Pregnancy-Specific Glycoprotein 9 Enhances Store-Operated Calcium Entry and Nitric Oxide Release in Human Umbilical Vein Endothelial Cells

**Background:** Preeclampsia is a hypertensive disorder of pregnancy affecting 2–8% of pregnancies globally, characterized by systemic vasospasm and vascular endothelial injury. Pregnancy-specific glycoproteins (PSGs) are secreted mainly from trophoblasts, with PSG9 known to promote angiogenesis. However, the association between PSG9 and preeclampsia, and the underlying mechanisms involving endothelial function, remained unclear. Store-operated Ca²⁺ entry (SOCE) mediated by Orai channel proteins is a key pathway for Ca²⁺ influx that can induce nitric oxide (NO) production from endothelial cells, but whether PSG9 regulates Orai channels and NO release was unknown.

**Methods:** Serum samples were collected from 4 patients with preeclampsia and 4 healthy pregnant controls at the Maternal and Child Health Hospital affiliated with Anhui Medical University (December 2018–September 2020). Preeclampsia diagnosis followed 2019 ACOG guidelines. PSG9 levels were measured by Western blotting. In vitro experiments used HUVECs treated with PBS or PSG9 (0.1 or 0.5 μg/mL) for 24 h. Western blotting assessed Orai1, Orai2, Orai3, and eNOS expression. SOCE was measured using Fluo-8/AM fluorescence after thapsigargin (2 μM)-induced Ca²⁺ store depletion in Ca²⁺-free solution, followed by addition of 2 mM Ca²⁺. NO production was measured using DAF-FM DA fluorescence probe. The Orai inhibitor BTP2 (5 μM) was used in blocking experiments. Statistical analysis used two-tailed Mann–Whitney test or two-way ANOVA with Games–Howell post hoc tests; p < 0.05 considered significant.

**Key Results:** Clinical data showed preeclampsia patients had significantly higher systolic blood pressure (167.50 ± 14.52 vs. 120.00 ± 8.04 mmHg, p < 0.05) and diastolic blood pressure (105.00 ± 11.28 vs. 74.5 ± 9.47 mmHg, p < 0.05) than controls, with no significant differences in age or gestational weeks. Western blotting confirmed serum PSG9 levels were significantly decreased in preeclampsia patients compared to controls (p < 0.05). In HUVECs, PSG9 (0.1 μg/mL) significantly enhanced Orai1 and Orai2 expression (p < 0.05) but not Orai3 (no significant difference). PSG9 treatment markedly increased SOCE evoked by TG-induced Ca²⁺ store depletion, and this increase was nearly completely abolished by BTP2 pretreatment (p < 0.05 for PSG9 vs. PBS; p < 0.05 for PSG9+BTP2 vs. PSG9). PSG9 at 0.1 μg/mL significantly enhanced eNOS expression compared to PBS (p < 0.05), but this effect was not observed at 0.5 μg/mL. BTP2 completely abolished the PSG9-induced eNOS enhancement. NO fluorescence intensity was significantly increased in PSG9-treated cells compared to PBS controls (p < 0.05), and BTP2 significantly abolished this PSG9-enhanced NO production (p < 0.05 for PSG9+BTP2 vs. PSG9).

**Clinical Implications:** This study demonstrates that PSG9 levels are reduced in preeclampsia and that PSG9 can enhance NO release from endothelial cells via Orai1/Orai2-mediated SOCE and the Ca²⁺/eNOS/NO signaling pathway. These findings suggest PSG9 may play a protective role in maintaining endothelial function during pregnancy, and its deficiency could contribute to the vascular dysfunction characteristic of preeclampsia. The results indicate PSG9 may warrant further investigation as a potential biomarker or therapeutic target for preeclampsia. However, the study has limitations including small sample size (n=4 per group), use of HUVECs rather than uterine artery endothelial cells, lack of resting [Ca²⁺]ᵢ measurements, and the authors acknowledge conflicting reports (Kandel et al. found increased PSG9 in preeclampsia), possibly due to racial, methodological, or sample differences. Future studies should examine PSG9 function in trophoblasts and assess resting [Ca²⁺]ᵢ.