Post-Transcriptional Silencing of PHD2 by CRISPR-Cas13d Enhances Pro-Angiogenic Signaling in Human Endothelial Cells
Sara Carolina Vieira Terra, Cynthia Silvia Bartolomeo, Sofia Mila Cantu de Sales, Daniel Casartelli de Santa Inez, Davi Coe Torres, Ricardo Weinlich, Roberta Sessa StilhanoIntroduction:
Peripheral artery disease (PAD) reduces tissue perfusion and causes ischemia, conditions in which hypoxia-responsive pathways have an important adaptive role. Hypoxia- inducible factor 1-alpha (HIF-1α), a central regulator of this response, is tightly controlled by prolyl hydroxylase domain protein 2 (PHD2), encoded by EGLN1. Here, we used an RNA-targeting CRISPR-Cas13d system to downregulate EGLN1 in human umbilical vein endothelial cells (HUVECs) under normoxia and hypoxia.
Methods:
A PHD2-knockdown (PHD2-KD) HUVEC line was generated by lentiviral delivery of an EGLN1-targeting RNA guide, alongside non-transduced (CTRL) and non-targeting gRNA (NTG) control lines. Molecular and functional assays assessed hypoxia-related signaling, metabolic activity, migration, and sprouting responses under normoxia and hypoxia.
results:
PHD2-KD HUVECs showed significantly increased VEGFA expression under both normoxia and hypoxia, at the mRNA level (7.0-fold and 5.4-fold, respectively) and protein level (6.6-fold and 9.4-fold, respectively). At early time points (3–12 h), HIF-1α gene and protein levels were elevated, accompanied by reduced inflammatory cytokine levels, with TNF-α becoming undetectable after 48 h. Functionally, PHD2-KD cells showed enhanced proliferation, faster wound closure (2.0-fold under normoxia and 2.2-fold under hypoxia), and increased sprouting activity, with up to 5.4-fold more sprouts per bead and sprouts up to 2.5-fold longer than controls.
Results:
Western blotting confirmed a marked reduction in PHD2 protein abundance in PHD2-KD cells relative to both CTRL and NTG cells. PHD2-KD HUVECs exhibited increased VEGFA transcript levels (6.5-fold under normoxia and 5.3-fold under hypoxia) and secreted VEGF concentrations (6.6-fold and 9.4-fold, respectively), together with early increases in HIF1A transcript and HIF-1α protein levels and lower TNF-α concentrations. Functional assays showed higher metabolic activity, faster wound closure (2.0-fold under normoxia and 2.2-fold under hypoxia), and enhanced sprouting, with up to 7.2-fold more sprouts per bead and sprouts up to 2.7-fold longer than those formed by CTRL.
Discussion:
CRISPR-Cas13d-mediated EGLN1 targeting reduced PHD2 protein abundance and was associated with higher endothelial metabolic activity, faster wound closure, enhanced sprouting behavior, and lower TNF-α secretion.
Conclusion:
CRISPR-Cas13d-mediated downregulation of EGLN1/PHD2 was associated with increased pro-angiogenic signaling and improved angiogenesis-related endothelial responses in vitro, supporting RNA-targeting strategies to modulate hypoxia-responsive pathways.