Recipient of the 2018 Alumni Distinguished Leadership Award

Dr. Imoukhuede\u27s award-winning research at the University of Illinois focuses on how the body controls blood vessel formation with the goal of developing treatments for more than 70 diseases, including breast cancer and cardiovascular disease. She is Assistant Professor in Bioengineering at University of Illinois at Urbana- Champaign. Dr. Imoukhuede earned a Bachelor of Science degree in Chemical Engineering from Massachusetts Institute of Technology and a PhD in Bioengineering from California Institute of Technology

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

Sex differences in cancer mechanisms

We now know that cancer is many different diseases, with great variation even within a single histological subtype. With the current emphasis on developing personalized approaches to cancer treatment, it is astonishing that we have not yet systematically incorporated the biology of sex differences into our paradigms for laboratory and clinical cancer research. While some sex differences in cancer arise through the actions of circulating sex hormones, other sex differences are independent of estrogen, testosterone, or progesterone levels. Instead, these differences are the result of sexual differentiation, a process that involves genetic and epigenetic mechanisms, in addition to acute sex hormone actions. Sexual differentiation begins with fertilization and continues beyond menopause. It affects virtually every body system, resulting in marked sex differences in such areas as growth, lifespan, metabolism, and immunity, all of which can impact on cancer progression, treatment response, and survival. These organismal level differences have correlates at the cellular level, and thus, males and females can fundamentally differ in their protections and vulnerabilities to cancer, from cellular transformation through all stages of progression, spread, and response to treatment. Our goal in this review is to cover some of the robust sex differences that exist in core cancer pathways and to make the case for inclusion of sex as a biological variable in all laboratory and clinical cancer research. We finish with a discussion of lab- and clinic-based experimental design that should be used when testing whether sex matters and the appropriate statistical models to apply in data analysis for rigorous evaluations of potential sex effects. It is our goal to facilitate the evaluation of sex differences in cancer in order to improve outcomes for all patients

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

Visualizing the Membrane Confinement, Trafficking and Structure of the GABA Transporter, GAT1

Transporter trafficking regulators can play an important role in maintaining the transporter density necessary for effective function. I determine interactions that confine GAT1 at the membrane by investigating GAT1 lateral mobility through fluorescence recovery after photobleaching (FRAP). I find that the mobility of GAT1 can be increased by depolymerizing actin or by blocking the GAT1 PDZ interacting domain. I also identify ezrin as the GAT1 adaptor to actin. Through fluorescence resonance energy transfer (FRET), the distance between GAT1-YFP and Ezrin-CFP is calculated as 64--68 Å, and it can be significantly increased by disrupting the actin cytoskeleton. Altogether, my data reveals that actin confines GAT1 to the plasma membrane via ezrin, an interaction mediated through the GAT1-PDZ interaction domain. Discoveries in the field of vesicle fusion provide direct ties to translational research. While the study of vesicle fusion classically has been applied to neurotransmitter and neuropeptide containing vesicles; there is evidence that secretory vesicles physiologically differ from vesicles trafficking membrane protein. For instance, GAT1 resides on a vesicle lacking neurotransmitter but containing some v-SNARE proteins. These differences in the vesicle composition suggest inherent differences in trafficking mechanisms, which can only be confirmed through further study of membrane protein trafficking. To this end, I apply total internal reflection fluorescence microscopy (TIRFM) to quantify the number of GAT1 molecules on vesicles and to observe the movement of vesicles containing fluorescently tagged GAT1 into the plasma membrane. I determine that these vesicles contain 3--7 molecules of GAT1 and uncover a population of GAT1 vesicles with ATP-dependent lateral displacement. The protein-protein interactions, trafficking, and oligomerization of mouse GAT1 were studied using fourteen different fusions of mGAT1 with fluorescent protein. We determine that a natural PDZ-interacting motif is minimally required for wild-type GAT1 behavior. Fusions with wild-type function yielded up to 21% FRET efficiency, indicating efficient GAT1 oligomerization. Additionally, 45% FRET was observed between a GAT1 construct and YFP-syntaxin-1A. Inserting XFP between R565 and L566, resulted in 33% FRET but impaired function, which indicated the "RL" motif in the proximal C terminus governs export from the endoplasmic reticulum but not transporter oligomerization.</p

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