Understanding the effects of oxytocin receptor variants on OXT-OXT receptor binding: A mathematical model.

Approximately half of U.S. women giving birth annually receive Pitocin, the synthetic form of oxytocin (OXT), yet its effective dose can vary significantly. This variability presents safety concerns due to unpredictable responses, which may lead to adverse outcomes for both mother and baby. To address the need for improved dosing, we developed a data-driven mathematical model to predict OXT receptor (OXTR) binding. Our study focuses on five prevalent OXTR variants (V45L, P108A, L206V, V281M, and E339K) and their impact on OXT-OXTR binding dynamics in two distinct cell types: human embryonic kidney cells (HEK293T), commonly used in experimental systems, and human myometrial smooth muscle cells, containing endogenous OXTR. We parameterized the model with cell-specific OXTR surface localization measurements. To strengthen the robustness of our study, we conducted a comprehensive meta-analysis of OXT- OXTR binding, enabling parameterization of our model with cell-specific OXT-OXTR binding kinetics (myometrial OXT-OXTR K d = 1.6 nM, kon = 6.8 × 10 5 M -1 min -1 , and koff = 0.0011 min -1 ). Our meta-analysis revealed significant homogeneity in OXT-OXTR affinity across experiments and species with a K d = 0.52 - 9.32 nM and mean K d = 1.48 ± 0.36 nM. Our model achieves several valuable insights into designing dosage strategies. First, we predicted that the OXTR complex reaches maximum occupancy at 10 nM OXT in myometrial cells and at 1 µM in HEK293T cells. This information is pivotal for guiding experimental design and data interpretation when working with these distinct cell types, emphasizing the need to consider effects for specific cell types when choosing OXTR-transfected cell lines. Second, our model recapitulated the significant effects of genetic variants for both experimental and physiologically relevant systems, with V281M and E339K substantially compromising OXT-OXTR binding capacity. These findings suggest the need for personalized oxytocin dosing based on individual genetic profiles to enhance therapeutic efficacy and reduce risks, especially in the context of labor and delivery. Third, we demonstrated the potential for rescuing the attenuated cell response observed in V281M and E339K variants by increasing the OXT dosage at specific, early time points. Cellular responses to OXT, including Ca 2+ release, manifest within minutes. Our model indicates that providing V281M- and E339K-expressing cells with doubled OXT dose during the initial minute of binding can elevate OXT-OXTR complex formation to levels comparable to wild-type OXTR. In summary, our study provides a computational framework for precision oxytocin dosing strategies, paving the way for personalized medicine.

Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells.

Oxytocin acts through the oxytocin receptor (OXTR) to modulate uterine contractility. We previously identified OXTR genetic variants and showed that, in HEK293T cells, two of the OXTR protein variants localized to the cell surface less than wild-type OXTR. Here, we sought to measure OXTR in the more native human myometrial smooth muscle cell (HMSMC) line on both the cell-surface and across the whole cell, and used CRISPR editing to add an HA tag to the endogenous OXTR gene for anti-HA measurement. Quantitative flow cytometry revealed that these cells possessed 55,000 ± 3200 total OXTRs and 4900 ± 390 cell-surface OXTRs per cell. To identify any differential wild-type versus variant localization, we transiently transfected HMSMCs to exogenously express wild-type or variant OXTR with HA and green fluorescent protein tags. Total protein expression of wild-type OXTR and all tested variants were similar. However, the two variants with lower surface localization in HEK293T cells also presented lower surface localization in HMSMCs. Overall, we confirm the differential surface localization of variant OXTR in a more native cell type, and further demonstrate that the quantitative flow cytometry technique is adaptable to whole-cell measurements.

Toward Blood-Based Precision Medicine: Identifying Age-Sex-Specific Vascular Biomarker Quantities on Circulating Vascular Cells.

Introduction: Abnormal angiogenesis is central to vascular disease and cancer, and noninvasive biomarkers of vascular origin are needed to evaluate patients and therapies. Vascular endothelial growth factor receptors (VEGFRs) are often dysregulated in these diseases, making them promising biomarkers, but the need for an invasive biopsy has limited biomarker research on VEGFRs. Here, we pioneer a blood biopsy approach to quantify VEGFR plasma membrane localization on two circulating vascular proxies: circulating endothelial cells (cECs) and circulating progenitor cells (cPCs). Methods: Using quantitative flow cytometry, we examined VEGFR expression on cECs and cPCs in four age-sex groups: peri/premenopausal females (aged < 50 years), menopausal/postmenopausal females (≥ 50 years), and younger and older males with the same age cut-off (50 years). Results: cECs in peri/premenopausal females consisted of two VEGFR populations: VEGFR-low (~ 55% of population: population medians ~ 3000 VEGFR1 and 3000 VEGFR2/cell) and VEGFR-high (~ 45%: 138,000 VEGFR1 and 39,000-236,000 VEGFR2/cell), while the menopausal/postmenopausal group only possessed the VEGFR-low cEC population; and 27% of cECs in males exhibited high plasma membrane VEGFR expression (206,000 VEGFR1 and 155,000 VEGFR2/cell). The absence of VEGFR-high cEC subpopulations in menopausal/postmenopausal females suggests that their high-VEGFR cECs are associated with menstruation and could be noninvasive proxies for studying the intersection of age-sex in angiogenesis. VEGFR1 plasma membrane localization in cPCs was detected only in menopausal/postmenopausal females, suggesting a menopause-specific regenerative mechanism. Conclusions: Overall, our quantitative, noninvasive approach targeting cECs and cPCs has provided the first insights into how sex and age influence VEGFR plasma membrane localization in vascular cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00771-1.

Axl and Vascular Endothelial Growth Factor Receptors Exhibit Variations in Membrane Localization and Heterogeneity Across Monolayer and Spheroid High-Grade Serous Ovarian Cancer Models.

Vascular endothelial growth factor receptors (VEGFRs) and Axl are receptor tyrosine kinases (RTK) that are targeted in ovarian cancer therapy. Two-dimensional monolayer culture and three-dimensional spheroids are common models for RTK-targeted drug screening: monolayers are simple and economical while spheroids include several genetic and histological tumor features. RTK membrane localization dictates RTK signaling and drug response, however, it is not characterized in these models. We quantify plasma membrane RTK concentrations and show differential RTK abundance and heterogeneity in monolayers versus spheroids. We show VEGFR1 concentrations on the plasma membrane to be 10 times higher in OVCAR8 spheroids than in monolayers; OVCAR8 spheroids are more heterogeneous than monolayers, exhibiting a bimodal distribution of a low-Axl (6200/cell) and a high-Axl subpopulation (25,000/cell). In addition, plasma membrane Axl concentrations differ by 100 times between chemosensitive (OVCAR3) and chemoresistant (OVCAR8) cells and by 10 times between chemoresistant cell lines (OVCAR5 vs. OVCAR8). These systematic findings can guide ovarian cancer model selection for drug screening.

Absolute Quantification of Plasma Membrane Receptors Via Quantitative Flow Cytometry.

Plasma membrane receptors are transmembrane proteins that initiate cellular response following the binding of specific ligands (e.g., growth factors, hormones, and cytokines). The abundance of plasma membrane receptors can be a diagnostic or prognostic biomarker in many human diseases. One of the best techniques for measuring plasma membrane receptors is quantitative flow cytometry (qFlow). qFlow employs fluorophore-conjugated antibodies against the receptors of interest and corresponding fluorophore-loaded calibration beads offers standardized and reproducible measurements of plasma membrane receptors. More importantly, qFlow can achieve absolute quantification of plasma membrane receptors when phycoerythrin (PE) is the fluorophore of choice. Here we describe a detailed qFlow protocol to obtain absolute receptor quantities on the basis of PE calibration. This protocol is foundational for many previous and ongoing studies in quantifying tyrosine kinase receptors and G-protein-coupled receptors with in vitro cell models and ex vivo cell samples.

Pharmacological chaperones for the oxytocin receptor increase oxytocin responsiveness in myometrial cells.

Oxytocin is a potent uterotonic agent administered to nearly all patients during childbirth in the United States. Inadequate oxytocin response can necessitate Cesarean delivery or lead to uterine atony and postpartum hemorrhage. Thus, it may be clinically useful to identify patients at risk for poor oxytocin response and develop strategies to sensitize the uterus to oxytocin. Previously, we showed that the V281M variant in the oxytocin receptor (OXTR) gene impairs OXTR trafficking to the cell surface, leading to a decreased oxytocin response in cells. Here, we sought to identify pharmacological chaperones that increased oxytocin response in cells expressing WT or V281M OXTR. We screened nine small-molecule agonists and antagonists of the oxytocin/vasopressin receptor family and identified two, SR49059 and L371,257, that restored both OXTR trafficking and oxytocin response in HEK293T cells transfected with V281M OXTR. In hTERT-immortalized human myometrial cells, which endogenously express WT OXTR, treatment with SR49059 and L371,257 increased the amount of OXTR on the cell surface by two- to fourfold. Furthermore, SR49059 and L371,257 increased the endogenous oxytocin response in hTERT-immortalized human myometrial cells by 35% and induced robust oxytocin responses in primary myometrial cells obtained from patients at the time of Cesarean section. If future studies demonstrate that these pharmacological chaperones or related compounds function similarly in vivo, we propose that they could potentially be used to enhance clinical response to oxytocin.

Naturally Occurring Genetic Variants in the Oxytocin Receptor Alter Receptor Signaling Profiles.

The hormone oxytocin is commonly administered during childbirth to initiate and strengthen uterine contractions and prevent postpartum hemorrhage. However, patients have wide variation in the oxytocin dose required for a clinical response. To begin to uncover the mechanisms underlying this variability, we screened the 11 most prevalent missense genetic variants in the oxytocin receptor (OXTR) gene. We found that five variants, V45L, P108A, L206V, V281M, and E339K, significantly altered oxytocin-induced Ca2+ signaling or ß-arrestin recruitment and proceeded to assess the effects of these variants on OXTR trafficking to the cell membrane, desensitization, and internalization. The variants P108A and L206V increased OXTR localization to the cell membrane, whereas V281M and E339K caused OXTR to be retained inside the cell. We examined how the variants altered the balance between OXTR activation and desensitization, which is critical for appropriate oxytocin dosing. The E339K variant impaired OXTR activation, internalization, and desensitization to roughly equal extents. In contrast, V281M decreased OXTR activation but had no effect on internalization and desensitization. V45L and P108A did not alter OXTR activation but did impair ß-arrestin recruitment, internalization, and desensitization. Molecular dynamics simulations predicted that V45L and P108A prevent extension of the first intracellular loop of OXTR, thus inhibiting ß-arrestin binding. Overall, our data suggest mechanisms by which OXTR genetic variants could alter clinical response to oxytocin.

Systems Biology Will Direct Vascular-Targeted Therapy for Obesity.

Healthy adipose tissue expansion and metabolism during weight gain require coordinated angiogenesis and lymphangiogenesis. These vascular growth processes rely on the vascular endothelial growth factor (VEGF) family of ligands and receptors (VEGFRs). Several studies have shown that controlling vascular growth by regulating VEGF:VEGFR signaling can be beneficial for treating obesity; however, dysregulated angiogenesis and lymphangiogenesis are associated with several chronic tissue inflammation symptoms, including hypoxia, immune cell accumulation, and fibrosis, leading to obesity-related metabolic disorders. An ideal obesity treatment should minimize adipose tissue expansion and the advent of adverse metabolic consequences, which could be achieved by normalizing VEGF:VEGFR signaling. Toward this goal, a systematic investigation of the interdependency of vascular and metabolic systems in obesity and tools to predict personalized treatment ranges are necessary to improve patient outcomes through vascular-targeted therapies. Systems biology can identify the critical VEGF:VEGFR signaling mechanisms that can be targeted to regress adipose tissue expansion and can predict the metabolic consequences of different vascular-targeted approaches. Establishing a predictive, biologically faithful platform requires appropriate computational models and quantitative tissue-specific data. Here, we discuss the involvement of VEGF:VEGFR signaling in angiogenesis, lymphangiogenesis, adipogenesis, and macrophage specification - key mechanisms that regulate adipose tissue expansion and metabolism. We then provide useful computational approaches for simulating these mechanisms, and detail quantitative techniques for acquiring tissue-specific parameters. Systems biology, through computational models and quantitative data, will enable an accurate representation of obese adipose tissue that can be used to direct the development of vascular-targeted therapies for obesity and associated metabolic disorders.