Role of KLF6 in endothelial pathology in pulmonary arterial hypertension

Abstract

Background: Loss of endothelial homeostasis is a key contributor to vascular remodelling in PAH. PAH shares many characteristics with cancer, including increased cell proliferation and apoptosis resistance. Accumulating evidence suggests that Krüppel-like factor 6 (KLF6), a transcription factor implicated in the progression of cancer, may play a contributory role. Hypothesis: Dysregulation of KLF6 signalling contributes to endothelial dysfunction in PAH. Aims: (1) To study KLF6 expression in PAH; (2) To characterise KLF6 effects on endothelial transcriptome and function; (3) To compare KLF6 with KLF2 and KLF4, two closely related transcription factors that are implicated in PAH. Methods: KLF6 expression was measured by qPCR in the lung tissues of hypoxic mice and MCT rats, ECFCs from PAH patients, and HPAECs exposed to hypoxia or TNF-α. Changes in the transcriptomic profile of KLF6-overexpressing HPAEC as well as lung tissues from patients with severe PAH were investigated. Changes in the transcriptional responses of HPAECs overexpressing KLF6 were compared with HPAECs overexpressing KLF2 or KLF4. Results: KLF6 expression was increased by hypoxic and inflammatory triggers in early preclinical PAH. KLF6 expression in HPAECs was upregulated under the “double hit” condition of hypoxia combined with TNF-α, while the expression of KLF2 and KLF4 were reduced. RNA-seq analysis showed that KLF6 unlike KLF2 and KLF4, activates pro- angiogenic signalling and increases the expression of genes that regulate endothelial identity and survival, including BMPR2, SOX17 and ENG. Spatial transcriptomic analysis revealed an increase in the expression of KLF6-regulated genes in the remodelled vessels. KLF6 was upregulated in PAH ECFCs, and an accumulation of KLF6+ ERG+ vWF+ cells was observed in plexiform lesions in human PAH. Conclusion: KLF6 uniquely orchestrates endothelial repair, but its sustained activation in selected subsets of ECs in advanced PAH suggests its potential involvement in the exaggerated repair processes seen in human PAH.