Abstract
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 Klf4 fl to a C57BL background. Male Klf4 fl/+ were crossed with female Klf4 fl/+ mice to generate Klf4 fl/fl (25%) offspring. In the next breeding scheme, Klf4 fl/fl mice were crossed with male tg- Cdh5 CreERT2 (tamoxifen inducible) mice to produce heterozygous Klf4 fl ::tg- Cdh5 CreERT2 offspring (100%). I then crossed male Klf4 fl ::tg-Cdh5 CreERT2 with female Klf4 fl ::tg- Cdh5 CreERT2 to produce offspring of Klf4 fl/fl ::tg- Cdh5 CreERT2 genotype (25%). Groups of these mice received tamoxifen everyday for 5 days, and on the 10 th day, (now called Klf4 ECKO 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 Klf4 ECKO lung, as compared to Klf4 fl/fl or tg- Cdh5 CreERT2 controls. Klf4 ECKO lung tissue showed increased recruitment of neutrophils and inflammatory cells. Trichrome staining revealed increased collagen deposition in the Klf4 ECKO versus the control mice. Importantly, I observed decreased lung alveoli (p<0.05 vs control, n=5), hemorrhage in Klf4 ECKO 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.
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