MiR-34a promotes choroidal neovascularization through modulating the KLF2/CXCR4 axis

Abstract

Purpose : Choroidal neovascularization (CNV) is the aberrant growth of blood vessels beneath the retina that characterizes the most severe form of age-related macular degeneration (AMD). A handful of microRNAs become dysregulated in ocular tissue during the progression of AMD and of these miR-34a may play a significant role due to its involvement in many gene pathways relating to inflammation, angiogenesis, and cellular senescence. Thus, we hypothesized that the upregulation of miR-34a in ocular tissue promotes CNV.

Methods : For this investigation, we used a well-established mouse model of laser induced CNV. Ocular tissue sections containing CNV lesions were stained via in-situ hybridization (ISH) and immunofluorescence (IF) to identify regions that express miR-34a. Female mice deficient for miR-34a and wild-type (WT) littermates were used for CNV lesion size measurements and transcriptomic analyses. Human umbilical vein endothelial cells (HUVECS) were used in several cell culture and transcriptomic assays to investigate the cellular and molecular mechanisms by which miR-34a promotes CNV.

Results : Upregulation of miR-34a coincides with CNV and its expression co-localizes with vascular endothelial cells. Lesion sizes of miR-34a^-/- mice were significantly reduced compared to WT controls (p=0.003). Bulk-cell RNA-seq of lasered choroids showed 44 genes related to angiogenesis were dysregulated due to miR-34a (|fc|>1.2). In vitro analyses of HUVECs treated with a miR-34a mimic showed increased proliferation (p=0.03), migration (p=0.0025), branch length (p=0.034), tube length (p=0.019), and branch number (p=0.035). Transcriptomic analyses of HUVECs treated with a miR-34a mimic and lasered mouse choroids (Rohlenova et al. 2020) identified KLF2 as a downstream target of miR-34a in CNV. CXCR4, a known target of KLF2, is upregulated in HUVECs after transfection with miR-34a mimic (p<0.001). IF staining revealed upregulation of KLF2 (p=0.004) and downregulation of CXCR4 (p=0.006) in the absence of miR-34a.

Conclusions : MiR-34a is upregulated in CNV and promotes angiogenesis. The potent anti-angiogenic transcription factor KLF2 is downregulated by miR-34a in choroidal vascular endothelial cells. Additionally, the pro-angiogenic factor CXCR4, known to be transcriptionally inhibited by KLF2, is upregulated in the presence of miR-34a. Thus, miR-34a may promote CNV in wet AMD through modulating the KLF2/CXCR4 axis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.