Introduction Pulmonary hypertension (PH) represents the vascular response to high blood pressure and is characterized by endothelial cell (EC) dysfunction, hyperproliferation of smooth muscle cells and inflammation, ultimately leading to vascular obstruction. Until now, treatment options are limited and focused solely on alleviating symptoms caused by right ventricular dysfunction. Krüppel like factor 4 (KLF4) is a transcription factor modulating anti-inflammatory responses in ECs and maintaining mitochondrial function under hypoxic conditions.

Purpose Our aim is to define a novel treatment option for pulmonary hypertension by targeted adeno-associated virus (AAV)-mediated overexpression of KLF4 in lung endothelial cells and its validation in a mouse model of the disease.

Methods KLF4 overexpression in ECs was achieved in endothelial cells by AAV9SLR application. Cells were further subjected to 1% O₂ for 2 days. Tight junction integrity was assessed by immunocytochemistry. Markers of endothelial-to-mesenchymal transition were measured by RT qPCR. In our in vivo study, mice were injected with AAV2-ESGHFY overexpressing KLF4 or EGFP as control two weeks after hypoxia induction. After 3 weeks, right ventricular function was measured by echocardiography, while right ventricular systolic pressure (RVSP) was assessed by right heart catheterization. Markers of pulmonary fibrosis were measured by RT qPCR and Sirius Red staining.

Results KLF4 overexpression led to improved tight junction expression and decreased mesenchymal markers in ECs in vitro. Moreover, the expression of pro-inflammatory markers was significantly decreased upon KLF4 overexpression. Mice subjected to AAV-KLF4 injection presented with a significantly improved right ventricular function and decreased RVSP. Importantly, we could detect amelioration of hypoxia-induced extracellular matrix deposition.