bluuebunny/crossref_metadata_2025 huggingface dataset:
Uncovering the Regulation of Fibrotic Signaling in Scleroderma by the Ubiquitin E3 Ligase KLHL42
Authors: Lear Travis , Lockwood Karina C. , Tuncer Ferhan , Larsen Mads , Evankovich John , Lafyatis Robert , Liu Yuan , Chen Bill B.
Published: April 2020

Systemic scleroderma (SSc) is an autoimmune disease of unknown cause that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive pro‐fibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three‐quarters of SSc patients develop pulmonary fibrosis within five years; excessive scarring in SSc lung fibrosis is the main driver of mortality. There are no approved medicines to treat lung SSc. Recent research suggests that TGFb signaling is directly tied to SSc disease, through its pro‐fibrotic transcription factor signaling. Previous studies have shown ubiquitin e3 ligases potently control TGFb signaling through the targeted degradation of key regulator proteins, however their role in SSc‐TGFb signaling remains unclear. We utilized primary SSc patient lung cells for High Throughput Screening of modulators of TGFb signaling through High Content Imaging of the fibrotic transcription factor SMAD2/3 nuclear translocation. We screened an RNAi library targeting Ubiquitin E3 ligases and observed that the knockdown of the E3 ligase KLHL42 impaired TGFb‐dependent fibrotic signaling. KLHL42 knockdown reduced the production of fibrotic material and decreased TGFb‐mediated SMAD activation. We utilized unbiased ubiquitin proteomics to identify the phosphatase PP2A regulator PPP2R5e to be a KLHL42 substrate. Mechanistic studies validated the ubiquitin‐mediated control of PPP2R5e stability through KLHL42. Knockdown of PPP2R5e exacerbated TGFb‐mediated fibrotic signaling, demonstrating a role for PPP2R5e in the regulation of fibrotic signaling in SSc. Finally, we observed KLHL42 affected overall PP2A phosphatase activity through its targeted degradation of the subunit PPP2R5e. Here we report the characterization of the KLHL42‐PPP2R5e axis regulating fibrotic signaling in SSc lung fibroblasts. Future studies will investigate the potential for chemical inhibition of KLHL42 to ameliorate fibrotic signaling in SSc.

Support or Funding Information

5F31HL143843, 1R01DK119627, 5P50AR060780‐08, 1K08HL144820, 5R01HL142777, 5R35HL139860, and 5R01HL133184.
Assembly of Hypothalamic Perineuronal Nets Contributes to Sustained Blood Glucose Lowering by FGF1 Action in the Brain
Authors: Alonge Kimberly M. , Mirzadeh Zaman , Scarlett Jarrad M. , Brown Jenny M. , Bentsen Marie A. , Logsdon Aric F. , Banks William A. , Guttman Miklos , Wight Thomas N. , Morton Gregory J. , Schwartz Michael W.
Published: April 2020

The mediobasal hypothalamus (MBH) plays a key role in the homeostatic control of blood glucose levels, and defective activity of glucoregulatory neurocircuits in this brain area is implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). We have identified irregularities in the abundance of perineuronal nets (PNNs), specialized extracellular matrix (ECM) lattices that regulate neurocircuit network interactions, surrounding MBH neurons in rodent models of T2DM. Specifically, diabetic Zucker Diabetic Fatty (ZDF) rats exhibit a 56% decrease in aggrecan+ PNN structures in the MBH compared to age‐matched normoglycemic WT controls, suggestive of destabilized glucose‐sensing neurocircuit interactions. To determine whether this effect is associated with changes in the relative abundance of chondroitin sulfate (CS)‐glycosaminoglycan (GAG) isomers (Δ0S‐, Δ4S‐, Δ6S‐, Δ4S6S, Δ2S6S‐CS), which constitute the glycan component of PNNs and influence the function of these matrices, we characterized CS‐GAG sulfation patterns using state‐of‐the‐art LC‐MS/MS + MRM analysis. Here, we report that diabetic ZDF rats exhibit a significant increase in Δ4S‐CS and corresponding decrease in Δ6S‐ and Δ2S6S‐CS isomers within the MBH that are suggestive of altered protein‐GAG interactions, axonal repulsion and ECM stiffening. We next sought to determine if these PNNs are targets for the effect of fibroblast growth factor 1 (FGF1) to induce sustained blood glucose lowering in ZDF rats following intracerebroventricular (icv) administration. We found that in ZDF rats, icv FGF1 injection normalized CS‐GAG composition while inducing a 2.2‐fold increase in aggrecan+ PNN structures. To determine whether this response is required for blood glucose lowering induced by central FGF1 treatment, ZDF rats received an icv injection of FGF1 followed immediately by bilateral microinjection of either vehicle or the CS‐GAG digesting enzyme, Chondroitinase ABC (ChABC), directed to the MBH to disassemble PNNs. Our finding that the duration of blood glucose lowering induced by icv FGF1 injection was markedly shortened by digestion of hypothalamic PNNs identifies PNNs as key targets for the action of FGF1 to induce sustained remission of diabetes. These data identify a key role for MBH PNNs in glucoregulatory neural network interactions involved in the homeostatic control of blood glucose.

Support or Funding Information

This project was supported by the National Institutes of Health Awards DK12266201 (to K.M.A) and DK083042 (to M.W.S). Research support also provided by Novo Nordisk, Inc (CMS‐431104).
Dissociation between reactive hyperemia measured in the macro‐ vs. microvasculature
Authors: Rosenberry Ryan , Trojacek Darian , Bartlett Miles , Nelson Michael D.
Published: April 2020

Reactive hyperemia (RH), the magnitude of tissue reperfusion following acute ischemia, has conventionally been interpreted as an index of microvascular function. However, RH is commonly measured using Doppler ultrasound in conduit arteries, well upstream of the microvascular tissue bed it purportedly represents. Near‐infrared diffuse correlation spectroscopy (DCS) measures tissue perfusion at the level of the microvasculature and therefore may more appropriately represent microvascular function, however these two end‐points have not yet been directly compared. Accordingly, this study assessed macro‐ versus microvascular RH in 12 young, healthy males (age 18±1, height 171.5 ± 6.8, weight 64.7 ± 12.2). Subjects were instrumented with an occlusive cuff placed on the upper arm and the DCS probe placed over the belly of the flexor digitorum profundus. The brachial artery was imaged just distal to the occlusive cuff for “macrovascular imaging”. Heart rate was monitored by ECG and blood pressure by continuous finger‐pressure in the contralateral limb. The occlusive cuff was inflated to 220 mmHg for 5 minutes, after which time the cuff was rapidly deflated and the hyperemic response recorded for an additional 3 minutes. The data show markedly delayed time to peak hyperemia measured in the microvasculature by DCS (28.8 ± 10.5s) compared to the conventional brachial artery velocity (9.4 ± 3.9s). These data raise several important questions regarding: (1) the interpretation of RH measured in conduit arteries, and (2) the interpretation of the slower DCS‐derived reperfusion kinetics, in relation to “microvascular function”. Further investigation is needed to elucidate the physiological phenomenon driving the macro‐ versus microvascular differences, and answer the aforementioned questions.
Mild Cognitive Impairment in the Presence of Depressive Symptoms Related to Impaired Cerebrovascular Function in the Obese Zucker Rat
Authors: Halvorson Brayden , Whitehead Shawn , Frisbee Jefferson
Published: April 2020

Metabolic syndrome is a highly prevalent constellation of pathologies that frequently combines overweight/obesity, moderate hypertension, atherogenic dyslipidemia, and type II diabetes mellitus, and is accompanied by a systemic vascular pro‐inflammatory, pro‐oxidant condition. The evolution of metabolic syndrome is associated with elevated cerebrovascular disease risk including elevations in both the occurrence and the severity of stroke and transient ischemic attacks within the cerebral circulation. Frequently, these impairments to cerebral vascular function also result in cognitive and behavioral impairments to the afflicted individual even in the absence of an acute pathological event.

Given the established elevation in depressive symptoms in OZR that develop in parallel to cerebrovascular dysfunction, we sought to determine the extent to which cognition was impaired.

In this study 17–18‐wk‐old male lean and (LZR) obese Zucker rats (OZR) were tested for learning, memory, and behavioral flexibility using an operant conditioning chamber (OCC) and the Morris Water Maze (MWM). Learning was assessed by number of trials to reach 8 correct consecutive responses in OCC or a 6 trial MWM protocol. Performance in a retrieval or probe session 24 h after learning was used to measure memory in OCC and MWM respectively. Behavioral flexibility was measured by analyzing error types in OCC and search strategy in the MWM probe test. Depressive symptoms were measured using coat score, novelty suppressed feeding response, and latency to start grooming and frequency of subsequent grooming after sucrose spray. Ex vivo pressurized middle cerebral arteries (MCA) were used to determine the extent to which metabolic syndrome in OZR alters key indices of vascular reactivity of cerebral resistance vessels and the mechanics of the vascular wall.

Endothelium dependent vasodilator reactivity was attenuated in OZR vs LZR in response to acetylcholine. Further impairments were demonstrated in OZR with an increase in depressive symptoms as measured by an increase in coat score and latency to start grooming after sucrose spray as well as mild cognitive impairment measured by increased time to reach the platform in MWM learning trials. In contrast, no signs of cognitive impairment were demonstrated in OCC. These results suggest that the changes in cerebrovascular function in OZR are associated with the development of depressive symptoms and mild cognitive impairment in MWM learning with no changes in learning, memory, or behavior flexibility in OCC at this age.

Support or Funding Information

CIHR (#130138 and #389769); NSERC (RGPIN‐2018‐05450)
Estrogen modulation of fate choice during kidney development
Authors: Wesselman Hannah , Pfaff Mairead , Wingert Rebecca
Published: April 2020

Vertebrate kidney formation is a nuanced process that directs renal progenitors to form the functional units of the kidney, known as nephrons, which are comprised of specialized cell types organized into discrete functional segments. The zebrafish embryo forms two nephrons by 24 hours post fertilization (hpf) and offers a simple but tractable genetic model to study nephron patterning. A chemical screen carried out by our lab coupled with whole mount in situ hybridization (WISH) identified 17ß‐estradiol (E2), the dominant form of estrogen in vertebrates, as a potential regulator of segmentation. Further investigation has revealed that E2 results in an expansion of the distal early (DE) and decrease of the distal late (DL) nephron segments due to alterations in cell number that were quantified with fluorescent ISH. Furthermore, E2 treated animals had expanded expression of the essential DE lineage transcription factor irx3b and its target, irx1a, suggesting E2 affects specification of DE precursors. To delineate the possible mechanism of action of E2’s renal effects, we conducted a targeted chemical screen with selective estrogen receptor modulators (SERMs) that exhibit preferential binding for known E2 receptors. The esr2 antagonist PHTPP similarly reduced the DE segment, suggesting that Esr2 may be involved in nephrogenesis. Consistent with this hypothesis, esr2a and esr2b transcripts are expressed during renal progenitor specification. To investigate which of these receptors (or both) are a major player in distal cell fate choice, we have created CRISPR/Cas9 mutants using guides designed for esr2a and esr2b, respectively. With additional genetic studies, we hope to further our understanding of the role of estrogen signaling in kidney development. Combined, these preliminary findings have relevance for understanding kidney disease models, as well as efforts to recapitulate development in vivo for drug discovery and regenerative therapies.

Support or Funding Information

R01DK100237
A link between the inflammatory mediator interleukin‐13, the transcriptional repressor REST, the KCNQ5 gene, and Kv7.5 potassium channel function in vascular smooth muscle cells
Authors: Byron Kenneth L. , Brueggemann Lyubov I. , Sandhu Gurpreet , Cabe Maleen , Cribbs Leanne L.
Published: April 2020

Inflammatory cytokines may contribute to development of allergic hyperresponsiveness in excitable tissues. In peripheral neurons, development of neuropathic pain was found to be associated with overexpression of the repressor element 1‐silencing transcription factor (REST) and subsequently linked to suppression of KCNQ gene transcription, which was associated with a reduction of M‐current and increased neuronal excitability. We previously found that Kv7.5 channels encoded by the KCNQ5 gene are important regulators of smooth muscle cell relaxation in both the vasculature and airways. We also found previously that both vascular and airway smooth muscle cells express REST, which translocated to the nucleus upon short term treatment with interleukin‐13 (IL‐13, 100 ng/ml for 24–48 hr). Chronic treatment of A7r5 vascular smooth muscle cells with IL‐13 for 5–6 days resulted in a 37±12% reduction of KCNQ5 mRNA level and a 34% reduction of endogenous whole cell Kv7.5 current amplitude. We performed a search for genomic sequences that match the REST consensus sequence and using chromatin immunoprecipitation techniques, we have now identified a REST binding region on the rat KCNQ5 gene. The link between IL‐13‐induced REST translocation and suppression of Kv7.5 functional expression was investigated using a REST knock‐out strategy in the A7r5 cell line, using CRISPR‐Cas9 technology. REST KO A7r5 cells had functional endogenous Kv7.5 currents that were insensitive to chronic treatment with IL‐13 and which trended to have higher current densities than those measured in A7r5 cells expressing non‐targeting guide RNA. In summary, REST appears to function as a link between the inflammatory cytokine IL‐13 and suppression of KCNQ5 gene expression/Kv7.5 current in vascular smooth muscle cells.

Support or Funding Information

The project described was supported by Award Number 01BX002344 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development.
Vascular ATP‐sensitive K+ (KATP) Channels: Sex and Fiber‐type Differences in the Support of Contracting Muscle Blood Flow and Interstitial PO2
Authors: Colburn Trenton D. , Weber Ramona E. , Schulze Kiana M. , Hageman K. Sue , Behnke Bradley J. , Musch Timothy I. , Poole David C.
Published: April 2020

Glibenclamide (GLI), prescribed to Type II diabetes patients, enhances insulin release by inhibiting pancreatic KATP channels. KATP channels support maximal aerobic capacity (V̇O2max; determined by cardiac output (Q̇) and O2 utilization within contracting skeletal muscle (arterial‐venous O2 difference)) and blood flow during treadmill running in male rats. High‐intensity exercise tolerance (critical speed, CS) is impaired by GLI but is not correlated with reductions in resting cardiac function (Q̇) which highlights the role for peripheral vascular KATP channel function to protect against fatigue. Whether muscle O2 delivery‐utilization matching (interstitial PO2, PO2is) is impaired by GLI in a fiber‐type specific manner, and whether sex differences exist in KATP function, are unknown.

PURPOSE

We hypothesized that local inhibition of KATP channels via GLI would decrease PO2is and blood flow within contracting fast‐twitch oxidative (mixed gastrocnemius (MG)) and slow‐twitch oxidative (soleus (SOL)) muscles with females (F) exhibiting the greatest reduction.

METHODS

Male (n=8), female (n=9, proestrus) and ovariectomized female (F+OVX; n=11) Sprague‐Dawley rats were utilized in these experiments. PO2is was determined, before and after GLI superfusion (5 mg kg−1), via phosphorescence quenching (G4) in the exposed MG and SOL muscles during electrically‐induced twitch contractions (180 s, 7V, 1Hz). Blood flow was assessed via infusion of fluorescent‐labeled microspheres (15 μm) at 180 s of contractions.

RESULTS

GLI reduced MG blood flow (female: 49 ± 9 vs 34 ± 5; male: 50 ± 5 vs 35 ± 4 ml min−1 100 g−1), nadir PO2is (female: 5.9 ± 0.3 vs 4.7 ± 0.3; male: 6.8 ± 0.7 vs 5.5 ± 0.4) and end‐contraction PO2is (female: 7.3 ± 0.5 vs 6.1 ± 0.5; male: 8.9 ± 1.1 vs 7.2 ± 0.5 mmHg), but did not impact SOL, of female and male rats (p<0.05). Conversely, in F+OVX, PO2is was reduced in the SOL (nadir: 12.5 ± 1.5 vs 8.8 ± 0.9; end‐contraction: 14.5 ± 1.5 vs 10.2 ± 1.1 mmHg; p<0.05), but not MG.

CONCLUSION

These data support the role of vascular KATP channels in supporting muscle O2 delivery and high‐intensity exercise tolerance by matching O2 delivery‐utilization with ovariectomy shifting KATP channel effects from fast‐ to slow‐twitch muscles. Thus GLI treatment in diabetic and other cardiovascular disease patients may potentiate exercise intolerance, especially for post‐menopausal females where compromised slow‐twitch fiber function impairs the ability or willingness to engage in low intensity exercise.

Support or Funding Information

Supported by NIH Grants: HL108328 (D.C.P and T.I.M) and F31HL145981 (T.D.C)
Does insulin resistance alter pulse transit time in the cerebral circulation?
Authors: Muer Jessica D. , Ward Aaron T. , Carter Katrina J. , Kellawan J. Mikhail , Harrell John W. , Roldan Alejandro , Wieben Oliver , Eldridge Marlowe W. , Schrage William G.
Published: April 2020

Approximately 80 million adults in the United States suffer from an insulin resistant state known as metabolic syndrome (MetSyn), associated with increased risk for strokes, cerebral infarcts and neurologic disease. Adults with MetSyn also exhibit increased peripheral arterial stiffness, but whether arterial stiffness occurs in the cerebral circulation remains unclear. We hypothesized adults with MetSyn would exhibit increased cerebrovascular stiffness, demonstrated as faster pulse transit time (PTT) compared to healthy adults. Participants were 32±7 years old, sedentary, non‐smoking, free of cardiovascular disease and unmedicated. Healthy normal‐weight adults (Control; n=11; weight=70±8 kg; BMI = 23±2 kg/m2) were normotensive and normolipidemic. Adults with MetSyn (n=6; BMI=38±8 kg/m2) met three or more of the five criteria according to American Diabetes Association (MAP=107±5 mmHg; glucose=82±17 mg/dL; triglycerides 167±123 mg/dL; HDL 39±9 mg/dL; waist circumference 119±16 cm). Subjects were instrumented to continuously monitor heart rate, blood pressure, and end‐tidal CO2. A 3T MRI using 4‐dimensional flow phase contrast vastly under‐sampled isotropic projection reconstruction (PC VIPR; ~6 minutes) was used to capture cerebral blood flow (CBF = blood velocity • vessel cross sectional area) in the internal carotid arteries (ICA) and superior sagittal sinus (SSS). Cerebral blood flow across 20 equal segments of the average cardiac cycle was used to determine the time to peak flow in the ICAs and SSS. Pulse transit time (PTT) was calculated as the difference in time to peak in the SSS from the time to peak in the ICAs. Data are presented as mean ± SD. Significance was determined using independent t‐tests and was set at p≤0.05. MAP, HR, SpO2, and end tidal CO2 were not different in magnet bore (p>0.05). ICA to SSS PTT was not different between Control and MetSyn (141±109 vs. 144±91 ms; p=0.743).

Conclusion

Pulse transit time was similar between healthy adults and those with MetSyn. Perhaps greater disease severity, aging, or their combination are required to negatively change PTT in MetSyn. Alternatively, the cerebral circulation may be resistant to vascular stiffening.

Support or Funding Information

ADA 1‐12‐IN‐39
Store‐Operated Calcium Entry Regulates Macrophage Chemotaxis
Authors: White Carl , Fresquez Adriana M.
Published: April 2020

The store operated calcium entry (SOCE) pathway is known to be important for cell migration; however, its involvement in regulating directed movement toward a chemotactic signal is less well understood. The aim of current study was to define the involvement of SOCE in macrophage chemotaxis. To achieve this aim, we used phase contrast microscopy to visualize primary mouse bone marrow derived macrophages migrating toward the source of the potent chemoattractant C5a (complement component 5a). A concentration gradient was established by pulsing C5a from a micropipette connected to a pressure regulating device. Chemotaxis was monitored for up to 90 minutes in the presence and absence of the SOCE blocker BTP2 (1 μM). We found that the chemotactic speed and total distance traveled was decreased in the presence BTP2. In addition, we quantified the morphological changes at the leading edge of the migrating cells and found that frequency and speed of protrusions was also reduced by BTP2. These studies establish the requirement for SOCE in macrophage chemotaxis and leading‐edge dynamics and suggest a role for SOCE in regulating the recruitment of macrophages to sites of inflammation in vivo.

Support or Funding Information

This work was supported by NIH Grant HL142906 to C.W.
Fusion protein EWS‐FLI1 partners with EWSR1 to regulate transcription in Ewing sarcoma
Authors: Ahmed Nasiha , Schwartz Jacob
Published: April 2020

Chromosomal translocation events drive many types of pediatric cancers. Ewing sarcoma is a pediatric bone cancer driven by a chromosomal translocation where the N‐terminal low‐complexity domain of EWSR1 fuses to the DNA‐binding domain of the ETS transcription factor FLI1. This fusion creates the aberrant transcription factor EWS‐FLI1, which drives tumor formation by altering the transcriptional landscape. Interestingly, constitutive expression of EWSR1 is retained in Ewing sarcoma cells. The low‐complexity domain found in EWSR1 and EWS‐FLI1 is intrinsically‐disordered and can mediate protein‐protein interactions with itself and other protein partners. Moreover, this domain has the propensity to phase separate. Despite the common domain between the two proteins, very little is known about the effect of EWSR1 on EWS‐FLI1 activity. We performed RNA‐sequencing to reveal that EWSR1 and EWS‐FLI1 coregulate a large pool of genes in Ewing sarcoma cells. Loss of EWSR1 in inhibits anchorage‐independent growth, suggesting that EWSR1 plays a role in cancerous properties of Ewing sarcoma cells. We have shown EWSR1 and EWS‐FLI1 bind each other directly in vitro and in vivo. Finally, we have found the existence of EWSR1, EWS‐FLI1, and RNA Pol II in a novel granular body in Ewing sarcoma cell lines. This direct interaction of EWSR1 and EWS‐FLI1 presents a straight‐forward model that merges wild‐type and fusion properties to alter transcriptional output and drive Ewing sarcoma biology.

Support or Funding Information

National Institutes of Health, American Cancer Society
Development and Characterization of Spontaneously Immortalized Cell Lines Derived from Mouse Endometriotic Lesions
Authors: Chawla Shivane , Lang Stephanie A. , Burns Katherine A.
Published: April 2020

Endometriosis affects approximately 1 in 10 women of reproductive age, where the tissue of the inner lining of the uterus, the endometrium, proliferates in and around the peritoneal cavity. Common symptoms of endometriosis include chronic pelvic pain, intense pain with menstruation, pain with urination and defecation, and infertility. Recently more attention is being brought to the links between endometriosis and other co‐morbidities such as heart disease, cancer, and autoimmune disorders. Other factors known to play a role in endometriosis are endocrine disrupting chemicals that are found in common household products that contain plasticizers. One of these environmental toxicants is a group of chemicals called phthalates. A challenge to the field of endometriosis is the availability of cell lines to study mechanism(s); therefore, we set forth to create cell lines from endometriosis lesions. In our model of endometriosis, uterine endometrial tissue expressing green fluorescent protein (GFP) is used to develop endometriosis lesions. From these lesions, multiple cell lines were generated, but for this study, the focus was on GFP positive (EmLe+) and GFP negative (EmLe‐) cell populations that emerged from the lesions. The EmLe+ line is phenotypically uterine derived exhibiting epithelial to mesenchymal transition (EMT) markers. The EmLe‐line phenotypically exhibits epithelial derived muscle markers. To characterize these lines and to determine their response to environmental toxicants, the cells were studied under normal conditions and exposure to a commonly found phthalate, bis (2‐ethylhexyl), also known as DEHP, to better understand the cellular properties in the development and progression of endometriosis. Cell proliferation, migration, and invasion assays were used to examine how endometrial cells cultured from a mouse model are able to proliferate, migrate, and invade under normal and DEHP exposure conditions. For the proliferation assay, each of the cell lines was plated at 5,000 cells per well in 24 well plates and counted each day under various conditions. To study cell migration and invasion, a 48‐well chemotaxis chamber was used with or without matrigel, respectively. From these studies, we find that the different cell types isolated from mouse endometriotic lesions differ in composition and behavior, and find that these cell lines may be a good model to study more in depth mechanisms of endometriosis development.

Support or Funding Information

R00 ES021737

P30 ES006096
Knockout of VDAC1 in H9c2 Cells Promotes tBHP‐induced Cell Apoptosis Through Decreased Mitochondrial HK II Binding and Enhanced Glycolytic Stress
Authors: Yang Meiying , Li Keguo , Sun Jie , Stowe David F. , Tajkhorshid Emad , Kwok Wai-Meng , Camara Amadou K.S.
Published: April 2020

VDAC1, the most abundant mitochondrial outer membrane protein, transports ions and metabolites between the cytoplasm and mitochondria. It is also involved in cellular energy production and apoptosis, but the role of VDAC1 in cell death remains controversial. Silencing or upregulation of VDAC1 may stimulate or inhibit cell apoptosis, growth and survival depending on cell types and stimuli. Using VDAC1 knockout (Vdac1−/−) embryonic stem cells (ES) and mouse embryonic fibroblast (MEF), it was revealed that VDAC1 was dispensable for both mitochondrial membrane permeability transition pore (mPTP) opening and Bax and Bid‐driven cell death. The effect of Vdac1−/− on oxidative stress‐induced cell death in a cardiac cell line has not been reported. We hypothesized that although VDAC1 is dispensable for mPTP and is not involved in mPTP‐mediated cell death, VDAC1 is an essential regulator of oxidative stress‐induced cell apoptosis in cardiac cells. To this end, we knocked out VDAC1 in the cardiac fibroblast cell line, H9c2 (Vdac1−/− H9c2) by the CRISPR‐cas9 technique and examined whether VDAC1 plays a role in cell death via oxidative stress induced by tert‐butylhydroperoxide (tBHP), an organic peroxide. We found that under physiologic conditions, Vdac1−/− did not affect H9c2 cell proliferation, and mitochondrial respiration. However, tBHP treatment enhanced the extracellular acidification rate (ECAR) and proton production rate (PPR) produced from glycolysis and promoted cell apoptosis in Vdac1−/− H9c2 cells compared to the wildtype. Vdac1−/− H9c2 cells also showed markedly reduced binding of mitochondria hexokinase II (HK II), a key glycolytic enzyme. Rescue of VDAC1 in Vdac1−/− H9c2 cells reinstated mitochondria HK II binding and concomitantly decreased tBHP‐induced cell death. Mitochondrial respiration remained the same after tBHP treatment in Vdac1−/− and wild type H9c2 cells. Our results suggest that Vdac1−/− in H9c2 cells enhances tBHP‐induced oxidative stress‐mediated cell apoptosis by reducing mitochondria HK II binding and by increasing ECAR and PPR produced from glycolytic stress.

Support or Funding Information

National Institutes of Health (R01 HL‐131673‐01A1), Veterans Administration (BX‐002539‐01), United States.
The exercise pressor reflex is inhibited by Brilliant Blue G in freely perfused and femoral artery ligated rats following intrathecal, but not hindlimb, treatment.
Authors: Estrada Juan , Ducrocq Guillaume , Kim Joyce , Kaufman Marc
Published: April 2020

The exercise pressor reflex (EPR) is mediated in part by purinergic(P)2X receptors which are compromised of seven subtypes. Blockade of the P2X3 subtype on the peripheral endings of group III and IV muscle afferents attenuates the EPR. We investigated whether other P2X receptors were involved in the EPR arc in decerebrate Sprague Dawley rats whose femoral arteries were either patent or were ligated. We antagonized P2X receptors with Brilliant Blue G (BBG), which has highest affinity for P2X7 receptors. We injected BBG in both group of rats either intrathecally (50mg/mL; 25–50uL) or into the superficial epigastric artery, which is a side branch of the femoral artery. Intrathecal injection of BBG attenuated the magnitude of the EPR in both rats with patent femoral arteries and in rats with ligated femoral arteries (P<0.05). Intrathecal injection of BBG had no effect on the pressor response to intra‐carotid injection of sodium cyanide (2ug), which is a potent and selective stimulant to carotid chemoreceptors. This latter finding indicates that intrathecal injection of BBG did not ascend to the medulla and did not impact the spinal sympathetic outflow. BBG injected either into the superficial epigastric artery or intravenously (5mg) had no appreciable effect on the EPR. In addition the effect of intrathecal BBG could not be explained by “neural fatigue” caused by repetitive contractions in rats with ligated femoral arteries. We conclude that intrathecal BBG treatment inhibits the EPR by antagonizing P2X4 or P2X7 receptors within the dorsal horn of the spinal cord.

Support or Funding Information

This study was funded by NIH grants R01 AR 059397 and P01 HL 134609.
A Comparative Analysis of the Vascular Effects of Clopidogrel Metabolites
Authors: Kuszynski Dawn S. , Christian Barbara D. , Dorrance Anne M. , Lauver D. Adam
Published: April 2020

Dual antiplatelet therapy using low‐dose aspirin with a purinergic receptor 2Y12 (P2Y12) antagonist is the most common preventative method for arterial thrombosis. Clopidogrel, a P2Y12 antagonist, effectively inhibits ADP‐induced platelet aggregation. However, it also increases adverse bleeding side effects which do not correlate with the antiplatelet effect. Of patients who have taken clopidogrel for at least one year, 31% have cerebral microbleeds and 47% of those patients also develop intracerebral hemorrhage. Through clopidogrel’s complex metabolism only 5% of the active metabolite responsible for the antiplatelet effect is generated. In order to avoid the complex metabolism associated with clopidogrel, DT678 was developed. DT678, a conjugate of the clopidogrel active metabolite, is completely converted into the active metabolite to inhibit platelet aggregation. We have previously reported that DT678 is equally as effective in preventing platelet aggregation however, it has a reduced bleeding risk compared to clopidogrel in a rabbit model. A more complete mechanistic understanding of the cellular pathways affected by clopidogrel and its metabolites are required in order to explain the difference in bleeding risk.

The purpose of this study was to determine the effects of clopidogrel on spontaneous myogenic tone generation, vasoconstriction or vasodilation in middle cerebral arteries (MCA). Rabbits were anesthetized and the jugular vein was cannulated for blood collection and intravenous drug administration of various doses of DT678, clopidogrel or vehicle. Blood collections were taken at baseline and two‐hour post IV drug administration. The blood was then used in a platelet aggregometry assay to determine the inhibition of platelet aggregation. Two‐hours post treatment the brain was collected and placed in Ca2+ free physiological salt solution. The MCA was isolated and cannulated on an arteriography chamber. The cannulated artery, in Ca2+ physiological salt solution, was pressurized to 80mmHg with a flow rate of 0.25ml/min through the lumen at 37°C. Spontaneous myogenic tone was allowed to develop before assessing the vascular effects of increasing concentrations (10−9–10−5M) of ADP or specific purinergic receptor agonists (P2Y1: MRS 2365; P2Y2 MRS 2768; P2Y4 MRS 4062; P2Y6: MRS 2693; P2Y11: NF 546; or P2Y14: MRS 2905). If spontaneous myogenic tone was not generated the MCA was preconstricted with 10−6M serotonin. Spontaneous myogenic tone and ADP‐induced vasodilation was inhibited in clopidogrel‐treated, but not DT678‐treated middle cerebral arteries. These data suggest that clopidogrel has non‐platelet mediated effects on purinergic receptors expressed in the MCA and may explain the cerebral bleeding side effects observed.

Support or Funding Information

This work was supported in part by MSU PHM/TOX and CVM Startup Funds, Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation Research Starter Grant and NIH/NHLBI R43HL139380.
Clarifying Ded1 Function in Translational Control using an in vivo Fluorescence‐Based Reporter Assay
Authors: Schuessler Peter Joseph , Srinivasan Aravind , Barbi Joseph , Walker Sarah E.
Published: April 2020

Eukaryotic translation initiation is a critical node at which gene expression is regulated. Among the factors that are known to regulate this process, DEAD‐box RNA helicases are believed to promote translation by resolving secondary structure in the 5′ UTR of an mRNA. These helicases include the protein Ded1 in budding yeast, which is required to remove cap‐distal structure in the 5′ UTR of an mRNA, thus enabling scanning of the preinitiation complex. Yet the role of Ded1 in translation is complex; a translationally repressive role has also been demonstrated by Ded1 overexpression. Further confounding investigations of Ded1 function are the large number of protein‐protein and protein‐RNA contacts made by Ded1 to regulate activity. To study the diverse roles that Ded1 plays in translation, we have developed a system that reports on Ded1‐specific changes in translation in vivo. This system relies on a fluorescence‐based reporter that simultaneously promotes the transcription of two mRNA transcripts: one encoding RFP with a 5′ UTR that is translated independent of Ded1 activity, and the other encoding GFP with a 5′ UTR that requires Ded1‐activity for translation. We are using this system in combination with a library of random Ded1 mutants to broadly interrogate Ded1 activities for effects on translation initiation and repression.

Support or Funding Information

This work was supported by NIH grant R00GM119173 and start‐up funds from SUNY at Buffalo, College of Arts and Sciences.
ATP‐sensitive K+ Channel Inhibition Diminishes Critical Speed in Male and Female Rats but V̇O2max in Females Only
Authors: Weber Ramona E. , Colburn Trenton D. , Schulze Kiana M. , Hageman K. Sue , Behnke Bradley J. , Musch Timothy I. , Poole David C.
Published: April 2020

Glibenclamide (GLI) stimulates pancreatic insulin secretion in patients with Type II diabetes by inhibiting ATP‐sensitive K+ (KATP) channels. KATP channels also support vasodilation, increasing blood flow and O2‐delivery‐utilization matching in contracting muscle. Whether KATP channels support high‐intensity exercise tolerance and the sex dependency of any such effect remains unknown.

Purpose

To determine the role of KATP channels in supporting maximal aerobic capacity (V̇O2max) and the determinants of the speed‐duration relationship (CS, critical speed, and D′, distance covered above CS). We hypothesized that KATP channel inhibition via GLI would impair V̇O2max and CS with females exhibiting the greatest reduction.

Methods

Male (n=11), female (n=10, proestrus), and ovariectomized female (F+OVX; n=12) Sprague‐Dawley rats were tested with and without GLI (10 mg kg−1 in DMSO i.p.). V̇O2max and CS were assessed using a motorized treadmill with an incline of 5%. V̇O2max was measured via plexiglass metabolic chamber connected to an O2 analyzer with treadmill speed increasing 5–10 m min−1 every minute until V̇O2 plateaued. Multiple (i.e., ≥ 4) constant speed runs to exhaustion on separate days were used to determine CS and D′.

Results

GLI reduced V̇O2max in female (71.5 ± 1.0 vs 67.9± 1.5 mL O2 min−1 kg−1; p<0.05) and F+OVX (76.8 ± 1.4 vs 74.4 ± 1.5 mL O2 min−1 kg−1; p<0.05) without affecting males (81.5 ± 2.0 vs 80.8 ± 2.0 mL O2 min−1 kg−1; p>0.05). In contrast, GLI reduced CS equivalently across groups (8–11%; p<0.05). D′ was unchanged (p>0.05).

Conclusion

Consistent with our hypothesis, KATP channel function supports exercise tolerance. Interestingly, whilst CS between sexes was not impacted differently, V̇O2max was diminished only in females and not males. These data demonstrate that the exercise intolerance in Type II diabetes patients may be exacerbated by sulphonylurea medication (i.e. GLI) with women being affected more severely in terms of O2 transport (V̇O2max).

Support or Funding Information

Supported by NIH Grants: HL108328 (D.C.P and T.I.M) and F31HL145981 (T.D.C)
Identifying Kinase Inhibitors as Potential Adjuvant Chemotherapeutics in Treatment for Ovarian Cancer
Authors: Anliker Clair , Corradi Anna , Wallert Mark A.
Published: April 2020

In the United States, 22,000 women are diagnosed with ovarian cancer annually resulting in 14,000 deaths. This makes ovarian cancer the fifth leading cause of cancer deaths in women despite its relatively low frequency. Plasma and ascites levels of lysophosphatidic acid (LPA) are elevated in ovarian cancer patients even at early stages of the disease. LPA is a bioactive lipid involved in signal transduction. The increased LPA within the peritoneal cavity causes a cascading effect with the activation of multiple different kinase pathways in ovarian cancer cells such as the Rho‐A/Rock, Ras/MAPK, and the PI3K/Akt pathways. The activation of these kinase pathways through elevated LPA levels are known to prevent apoptosis, increase cell proliferation and metastasis, and decrease sensitivity to platinum‐based therapy. In efforts to increase the overall survival, enhance progression‐free intervals, and to improve the overall quality of life for patients with ovarian cancer, it has been hypothesized that combining traditional chemotherapeutics with kinase inhibitors would slow tumor cell proliferation and decrease disease progression. In these studies, two ovarian adenocarcinoma cell lines SKOV‐3 and CAOV‐3 are used to evaluate cell proliferation to determine IC50 values of common platinum‐based chemotherapeutics, Cisplatin, and Carboplatin, as well are the PARP inhibitors, Olaparib and Rucaparib, independently and in conjunction with of one of four kinase inhibitors; BI‐D1870 (Rsk inhibitor), MK‐2206 (Akt inhibitor), SCH772934 (Erk inhibitor), and Y27632 (Rock inhibitor). We demonstrate that the addition of kinase inhibitors to traditional and modern ovarian cancer chemotherapeutics decreases the IC50 values for these chemotherapeutics thus providing a potential pathway for improved ovarian cancer therapy.
Binding of the exocyst complex to the SNARE regulator Sec1
Authors: Forson Jacqueline , Martínez-Núñez Leonora , Lepore Dante , Munson Mary
Published: April 2020

Vesicles in eukaryotic cells act as intracellular carriers to transport cargo between membrane‐bound organelles and the plasma membrane. Cellular trafficking is a highly regulated process that is critical for maintaining cellular growth, homeostasis, signaling and division. These cellular trafficking events ultimately culminate in membrane fusion, where secretory vesicles release their contents to the extracellular space. However, the molecular and structural underpinnings of trafficking and membrane fusion still remains misunderstood. The process of intracellular membrane fusion relies on several conserved protein families, including SNAREs, Sec1/Munc18 (SM) proteins, multi‐subunit tethering complexes (e.g. exocyst), and Rab GTPases. Each of these regulatory factors plays multiple roles in assisting vesicle docking and SNARE assembly, indicating a trafficking system with overlapping sets of reactions. Exocyst, a hetero‐octameric protein complex, is proposed to tether vesicles to the plasma membrane and promote specific SNARE‐mediated membrane fusion. Previous binding experiments using recombinant proteins revealed interactions between two exocytic SNARE proteins and Sec1 with a single subunit of exocyst, Sec6. Currently, Sec6 is proposed to bind both binary (Sso1:Sec9), and ternary (Sso1:Sec9:Snc2) SNARE complexes during SNARE complex assembly. The timing in which Sec1 binds to the ternary SNARE complex and exocyst to mediate membrane fusion is unknown. Temperature sensitive sec1 yeast cells are defective for cell growth, SNARE complex assembly, and secretion of protein and lipid cargo at the restrictive temperature. This evidence suggests that Sec1 may play both a general and a specific role during membrane trafficking. Recent structural evidence from a mutant “activated” yeast exocyst complex shows increased flexibility and dynamics of two subunits of exocyst, Exo70 and Sec6, which lead to increased binding to SNAREs compared to wild‐type exocyst. Our study aims to further investigate the mechanisms of SNARE assembly and membrane fusion, using biochemical and EM methods to characterize the interactions of yeast exocyst, Sec1 and SNARE proteins. Using purified forms of each of the proteins and complexes, interactions between Sec1, SNARE proteins, and exocyst (wild type and activated mutant) are being analyzed and quantified.
Synthesis and secondary structure characterization of sialyl‐Tn bearing MUC1 glycopeptides
Authors: Ormaza David , Cudic Mare
Published: April 2020

Objective

To synthesize carbohydrate building blocks bearing the sialyl‐Tn antigen, and incorporate them into a MUC1 peptide backbone for structural and functional studies with endogenous lectins.

Mucin 1 (MUC1), a transmembrane mucin protein, is a well‐known biomarker of different cancer types. The aberrantly glycosylated forms of MUC1 display specific truncated tumor‐associated carbohydrate antigens (TACAs) on the surface of the cell such as: Tn (GalNAc), T (Galβ1,3GalNAc), sTn (NeuAcα2,6GalNAc), and sT (2,3‐sT and 2,6‐sT). These truncated glycan structures, often capped by sialic acid, play a key role in tumor initiation, progression, and metastasis, and accumulating evidence suggests their involvement in tumor escape from immune defenses through interaction with endogenous carbohydrate‐binding proteins (lectins). Macrophage galactose binding lectin (MGL) is a C‐type lectin receptor found on antigen‐presenting cells (APCs) which facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. The main goal of this research is to evaluate the interactions between Tn and sTn antigen of tumor‐associated MUC1 with MGL and to characterize the molecular origin of the high affinity and selectivity of this endogenous lectin for its natural MUC1 ligands. This interaction plays a crucial role in tumor immune evasion. Nonetheless, MGL targeting is an attractive approach to enhance cancer vaccine efficacy. It is of crucial importance to understand the fine‐intricate nature of immune cells to either augment the anti‐tumorigenic effects or minimize the pro‐tumorigenic effects. The aim of this research is to synthesize structurally well‐defined chemical probes, mono‐ and multiple‐glycosylated MUC1 peptide models carrying the Tn or sTn O‐glycans, that allow for the control of the complexity of the chemical space of the multivalent ligands. For this purpose, a concise scheme was developed for the synthesis of the sTn antigen building blocks to be used in the solid‐phase synthesis of glycopeptide models. We employed the use of orthogonally protected phenyl thioglycoside glycosyl donors in the presence TfOH/NIS or TMSOTf/NIS as promoter system. The glycosylated threonine and sialylated glycosylated threonine were synthesized in a relatively high yield, 85% and 60%, but with moderate stereoselectivity, α/β ratio 3.5:1 and 2:1, respectively. The subsequent reduction and N‐acetylation of an azido group resulted in targeted glycosylated amino acid building block that was used in the solid‐phase peptide synthesis of MUC1 sTn bearing glycopeptides. The secondary structure of synthesized peptides was determined by circular dichroism (CD) spectroscopy. The synthetic efforts will be followed by mechanistic studies including isothermal titration calorimetry (ITC) and AFM.

Support or Funding Information

This work was supported by the National Institutes of Health Grant CA242351 to M. Cudic.
Glucagon‐like peptide‐1 mediated renal sympathetic activation in obese rats: role of intrarenal mechanisms
Authors: Zheng Hong , Liu Xuefei
Published: April 2020

Accumulated evidence indicates that obesity is associated with enhanced sympathetic activation. Chronic sympathetic activation increases the risk of cardiovascular complications and subsequent mortality. The mechanisms linking obesity with sympathetic activation are complex and not completely understood. Gut produced hormone glucagon‐like peptide‐1 (GLP‐1) affects cardiovascular function via regulating blood pressure and sympathetic nerve activity. The aim of the study was to determine the role of intrarenal GLP‐1 mediated renal sympathetic activation in obese rats. In anesthetic Sprague Dawley rats, renal intra‐pelvic infusion of GLP‐1 receptor agonist exendin‐4 increased afferent renal sympathetic nerve activity (A‐RSNA), efferent renal sympathetic nerve activity (T‐RSNA), arterial pressure and heart rate. All the sympathetic and hemodynamic responses to the exendin‐4 infusion were significantly enhanced in high fat diet (HFD, 42% of calories from fat for 12 weeks) induced obese rats. The natriuretic and diuretic effects mediated by intrarenal exendin‐4 infusion was significantly blunted in the obese rats. Furthermore, ablation of renal sympathetic nerves significantly improved the diuretic and natriuretic responses to exendin‐4 infusion in obese rats. We also found significant altered GLP‐1 level and GLP‐1 receptor expression in the kidney of obese rats. The results suggest that altered intrarenal GLP‐1 mediated mechanisms may contribute to the development of obesity‐related high sympathetic activation and obesity hypertension.

Support or Funding Information

The study was supported by the author’s faculty start‐up fund from Sanford School of Medicine of the University of South Dakota Department of Basic Biomedical Sciences.