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

Cell surface expression of VEGFR1 and VEGFR2.

<p>(A) Receptor levels from mouse ex vivo endothelial cells, human in vitro endothelial cells and mouse in vitro fibroblasts are plotted on a log-scale. Mouse endothelial cells freshly isolated from skeletal muscle have an average surface expression of 2,800 VEGFR1/endothelial cell and 1,600 VEGFR2/endothelial cell, representing significantly higher VEGFR1 relative to VEGFR2 (p<0.001). Cultured, human endothelial cells display 1,800 VEGFR1/endothelial cell and 5,800 VEGFR2/endothelial cell, a significant difference in surface expression (p<0.001). The fibroblasts have an average surface expression of 36,000 VEGFR1/fibroblast and 700 VEGFR2/fibroblast, representing significant differences between VEGFR1 and VEGFR2 surface levels (p<0.001). (B) Endothelial cells from C57BL/6 have an average endothelial surface expression of 2,000 VEGFR1/cell and 1,300 VEGFR2/cell within the gastrocnemius and 3,200 VEGFR1/cell and 1,700 VEGFR2/cell within the tibialis anterior. BALB/c have an average endothelial surface expression of 2,600 VEGFR1/cell and 1,600 VEGFR2/cell within the gastrocnemius and 3,700 VEGFR1/cell and 2,000 VEGFR2/cell within the tibialis anterior.</p

Expression of VEGF Receptors on Endothelial Cells in Mouse Skeletal Muscle

<div><p>VEGFR surface localization plays a critical role in converting extracellular VEGF signaling towards angiogenic outcomes, and the quantitative characterization of these parameters is critical for advancing computational models; however the levels of these receptors on blood vessels is currently unknown. Therefore our aim is to quantitatively determine the VEGFR localization on endothelial cells from mouse hindlimb skeletal muscles. We contextualize this VEGFR quantification through comparison to VEGFR-levels on cells in vitro. Using quantitative fluorescence we measure and compare the levels of VEGFR1 and VEGFR2 on endothelial cells isolated from C57BL/6 and BALB/c gastrocnemius and tibialis anterior hindlimb muscles. Fluorescence measurements are calibrated using beads with known numbers of phycoerythrin molecules. The data show a 2-fold higher VEGFR1 surface localization relative to VEGFR2 with 2,000–3,700 VEGFR1/endothelial cell and 1,300–2,000 VEGFR2/endothelial cell. We determine that endothelial cells from the highly glycolytic muscle, tibialis anterior, contain 30% higher number of VEGFR1 surface receptors than gastrocnemius; BALB/c mice display ∼17% higher number of VEGFR1 than C57BL/6. When we compare these results to mouse fibroblasts in vitro, we observe high levels of VEGFR1 (35,800/cell) and very low levels of VEGFR2 (700/cell), while in human endothelial cells in vitro, we observe that the balance of VEGFRs is inverted, with higher levels VEGFR2 (5,800/cell) and lower levels of VEGFR1 (1,800/cell). Our studies also reveal significant cell-to-cell heterogeneity in receptor expression, and the quantification of these dissimilarities ex vivo for the first time provides insight into the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) signaling.</p> </div

Dissociation enzymes.

<p>0.2% collagenase or 0.2% dispase was applied to HUVECs for 30 min at 37°C with intermittent vortexing to determine whether VEGFR1, VEGFR2, and NRP1 surface levels are sensitive to dissociation enzymes. Collagenase 4 did not affect VEGFR1 or VEGFR2 density. Each enzyme affected NRP1 density.</p

<b>Surface receptor statistics.</b>

<p>Endothelial cells from gastrocnemius (mixed muscle) and tibialis anterior (white/glycolytic muscle) were isolated from 31 C57BL/6 and 20 BALB/c mice. 3T3 fibroblasts, obtained from ATCC, represent a mouse in vitro sample.</p

Cell-by-cell analysis of VEGFR1 and VEGFR2 distribution on ex vivo and in vitro cells.

<p>(A) Human and mouse endothelial cells display similar distributions of VEGFR1, the magnified traces show the distributions at the range encompassing a majority of the endothelial cells expressing VEGFR1, and it shows that the histograms overlap considerably, while the higher-VEGFR1-expressing fibroblasts have a much broader distribution of VEGFR1. (B) The histograms representing VEGFR2 levels on human in vitro endothelial cells, mouse ex vivo endothelial cells, and mouse in vitro fibroblasts have distinct profiles. The mouse endothelial cells have a large population expressing low levels of VEGFR2, and a broader range of cells expressing high levels of VEGFR2 relative to the mouse fibroblasts, which are more homogenous in their surface expression of VEGFR2. The human endothelial cells have much higher numbers of VEGFR2 on the cell surface, and the greatest heterogeneity in endothelial, VEGFR2 surface numbers.</p

Cell-by-cell analysis of VEGFR1 and VEGFR2 distribution on mouse endothelial cells.

<p>The distributions show that there is significant heterogeneity in endothelial surface expression of VEGFRs, with 90% of endothelial cells expressing up to 10,000 receptors. The distributions also reveal a significant population of endothelial cells expressing low numbers of VEGFR2 (≤2,500 receptors/cell), thus lowering the average number of VEGFR2/cell relative to VEGFR1. (A) C57BL/6 gastrocnemius, (B) C57BL/6 tibialis anterior, (C) BALB/c gastrocnemius, (D) BALB/c tibialis anterior.</p

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

Plasmonic Optical Trapping in Biologically Relevant Media

<div><p>We present plasmonic optical trapping of micron-sized particles in biologically relevant buffer media with varying ionic strength. The media consist of 3 cell-growth solutions and 2 buffers and are specifically chosen due to their widespread use and applicability to breast-cancer and angiogenesis studies. High-precision rheological measurements on the buffer media reveal that, in all cases excluding the 8.0 pH Stain medium, the fluids exhibit Newtonian behavior, thereby enabling straightforward measurements of optical trap stiffness from power-spectral particle displacement data. Using stiffness as a trapping performance metric, we find that for all media under consideration the plasmonic nanotweezers generate optical forces 3–4x a conventional optical trap. Further, plasmonic trap stiffness values are comparable to those of an identical water-only system, indicating that the performance of a plasmonic nanotweezer is not degraded by the biological media. These results pave the way for future biological applications utilizing plasmonic optical traps.</p></div