(20) HIF-1α mediates hypoxia-induced angiogenic and fibrotic phenotypes in uterine fibroid cells

Md Soriful Islam¹, Valeria I. Macri¹, Eslam E. Abd El-Fattah¹, Joscelyn Mejias², Rachel Michel¹, Samya El Sayed¹, Casie Ingley¹, Bhuchitra Singh¹, Joshua T. Brennan¹, Mostafa A. Borahay¹, Jennifer H. Elisseeff², James H. Segars¹

¹Department of Gynecology and Obstetrics, Division of Reproductive Sciences & Women’s Health Research, Johns Hopkins Medicine, Baltimore, Maryland 21205, USA.

²Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA

Abstract Text:

Uterine fibroids, the most common benign tumors in reproductive-age women, exhibit dysregulated states of fibrosis and angiogenesis. Hypoxia, a defining feature of the fibroid microenvironment, is suspected to drive these pathological processes, yet the underlying molecular mechanisms remain unclear. HIF-1α, a transcription factor activated under low-oxygen conditions, may regulate genes involved in angiogenesis and fibrosis. However, its precise role in hypoxia-mediated fibroid pathogenesis remains unexplored. We hypothesized that hypoxia activates HIF-1α signaling, leading to enhanced angiogenic and fibrotic responses in fibroid cells. To test this hypothesis, we examined the effects of hypoxia and HIF-1α inhibition on angiogenic and fibrotic markers in uterine fibroid cells.

Primary uterine fibroid cells and P57 immortalized fibroid cells were cultured under hypoxic (1% O₂) and normoxic (20% O₂) conditions for 48 hours. Cells were treated with the HIF-1α inhibitor KC7F2 to assess its role in hypoxia-mediated responses. Protein levels of HIF-1α were quantified by western blotting. Angiogenic (ADM, ET1, VEGF, PDGF) and fibrotic (COL1A1, COL5A1, LOX1, P4HA1, POSTN, TNC) gene expression was analyzed by qRT-PCR.

Fibroid cells under hypoxia exhibited a significant increase in angiogenic gene expression. ADM and ET1 mRNA levels were upregulated by 1.6-fold and 1.5-fold, respectively. Hypoxia also increased the expression of VEGFA (2.2-fold), VEGFB (1.3-fold), and VEGFC (1.4-fold). Similarly, PDGFB (1.6-fold) and PDGFC (1.4-fold) were elevated, while PDGFA remained unchanged. HIF-1α protein levels were significantly increased (3.2-fold) under hypoxia, suggesting its activation in response to low oxygen. To determine whether HIF-1α mediates hypoxia-induced angiogenesis, cells were treated with KC7F2, which effectively reduced HIF-1α protein levels. KC7F2 treatment significantly downregulated hypoxia-induced ADM and ET1 expression. Similarly, VEGFB and VEGFC levels were decreased, while VEGFA remained unaffected. Among PDGF isoforms, KC7F2 reduced PDGFB expression (2.6-fold under hypoxia). These findings suggest that HIF-1α plays a central role in hypoxia-driven angiogenesis in fibroid cells. To investigate the impact of hypoxia on fibrosis, fibroid cells were exposed to 1% O₂ for 48 hours. Hypoxia induced changes in collagen expression. COL1A1 (1.6-fold) and COL5A1 (1.7-fold) mRNA levels were significantly upregulated, while COL3A1 remained unchanged. Additionally, hypoxia increased the expression of LOX1 (1.6-fold), P4HA1 (1.9-fold), and P4HA2 (1.9-fold), enzymes involved in collagen synthesis and cross-linking. Fibrotic remodeling markers POSTN and TNC were modestly upregulated (1.2-fold each). To assess the role of HIF-1α in hypoxia-driven fibrosis, fibroid cells were treated with KC7F2. Collagen and matrix remodeling genes were affected by HIF-1α inhibition. KC7F2 treatment significantly reduced COL1A1 and COL3A1 mRNA levels but had no effect on COL5A1. Hypoxia-induced LOX1 expression was diminished by KC7F2, whereas P4HA1 levels were not significantly altered. KC7F2 also decreased the expression of POSTN (1.7-fold) and TNC (1.3-fold) under hypoxia. These findings indicate that HIF-1α is an important mediator of hypoxia-induced fibrosis in fibroid cells.

Our findings demonstrate that hypoxia promotes angiogenic and fibrotic phenotypes in uterine fibroid cells via HIF-1α activation. Targeting HIF-1α may serve as a potential therapeutic strategy to regulate hypoxia-driven fibrosis and angiogenesis in fibroids.

Support: This research was supported by SRI and Bayer Discovery/Innovation Grant and, in part, by R01HD111243 and R01HD100365.