf4fl::tg-Cdh5CreERT2 offspring (100%). I then crossed male Klf4fl::tg-Cdh5CreERT2 with female Klf4fl::tg-Cdh5CreERT2 to produce offspring of Klf4fl/fl::tg-Cdh5CreERT2 genotype (25%). Groups of these mice received tamoxifen everyday for 5 days, and on the 10th day, (now called Klf4ECKO mice) all mice were sacrificed. To determine the loss-of-EC-Klf4 protein and to examine the phenotypic alterations, lungs were collected, subjected to biochemical and molecular experiments, fixed and stained for microscopic analyses. Tamoxifen treatment decreased EC-Klf4-protein expression significantly in the Klf4ECKO lung, as compared to Klf4fl/fl or tg-Cdh5CreERT2 controls. Klf4ECKO lung tissue showed increased recruitment of neutrophils and inflammatory cells. Trichrome staining revealed increased collagen deposition in the Klf4ECKO versus the control mice. Importantly, I observed decreased lung alveoli (p<0.05 vs control, n=5), hemorrhage in Klf4ECKO mice, compared to the control groups. Anti-CD31 and anti-von Willebrand Factor (vWF) staining showed significant decrease (p<0.5 versus control) in microvessel densities in the lung.
Summary: These compelling sets of results predict the ability of endothelial-Klf4 to regulate EC regeneration, and its loss in lung fibrosis.
Author Disclosures: T. Perkins: Other Research Support; Modest; NIH-T32 training fellowship to Tina Perkins, American Heart Association Pre-doctoral fellowship to Tina Perkins. K. Wary: Research Grant; Modest; American Heart Association grant in aid to Kishore Wary.
This research has received full or partial funding support from the American Heart Association
- © 2014 by American Heart Association, Inc.