Prolyl 3-hydroxylase 2 (P3H2) catalyzes the post-translational formation of 3-hydroxyproline on collagens, mainly on type IV. Its activity has never been directly associated to angiogenesis. Here, we identified P3H2 gene through a deep-sequencing transcriptome analysis of human umbilical vein endothelial cells (HUVECs) stimulated with vascular endothelial growth factor A (VEGF-A). Differently from many previous studies we carried out the stimulation not on starved HUVECs, but on cells grown to maintain the best condition for their in vitro survival and propagation. We showed that P3H2 is induced by VEGF-A in two primary human endothelial cell lines and that its transcription is modulated by VEGF-A/VEGF receptor 2 (VEGFR-2) signaling pathway through p38 mitogen-activated protein kinase (MAPK). Then, we demonstrated that P3H2, through its activity on type IV Collagen, is essential for angiogenesis properties of endothelial cells in vitro by performing experiments of gain- and loss-of-function. Immunofluorescence studies showed that the overexpression of P3H2 induced a more condensed status of Collagen IV, accompanied by an alignment of the cells along the Collagen IV bundles, so towards an evident pro-angiogenic status. Finally, we found that P3H2 knockdown prevents pathological angiogenesis in vivo, in the model of laser-induced choroid neovascularization. Together these findings reveal that P3H2 is a new molecular player involved in new vessels formation and could be considered as a potential target for anti-angiogenesis therapy.

Prolyl 3-hydroxylase 2 (P3H2) is a key enzyme involved in the architecture of the extracellular matrix (ECM). While previously shown to be regulated by VEGF-A and to play a role in angiogenesis, its function in cancer remains ambiguous. While characterized as a tumor suppressor, its precise function in colorectal cancer (CRC) progression is poorly defined. Bioinformatic analysis and patient data reveal that P3H2 transcript levels are significantly reduced in colon adenocarcinoma tissues, showing a progressive decline in metastatic lesions. Furthermore, VEGF-A exposure upregulates P3H2 transcripts in the HCT116 CRC cell line. To investigate its impact in CRC, we generated a stable HCT116 clone overexpressing P3H2. In vitro studies demonstrated that while P3H2 overexpression inhibited anchorage-independent growth, it significantly enhanced cellular invasion without altering cell proliferation. In vivo, however, P3H2-overexpressing tumors exhibited accelerated tumor growth and a statistically significant increase in lung metastases. P3H2 overexpression remodeled the tumor microenvironment (TME) by modifying its main substrate, Collagen IV, resulting in the induction of increased vessels density. Our study repositions P3H2 as a dynamic enzymatic switch within the TME. This work identifies P3H2-driven ECM remodeling as a promising therapeutic axis in advanced CRC, with particular relevance for combination strategies targeting angiogenesis.