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

Quantitative Flow Cytometry-Guided Protein Biomarker Characterization for Translational Proteomics and Precision Medicine

Angiogenesis, the growth of blood vessels from pre-existing vasculature, is primarily regulated by vascular endothelial growth factor receptors (VEGFRs). Dysregulated angiogenesis is associated with cancers, obesity, and over 70 vascular diseases. Upregulated VEGFR protein expressions in diseased vasculature are promising biomarkers for predicting clinical outcomes, as indicated by non-quantitative immunohistochemical studies in patients with impaired vascularization or tumor angiogenesis. While the quantitative characterization of VEGFRs is critical in identifying biomarkers for anti-angiogenic therapies, VEGFR biomarker development presents two particular challenges: (1) The invasive tissue biopsy needed limits the amount of VEGFR data that can be collected from both normal and diseased vasculatures, and (2) we poorly understand the significance of endothelial and various non-endothelial VEGFR-expressing cells in angiogenic therapies. To address these challenges, here I pioneer a blood biopsy-based proteomic approach that allows non-invasive VEGFR quantification. More significantly, I identify and establish age- and sex-specific basal levels of VEGFRs on endothelial cells and bone marrow-derived progenitor cells (Chapter 2). In recent years, blood biopsies have expanded our knowledge of vascular pathology. In particular, circulating angiogenic cells, such as circulating endothelial cells (cECs) and circulating progenitor cells (cPCs), are isolated and counted, and their elevated abundances are often correlated with vascular disease progression and cancer prognosis. However, cECs and cPCs have been overlooked as accessible proxies for profiling vascular biomarker expressions by activated or damaged vasculatures. For the first time, I show that cPCs and cECs exhibit heterogeneous plasma membrane expression of VEGFRs, which are correlated with donor sexes and ages, particularly pre- vs. post-menopausal status. Menopause is known to reduce regenerative and angiogenic capacities, as manifested by decreased capillary growth in skeletal muscle and increased risks for cardiovascular diseases. Here I provide baseline VEGFR expression ranges for these cells, showing that ~50% of cECs in premenopausal females exhibit intermediate-to-high plasma membrane expression (138,000 VEGFR1 and 39,000-236,000 VEGFR2/cell) and ~25% of cECs in males exhibit high VEGFR plasma membrane expression (206,000 VEGFR1 and 155,000 VEGFR2/cell). In marked contrast, nearly all cECs in postmenopausal females are VEGFR-low (2,900 VEGFR1 and 3,400 VEGFR2/cell), agreeing with the reduced angiogenic capacities after menopause. Additionally, VEGFR1 signaling is critical for cPC localization to activated or damged blood vessels. My data show that VEGFR1 plasma membrane localization in cPCs occurs only in postmenopausal females, suggesting menopause activates VEGFR1 signaling pathways in cPCs. Therefore, my data offer quantitative insights into how VEGFR-regulated regenerative and angiogenic capacities are altered due to menopause. Overall, these findings provide the first insights into how sex and age interactions, particularly menopause, influence VEGFR plasma membrane localization in circulating angiogenic cells. More importantly, the findings help establish age- and sex-specific VEGFR baselines for predicting vascular disease progression and therapeutic outcomes. The second challenge is quantitatively characterize how endothelial and non-endothelial VEGFR-expressing cells contribute to angiogenic regulation. Here, I quantitatively elucidate the changes in VEGFR expressions by endothelial cells and non-enodthelial cells in adipose tissues, and identify biomarkable adipose tissue cells that show altered VEGFR membrane expressions in normal versus high-adiposity states (Chapter 3). Obesity is a major risk factor for vascular disorders, including peripheral artery disease, critical limb ischemia, and several cancers. I hypothesize that VEGFR membrane expression by adipose tissue cells is altered as body fat accumulates (increased adiposity). The VEGFR quantification data presented here indicate that ~ 20% of activated lymphocytes upregulate their membrane expressions of VEGFR1 and VEGFR3 by tenfold in response to increased subcutaneous adiposity induced by lipedema, which is very commonly accompanied by impaired vascularization and chronic inflammation. On the other hand, in murine visceral adipose tissue, myeloid progenitor cells exhibit the highest VEGFR membrane expressions (16,000 ± 4,700 VEGFR1, 50,000 ± 6,200 VEGFR2, and 2,100 ± 460 VEGFR3/cell). Compared to myeloid progenitor cells, visceral endothelial cells exhibit an order of magnitude lower VEGFR1 and VEGFR2 levels (2,400 ± 710 VEGFR1/cell, 1,100 ± 190 VEGFR2/cell, and 1,200 ± 220 VEGFR3/cell, respectively). My approach and findings are foundational to a systematic understanding of how VEGFR-expressing adipose cells regulate adipose angiogenesis and adipogenesis. Future studies are warranted to compare how VEGFR membrane expressions differ in chow-fed and high fat-fed mice, and the quantitative proteomic findings will guide therapies for visceral obesity-associated vascular disorders. Last but not least, unlike VEGFRs, many receptors of clinical interest, particularly the oxytocin receptor (OXTR) and its genetic variants, do not have specific antibodies that enable quantitative characterization. To overcome this issue, I have designed a transfected cell model that is engineered to express HA-OXTR-GFP protein complexes, in which an N-terminal HA acts as a proxy for membrane OXTR detection and a C-terminal GFP acts as an indicator in selecting transfected cells from untransfected cells (Chapter 4). This transfected cell model is applied to characterize the varied dose-response profiles of OXTR wild-type and variant cells to oxytocin, a common labor induction drug. My OXTR quantification data show clear correlations to oxytocin-induced functional outcomes, including calcium release and cell desensitization, suggesting that the quantities of different OXTR variants are predictive of cell responses to administered oxytocin and should be considered when making personalized oxytocin dosing decisions. Overall, my results demonstrate that membrane expression of VEGFRs is significantly associated with physiological factors such as sex, age, and menopause, and with pathological adipose tissue expansion. Although VEGFR protein expression is a promising biomarker for many vascular diseases and cancers, quantitative and baseline VEGFR data are still needed for VEGFR-driven pathology. My work on both VEGFRs and other biomarkable receptors, such as OXTR, provides much-needed standardized approaches and quantitative data, a first step towards proteomic biomarker-driven precision medicine

All-Weather and Superpixel Water Extraction Methods Based on Multisource Remote Sensing Data Fusion

The high spatial and temporal resolution of water body data offers valuable guidance for disaster monitoring and assessment. These data can be employed to quickly identify water bodies, especially small water bodies, and to accurately locate affected areas, which is significant for protecting people’s lives and property. However, the application of optical remote sensing is often limited by clouds and fog during actual floods. In this paper, water extraction methods of the multisource data fusion model (MDFM) and superpixel water extraction model (SWEM) are proposed, in which the MDFM fuses optical and synthetic aperture radar (SAR) images, and all-weather water extraction is achieved by using spectral information of optical images, texture information and the good penetration performance of SAR images. The SWEM further improves the accuracy of the water boundary with superpixel decomposition for extracted water boundaries using the fully constrained least squares (FCLS) method. The results show that the correlation coefficient (r) and area accuracy (Parea) of the MDFM and SWEM are improved by 2.22% and 9.20% (without clouds), respectively, and 3.61% and 18.99% (with clouds), respectively, compared with the MDFM, and 41.54% and 85.09% (without clouds), respectively, and 32.31% and 84.31% (with clouds), respectively, compared with the global surface water product of the European Commission Joint Research Centre’s Global Surface Water Explorer (JRC-GSWE). The MDFM and SWEM can extract water bodies with all weather and superpixel and improve the temporal and spatial resolution of water extraction, which has obvious advantages

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