Targeting YAP signaling attenuates hypoxia-induced angiogenic and fibrotic responses in uterine fibroid cells

Introduction: : Uterine fibroids (leiomyomas) are the most common benign tumors in women of reproductive age and are characterized by excessive extracellular matrix (ECM) deposition and altered vascularization. Despite their high prevalence and associated morbidity, effective non-hormonal treatment options remain limited. Hypoxia is a prominent feature of the fibroid microenvironment and is thought to drive both fibrotic and angiogenic processes. However, the molecular mechanisms linking hypoxia to these pathological features remain poorly defined. Yes-associated protein (YAP), a transcriptional coactivator regulated by mechanical and environmental cues, is implicated in tissue fibrosis and tumor growth. Although YAP is overexpressed in fibrotic conditions, including uterine fibroids, its functional role in hypoxia-induced remodeling has not been fully elucidated. Preliminary evidence suggests that YAP activation may integrate hypoxic signaling with downstream pro-fibrotic and pro-angiogenic gene expression. In this study, we examined the role of YAP in mediating angiogenic and fibrotic responses to hypoxia in uterine fibroid cells. Our findings revealed that hypoxia activated YAP signaling, leading to upregulation of angiogenic and fibrotic markers. Inhibition of YAP suppressed these hypoxia-induced effects, supporting the therapeutic potential of targeting the hypoxia-YAP signaling axis in uterine fibroids.

Materials and

Methods: : Primary uterine fibroid cells were isolated from surgical tissue specimens obtained from three patients (n = 3). Fresh fibroid samples were collected immediately after surgery under approved IRB protocols. Primary cultures were established using a combination of mechanical dissociation and enzymatic digestion with collagenase and DNase. Early passage cells (passages 1–5) were used for all experiments. Fibroid cells were cultured under either normoxic (20% O₂) or hypoxic (1% O₂) conditions for 48 hours. To investigate the role of YAP in hypoxia-induced angiogenic and fibrotic responses, cells were treated with verteporfin (1 µM), a pharmacological inhibitor of YAP signaling, at a concentration previously validated for functional inhibition. Protein expression levels of YAP, the YAP target connective tissue growth factor (CTGF), and the fibrotic marker plasminogen activator inhibitor-1 (PAI-1) were quantified by western blotting. Expression of hypoxia-inducible factor 1-alpha (HIF-1α) was assessed by both western blotting and immunofluorescence. Gene expression of angiogenic markers (ADM, ET1, VEGFA, VEGFB, VEGFC, PDGFA, PDGFB, PDGFC) and fibrotic markers (COL1A1, COL3A1, COL5A1, LOX1, P4HA1, VCAN, FN1) was assessed by real-time PCR. Gene expression levels were normalized to the housekeeping gene ARF1 and analyzed using the Delta-Delta CT method. Experiments were conducted with 3–9 biological replicates from independent cell cultures. Statistical analyses were performed using GraphPad Prism 10 (version 10.2.0, build 392). Data normality was assessed using the Shapiro-Wilk test. Differences among groups were analyzed by ordinary one-way ANOVA or the Brown-Forsythe ANOVA test based on normality, with significance set at p ≤ 0.05.

Results, Conclusions, and Discussions:: Exposure of uterine fibroid cells to hypoxia (1% O₂ for 48 hours) increased the activation of YAP signaling, as evidenced by elevated levels of active YAP protein and its downstream target CTGF (Fig 1A-C). Treatment with the YAP inhibitor verteporfin significantly reduced hypoxia-induced YAP and CTGF expression (Fig 1A-C), supporting a role for YAP in mediating hypoxic responses. To determine whether YAP regulates angiogenesis, we assessed HIF-1α levels following verteporfin treatment. Hypoxia markedly elevated HIF-1α protein levels (Fig 1D-E), which were substantially reduced by verteporfin (Fig 1D-E), as confirmed by both western blotting and immunofluorescence. Hypoxia also increased the expression of multiple angiogenic factors, including ADM, ET1, and several VEGF and PDGF isoforms (Fig 1F), all of which were significantly suppressed by verteporfin treatment (Fig 1F), implicating YAP signaling in the regulation of angiogenesis. In addition to promoting angiogenic responses, hypoxia induced a fibrotic phenotype, characterized by upregulation of PAI-1 (Fig 1G-H), specific collagen subtypes (COL1A1 and COL5A1), and collagen-processing enzymes such as LOX1 and P4HA1 (Fig 1I). Verteporfin treatment attenuated the expression of these fibrotic markers (Fig 1G-I), suggesting that YAP contributes to hypoxia-driven extracellular matrix remodeling. Furthermore, fibrotic ECM components, including fibronectin (FN1) and the proteoglycan versican (VCAN) (Fig 1I), were elevated under hypoxia and reduced following YAP inhibition (Fig 1I).
Together, these findings establish YAP as a key regulator of hypoxia-induced angiogenesis and fibrosis in uterine fibroid cells. These results provide new mechanistic insight into fibroid biology and support further investigation of YAP-targeted strategies as potential therapeutic approaches for fibroid treatment.

Acknowledgements and/or References (Optional):: NIH grant R01HD111243 to JS, MAB, JHE.