bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Transgenic Mouse Models for the Expression of Optical Biosensors of Endogenous G Protein Activity
Authors: Janicot Remi , Maziarz Marcin , Park Jong‐Chan , Garcia‐Marcos Mikel
Published: May 2022

G protein‐coupled receptors (GPCRs) are prime pharmacological targets, which is illustrated by the fact that they are the target for ~35% of FDA‐approved drugs and for countless drugs under investigation. These receptors initiate signaling pathways primarily by activating heterotrimeric G‐proteins (Gαβγ) inside the cell. Biosensors that directly measure G protein activation (i.e., formation of Gα‐GTP or Gβγ dissociation) have proven of great value as their readouts are not distorted by signal amplification or pathway cross‐talk, which is in stark contrast to what occurs by indirect measurements of downstream readouts like second messengers. However, these biosensors often require expression of exogenous G proteins which can distort readouts, and are also difficult to implement in many physiologically‐relevant systems, like primary cell cultures. To start addressing these issues, our laboratory has recently developed biosensors based on bioluminescence resonance energy transfer (BRET), called BRET biosensor with ER/K linker and YFP (or BERKY), that can detect endogenous levels of Gα‐GTP or free Gβγ by expressing a single polypeptide chain construct in cells. Here, we set out to further improve the applicability and reproducibility of this technology by generating transgenic mice for the conditional expression of BERKY sensors in virtually any cell type. Since BERKY sensors consist of the same core sequence and only differ in their detector modules, we envisioned a strategy to express two different biosensors from a single transgenic insert by leveraging a Flp recombination of FRT5 sites engineered within the constructs. We confirmed that the introduction of the FRT5 sequence did not interfere with the performance of any of the four biosensors (Gβγ, Gαq‐GTP, Gα13‐GTP, and Gαi‐GTP). Two mouse lines were initially engineered: in one, the Gβγ sensor is expressed before Flp recombination, and the Gαq‐GTP sensor is expressed after, whereas in the second Gα13‐GTP and Gαi‐GTP are expressed before and after Flp‐mediated recombination, respectively. To allow for precise control of the expression of the sensors, the ubiquitous CAG promoter was followed by a loxP‐STOP‐loxP cassette to prevent expression until Cre mediated recombination. After a series of breeding crosses following a specific genotyping strategy, we have successfully established four mouse lines, one for each of the biosensors described above. In conclusion, we have generated mouse lines for the expression of optical biosensors for the detection of endogenous G protein activation. We propose that this experimental platform should prove useful for investigating complex regulatory mechanisms with high fidelity and in any primary cell type fit for cell culture.
Selective mGlu1 Potentiation Reverses Cortical Disinhibition and Schizophrenia‐like Social and Cognitive Deficits
Authors: Gallinger Isabel G. , Luessen Deborah , Conn Jeffrey
Published: May 2022

There is a critical need to develop novel therapeutic strategies for addressing the negative and cognitive symptom domains in schizophrenia (SCZ). Multiple clinical and preclinical studies support the hypothesis that loss of GABAergic inhibitory transmission in the prefrontal cortex (PFC) and other forebrain regions may play a critical role in the pathophysiological changes underlying cognitive and social behavioral deficits in SCZ patients. Recently, we demonstrated that activation of the mGlu1 subtype of metabotropic glutamate receptor increases inhibitory transmission in the PFC by selective excitation of somatostatin‐expressing GABA interneurons. Genetic studies in humans reveal an association between deleterious non‐synonymous single nucleotide polymorphisms (nsSNPS) in of the human gene encoding mGlu1 (GRM1) and SCZ, raising the possibility that mGlu1 signaling is critical to the function of brain circuits underlying symptoms associated with this disorder.

Here, we examine whether novel positive allosteric modulators (PAMs) selective for mGlu1 reverse electrophysiological effects and behavioral deficits in an MK‐801‐induced animal model of SCZ. We used ex vivo whole‐cell patch‐clamp electrophysiology to show that MK‐801 decreased the frequency of spontaneous inhibitory postsynaptic currents onto layer V pyramidal cells of the PFC, and this cortical disinhibition was reversed by mGlu1 activation. Furthermore, acute MK‐801 treatment induces inhibitory deficits onto PFC‐basolateral amygdala (BLA), but not PFC‐nucleus accumbens, projecting layer V pyramidal cells that can be reversed by selective mGlu1 activation. PFC‐BLA projections are known to contribute functionally to social‐approach behavior, while the BLA plays a critical role in object recognition and cognition broadly. We found that the mGlu1 PAM VU6004909 effectively reversed MK801‐induced deficits in social interaction and social novelty preference in a three‐chamber assay. Additionally, VU6004909 restored novel object recognition following MK‐801 treatment. In combination with our previous reports that mGlu1 PAMs reverse deficits in inhibitory transmission in the PFC, the current study provides strong evidence for mGlu1 PAMs as a promising novel approach for the treatment of social and cognitive deficits in SCZ patients.
Using Google Tools to Increase Student Learning, Engagement, and Collaboration in Remote, Hybrid, and In‐person Courses
Authors: Stokes Jennifer A.
Published: May 2022

The COVID‐19 global pandemic turned the educational community upside‐down. As all in person coursework quickly transitioned to online delivery, faculty had to formulate new ways to not only disseminate course content but also assess student learning and engage students in the virtual classroom. As the pandemic state continues to affect higher education, the use of online collaborative tools has become essential to many college classrooms. This poster reviews the implementation of a selection of Google tools (e.g. Docs, Forms, Jamboard) in remote, hybrid, and in‐person courses.

Initially, these online tools were added to engage students when strict COVID‐19 guidelines were in place in the hybrid and remote classroom setting as a way to promote student collaboration and group learning. In class activities, homework, and other group assignments were modified such that both in‐person and remote students could collaborate in real time during the synchronous class session and outside of scheduled class time. Student feedback on course evaluations indicate that the learning environment was positive, collaborative, and engaging. Student comments specifically pointed to the success of these activities. Students ranked the course greater than 4.0 (out of 5.0) across all evaluative questions.

In the spring 2020 hybrid version of the course, summative assessments were modified to include open‐book take‐home case studies in addition to the timed objective exam. Required biweekly virtual small group sessions allowed students to collaborate on these challenging case‐based critical thinking problems. The case study exams were designed in Google Docs and a template file (copy function) was created for student use. Overall student perception of the case study exams was very high as indicated by the students' ranking of this activity on a survey and written feedback. Additionally, student performance improved on the case study exams (p < 0.001) as their critical thinking, analytical, and scientific writing skills improved, while their performance on the timed objective exams remained relatively consistent (p = 0.157).

Examples of how these online tools can be set‐up for use as formative and summative assessments as well as interactive, collaborative activities will be presented and shared using QR codes at the poster session. As coursework has returned to in‐person learning, many of these tools are still used due to ease of use, positive feedback, and benefits to student learning.
Coupling auxin‐inducible degradation with quantitative proteomics for in‐vivoprotein functional characterization
Authors: DeMarco Andrew G. , Hall Mark C.
Published: May 2022

Functional characterization of proteins typically involves disrupting expression by genetic deletions, mRNA knockdowns, or by inhibiting function with conditional mutations, followed by observation of concomitant biological effects. These methods, however, are prone to indirect effects resulting from the permanent or long‐term changes to the target protein expression/function. In contrast, the auxin‐inducible degradation (AID) system allows very rapid depletion of endogenous target protein levels, thereby minimizing deleterious effects from long‐term perturbation caused by some traditional methodologies. Combining the AID system with quantitative proteomics in applicable model systems allows detection of proteome changes immediately following target protein degradation. The rapid depletion achieved by AID is well‐suited to studying the biological specificity of regulatory enzymes and confidently identifying their direct substrates. Our lab is interested in understanding the role of phosphatases in the regulation of the cell cycle. We demonstrate the power of combining AID and proteomics in the budding yeast, S. cerevisiae, by targeting components of protein phosphatase 2A (PP2A), an abundant heterotrimeric phosphatase with numerous substrates involved in multiple cellular processes, including the cell cycle. The AID system achieves ≥ 85% reduction in the abundance of PP2A components within 10‐15 minutes after auxin addition, resulting in detectable phosphoproteome changes by 20 minutes. The rapid response time allows us to selectively probe the proximate phosphoproteome changes enriched with direct PP2A substrates while minimizing long‐term, indirect effects resulting from slower transcriptional changes and cellular adaptation responses. Current work is focused on applying this system to studying the contributions of different PP2A complexes at different cell cycle stages, providing novel information on PP2A regulatory contributions to this fundamental biological process. The AID system coupled with proteomics is broadly applicable for studying the role of almost any protein in diverse physiological settings and model organisms.
Lipid Expansion Microscopy
Authors: White‐Mathieu Brittany M. , Conwell Amanda , Wu Kane , Baskin Jeremy
Published: May 2022

Membrane architectures whose dimensions and features are smaller than the diffraction limit of light orchestrate diverse cellular events such as lipid transport, vesicle formation, and calcium signaling. These structures, which include membrane invaginations, organelle contact sites, and membrane microdomains, are primarily composed of phospholipids, making methods to visualize these biomolecules vital to our understanding of cellular function. Techniques to accurately image phospholipid‐containing structures with fluorescence microscopy are challenged by the diffusion of lipids within the bilayer, even in fixed samples. Expansion microscopy (ExM) utilizes hydrogel formation to fix biomolecules in place and swell samples to produce high‐resolution images of protein‐ and nucleic acid‐containing cellular structures 30‐70 nm in size. Using chemical reporter metabolites and a novel multifunctional fluorophore probe, here we present Lipid Expansion Microscopy (LExM), which enables the high‐resolution imaging of phospholipids with a tunable expansion factor using the principles of ExM. We will present key synthetic and technological advances critical to the development of LExM as well as its application to visualize nanoscale membrane structures, phospholipid‐organelle colocalization, and the spatial component of flux through specific lipid biosynthetic pathways within intact cells.
Mapping myocyte microdomains with optogenetic cAMP signaling
Authors: Haar Lauren , Kinney William , Lawrence David
Published: May 2022

Recent advances in biosensor and optically based techniques have provided a map of the subcellular regions that dictate myocyte behavior. Microdomain level changes of cyclic adenosine 3’5’ monophosphate (cAMP) signaling are known to impact the progression of cardiovascular disease. Yet, tools to direct these site‐specific interactions are limited. To address this issue, we have applied a set of optogenetic protein species with the spatiotemporal resolution necessary to direct cAMP signaling at distinct intracellular sites. We hypothesize that applying these proteins to a cardiac cell system will help resolve the relationship between cAMP microdomain signaling and cardiac cell behavior.

Photoactivation studies have been conducted using optogenetic analogues of adenylate cyclase and protein kinase A (PKA) in the H9c2 cell model. Expression and subcellular localization of these light responsive proteins is characterized by live cell microscopy in this cardiac cell line. Site‐specific impacts of signaling are quantified using a genetically encoded PKA activity biosensor as well as pharmacological agonists/antagonists for cAMP signaling mediators and effectors.

Results indicate that global cAMP production, through a light activatable adenylate cyclase (bPAC), leads to phosphorylation of a PKA reporter anchored at the outer mitochondrial membrane and plasma membrane. Reporters with nuclear localization do not show this phosphorylation. Experiments validate the expression and recruitment of a light directed protein kinase A ‘optogenetic switch’ analog in cardiac cells. The expression and recruitment are confirmed with live cell microscopy and demonstrate a unique ability to direct PKA activity and retention to specific intracellular subdomains. The impact of this signaling on microdomain organization as well as the site‐specific impacts of light directed PKA microdomain recruitment on cell proliferation, size as a function of biomass, and cell viability compared are considered. This is done in relationship to non illuminated (dark) and catalytically dead optogenetic protein analog controls.

We conclude from these results that light directed signaling in cardiac myocytes can provide a site‐specific view of intracellular signaling cascades. The application of this approach represents a ‘first step’ in creating therapies that address the microdomain signaling disruptions associated with cardiac disease.
Inhibition of SUMO‐mediated Protein‐Protein Interactions in DNA Damage Repair
Authors: Jones Emily E. , Thu Yee Mon
Published: May 2022

Maintenance of genomic integrity requires functional repair mechanisms. Some mechanisms include the use of sumoylation, a process in which proteins are post‐translationally modified with a small peptide SUMO (small ubiquitin‐like modifier). In response to DNA damage, multiple proteins become sumoylated. Current evidence suggests that sumoylation in this context mediates protein‐protein interactions to promote DNA damage repair. SUMO‐interacting motifs (SIMs) play an essential role in SUMO‐mediated protein‐protein interactions. The SIM on a protein is a region that allows it to recognize and bind the SUMO attached to another protein. Previous studies have shown that inhibiting this type of interaction can lead to sensitized cancer cells by impairing DNA damage repair and response. Thus, this project aims to study a similar inhibition by overexpressing SIMs naturally found in two proteins, Slx5 and Sgs1, in the eukaryotic model organism, Saccharomyces cerevisiae. Both proteins are a part of their respective protein complexes, and their SIMs serve as a functional way for the complexes to interact with sumoylated proteins during DNA repair. We hypothesize that overexpressed SIMs will disrupt endogenous SUMO‐SIM interactions important for DNA damage repair and that cells with overexpression of the SIM would be sensitized to DNA damaging agents. We observed that cells overexpressing SIMs from Slx5 exhibited a growth defect compared to cells without overexpression. However, when treated with DNA damaging agents, such as methyl methanesulfonate and UV, cells with SIM overexpression exhibited little to no sensitivity compared to the wild type.

To further explore the effect of SUMO‐SIM interaction, we have designed another protein containing the DNA‐binding (SAP) domain of Siz2, a SUMO protein ligase specific to DNA damage, and the SUMO‐binding domain of Ulp1, a SUMO isopeptidase, to potentially disrupt SUMO:SIM interactions involved in DNA repair. Inclusion of the SAP domain along with a nuclear localization signal (NLS) is hypothesized to increase the nuclear localization of the protein during DNA repair, which will be observed using fluorescent microscopy of a GFP tag incorporated into the protein. With increased nuclear localization and inclusion of the SUMO‐binding domain, we hypothesize that there will be disruption to the SUMO:SIM interactions involved in DNA damage repair, resulting in an increase in the cells’ sensitivity to DNA damaging agents.
Deletion of Myostatin Resolves Myosteatosis and Improves Angiogenesis in Obese Mice
Authors: Speese Andrew , Padgett Caleb , Bridgewater Cody , Mintz James , Corley Zachary , Fulton David , Stepp David
Published: May 2022

Andrew C. Speese, B.S.1, Caleb A. Padgett M.S.1, Cody L. Rosewater M.S.1, Zachary L. Corley B.S.1, James D. Mintz M.B.A.1, David J. Fulton Ph.D.1,2, and David W. Stepp Ph.D.1,3

1Vascular Biology Center, 2Department of Pharmacology and Toxicology, and 3Department of Physiology, Medical College of Georgia, Augusta University, Augusta, GA

Objective

To test the hypothesis that increased muscle mass achieved through deletion of myostatin improves ischemic angiogenesis in obese mice.

Methods

db/db mice, a well‐described model of obesity, were crossed with mice lacking myostatin (MSTN KO), a myokine that negatively regulates muscle differentiation and growth, to generate lean and obese mice with and without elevated muscle mass (MSTN KO). We utilized confocal and electron microscopy (EM) and nuclear magnetic resonance (NMR) spectroscopy to assess morphology and histology in the skeletal muscle (SKM) of db +/‐_MSTN +/‐ mice. Muscle lipid contents were assessed via mass spectrometry. Femoral artery ligation was used to stimulate in vivo angiogenesis in response to ischemia. Gene expression was assessed by RNA‐Seq with mRNA and protein expression follow‐up in isolated gastrocnemius skeletal muscle via RT‐qPCR and western blotting.

Results

As previously described, myostatin deletion results in significantly increased muscle mass without altering whole‐body mass, fat percentage, or activity levels in obese db/db mice. Blood glucose is markedly improved but plasma lipids remain elevated. In parallel with these improvements, we observed that obesity inhibits and MSTN deletion restores vascularization following hind limb ischemia. Despite persistent plasma lipedema, ectopic lipid deposition in skeletal muscle was largely ameliorated in obese mice lacking myostatin, a reduction largely explained by a decrease in tissue triglyceride levels. EM and confocal microscopy revealed accumulation of lipid in obese muscle cells that was lacking in obese mice with MSTN KO. The resolved deposition occurred predominantly inside muscle cells with fat cell infiltration in external spaces largely unaffected. RNA‐Seq analysis of skeletal muscle revealed marked upregulation of lipogenesis pathways, notably the expression of stearoyl Co‐A desaturase 1 (SCD1), the rate limiting enzyme for the oleate synthesis and subsequent triglyceride production. SCD1 was elevated in muscle from normal obese mice but not those lacking myostatin at both the protein and RNA level.

Conclusion

In summary, increased muscle mass in obese mice provides potent protection to vascular and metabolic health. A potent correlate of this improvement is the resolution of myosteatosis in obese, hypermuscular mice. These data suggest that resolution of myosteatosis, independent of muscle mass, may afford similar protection, possibly by targeted deletion or blockade of SCD1.
Derivatives of 3,4‐dihydroxyhydrocinnamic acid at the 6‐position as mechanistic probes of L‐DOPA dioxygenase
Authors: Steiner Jessica , Xhafkollari Gisella , Strzeminski David J. , Leyes Porello Sebastian , Colabroy Keri L. , Peterson Larryn W.
Published: May 2022

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make antibiotics and other bioactive materials as well as degrade plant material, but we have a limited understanding of the breadth and depth of substrate space for these powerful enzymes. To this end, we report the syntheses, redox potentials and pKa s of 3,4‐dihydroxyhydrocinnamic acid (DHHCA) derivatives substituted at the 6‐position and their characterization as substrates of L‐DOPA dioxygenase from Streptomyces lincolnensis. The cleavage of diverse catecholic substrates is an important element of bioremediation. Extradiol dioxygenase cleavage of DHHCA derivatives also promises to yield insight into mechanism and provide synthons for various applications.
Intermittent Hypoxia Destabilizes Histone Deacetylase 5 via Reactive Oxygen Species Dependent Dephosphorylation
Authors: Nanduri Jayasri , Wang Ning , Prabhakar Nanduri R.
Published: May 2022

We recently identified histone deacetylase (HDAC5) protein as an early epigenetic regulator of HIF‐1 mediated transcription of pro‐oxidants resulting in intermittent hypoxia (IH)‐induced sympathetic activation (Wang et al Frontiers Physiology 2021). However, underlying molecular mechanisms of decreased HDAC5 protein by IH remain unknown. In this study, we show that reactive oxygen species (ROS) generated by NADPH oxidases contribute to IH‐induced HDAC5 destabilization in pheochromacytoma 12 (PC12) cells. Treating IH exposed cells with phosphatase but not kinase inhibitors stabilized HDAC5 protein. Furthermore, overexpression of mutant HDAC5 by substituting S259 with alanine decreased HDAC5 protein expression. Pharmacological treatment with Calyculin A, a serine/threonine protein phosphatase (PP1 and PP2A) inhibitor or overexpression of NIPP1 (endogenous nuclear PP1 inhibitor) restored HDAC5 protein in IH exposed cells. Taken together these data suggest that dephosphorylation of S259 by PP1 destabilizes HDAC5 protein in response to IH. Our results demonstrate that ROS‐dependent activation of phosphatases constitute another layer of cellular responses to gene activation by IH distinct from previously identified protein kinase mechanisms contributing to IH induced autonomic morbidities.
Gene‐to‐gene damaging variant rate analysis uncovers novel Alzheimer’s disease‐associated genes
Authors: Zhang Jianhua , Mritunjay Pandey , Emma Fikse , Adam Awe , Nicole Lue G. , Claire Kittock M. , Chen Weiping , Yang Yanqin , Poorni Adikaram R. , Nirmal Jacob , Greenfest‐Allen Emily , Rachel Thomas , Zhang Bomeny L. , Wang Xiaowen , Li Yulong , William Simonds F.
Published: May 2022

Alzheimer’s disease (AD) is a progressively debilitating neurodegenerative disease that exacts a heavy toll on our social and health care systems. AD is presumably caused by pathogenic variants in a large number of genes working in concert with environmental factors and personal behaviors. However, despite years of worldwide efforts, only a few dozen AD‐associated genes have been identified to date. Employing the gene‐to‐gene damaging variant rate (g2gDVR) analysis of published datasets containing 181,388 subjects we discovered 2114 potentially AD‐associated genes, of which 711 were linked to the higher AD prevalence among African‐Americans. The DVRs of these genes in AD population (55,460 subjects found in NIAGADS) were at least two‐fold higher than those in the general population (125,748 subjects in gnomAD) with false discovery rate less than 1x10‐12. The advantage of the g2gDVR method for studies of disease is that it focuses on variants expected to impact gene function, in contrast to methods based on SNP analysis, such as GWAS, that are gene‐agnostic. In combination with transcriptome profiling of 21 brain regions from 2728 human AD and control samples (collected from various GEO datasets) we identified three of the 711 genes (GNB5, SNAP91 and AKAP11) that were candidate causative genes based on their reduced expression in both hippocampus and entorhinal cortex regions of AD patient brains. In the context of the APP/PSEN1 mouse model of AD, we further confirmed that Gnb5 heterozygosity exacerbates the formation of both amyloid plaques and neurofibrillary tangles, the hallmarks of AD brain neuropathology. These results suggested that the g2gDVR analysis method is highly effective in identification of AD‐associated genes and is expected to be broadly applicable to other polygenic diseases.
Establishing Dietary Iron Requirements for Mouse and Rat Models of Hereditary Hemochromatosis
Authors: Lee Jennifer K. , Yu Yang , DeLellis Regina R. , Ebea Pearl , Zhu Sean , Theophilus Rufus , Collins James F.
Published: May 2022

The established iron requirement for laboratory rodents is 35‐50 ppm, but standard rodent chows contain up to 350 ppm iron. Excess dietary iron exacerbates iron accumulation in hepcidin (Hamp) knockout (KO) mice and rats, which model the iron‐overload disorder hereditary hemochromatosis (HH) in humans. Establishing iron requirements is important to allow for proper experimental design when studying Hamp KOs since WT littermates are not iron‐loaded (and thus not an appropriate control group for iron‐loaded Hamp KOs). We hypothesized that Hamp KOs have iron requirements at least 50% below those of WT animals, given that intestinal iron absorption is inappropriately elevated (by 2‐3‐fold) in the KOs. In the present study, WT and Hamp KO mice and rats of both sexes were weaned onto one of four AIN‐93G purified rodent diets, with the following iron concentrations (in ppm): 5‐7 (low), 17‐20 (low marginal), 24‐27 (high marginal), or 45‐49 (adequate). After 6 weeks on the diets, bioindicators of iron status were assessed, including blood hemoglobin content and hematocrit, serum ferritin and nonheme iron content, tissue nonheme iron concentration, and blood transferrin saturation. Outcomes showed that significant iron loading occurred in Hamp KO animals when fed the high‐marginal and adequate iron diets, as indicated by higher serum ferritin and nonheme iron content, elevated hepatic iron levels and increased TSAT. Iron loading, however, did not occur when the KO rats and mice were fed the low‐marginal iron diets. Animals fed the low‐iron diet, however, developed IDA. We thus conclude that the optimal amount of dietary iron that will not result in iron loading post‐weaning in Hamp KOs, while also not causing iron depletion, is ~22 ppm. Consistent with our hypothesis then, Hamp KO rodents require about half as much iron as WT controls.
Structure Activity Relationships of Illicit Fentanyl Analogs
Authors: Moore Sierra , Anand Jess , Dixon Emma , Perrien‐Naccarrato Carmelita , Traynor John R.
Published: May 2022

Fentanyl is a highly potent and efficacious mu opioid receptor (MOR) agonist with significant abuse potential. More than 70% of opioid overdose deaths involve fentanyl or one of its analogs; recently the DEA announced that 2 in 5 counterfeit prescription pills contain a lethal dose of fentanyl. However, little is known about the novel fentanyl analogs (fentalogs) flooding the illicit drug market. It is presumed that these novel fentalogs are also potent, highly efficacious MOR agonists and they are driving the increase in opioid overdoses, but pharmacological data on these compounds are scarce. We have determined the affinity for and agonist activity of a library of novel fentanyl analogs in vitro. We found that structure greatly alters affinity and agonist activity; in this library, affinity at MOR ranged from 0.4 nM to almost 400 nM – fentanyl displays 1.6 nM affinity for MOR. Based on the data collected from this library, we have been able to construct structure‐activity relationships which may allow us to predict which fentalogs are potent agonists and are likely to carry significant abuse liability. Conversely, we have discovered new high affinity, low efficacy compounds which could be developed into novel rescue therapies.
Canagliflozin Regulates Human Endothelial Cell Function: Role of Heme Oxygenase‐1
Authors: Durante William , Behnammanesh Ghazaleh , Durante Giovanna L. , Peyton Kelly J.
Published: May 2022

Sodium‐glucose cotransporter‐2 (SGLT2) inhibitors are the newest class of glucose lowering drugs that act by inhibiting glucose reabsorption in the proximal tubule of the kidney. Recent clinical trials found that SGLT2 inhibitors reduce cardiovascular disease and mortality in patients with diabetes. The mechanism underlying the salutary actions of these drugs is not completely clear; however, it does not involve differential improvements in glycemic control. Given the critical role that endothelial cells (ECs) play in maintaining vascular homeostasis, the present study investigated the effect of the SGLT2 inhibitor canagliflozin on EC function. It also determined if the vasoprotective protein heme oxygenase‐1 (HO‐1) contributes to the cellular actions of the drug. Treatment of human umbilical vein ECs with clinically relevant concentrations of canagliflozin blocked cell proliferation, and this was associated with a pronounced decline in DNA synthesis. Canagliflozin also inhibited the migration of ECs following scrape injury in a concentration‐dependent manner. Incubation of ECs with a high concentration of glucose (25mM) and oxidized low‐density lipoprotein (oxLDL;50mg/L), which mimics the diabetic milieu, stimulated the adhesion of monocytes to ECs. However, treatment of ECs with canagliflozin markedly reduced the binding of monocytes to these cells. In addition, canagliflozin increased the expression of HO‐1 in a time and concentration‐dependent fashion, leading to a significant rise in endothelial HO‐1 activity. Finally, inhibition of HO‐1 activity or expression increased the adhesion of monocytes to glucose/oxLDL‐challenged ECs exposed to canagliflozin but had no effect on the proliferative or migratory response of canagliflozin‐treated ECs. In conclusion, the present study identifies canagliflozin as a key regulator of EC function. Moreover, it demonstrates that the induction of HO‐1 by canagliflozin contributes to the anti‐inflammatory action the drug. Thus, canagliflozin may exert some of its favorable effects in diabetes by limiting endothelial inflammation via the upregulation of HO‐1.
Low Blood Pressure is Independent of Plasma Renin in the Bmal1 Knockout Rat
Authors: Rhoads Megan K. , Pollock David M.
Published: May 2022

Blood pressure (BP) is a crucial physiological parameter that exhibits a circadian rhythm in healthy individuals. Loss of this rhythm is associated with morbidity and mortality. To determine the contribution of the molecular clock to BP regulation, we developed a rat strain with whole body loss of Bmal1 gene expression,a core component of the transcription/translation feedback loop known as the molecular clock. We previously reported that Bmal1‐/‐ rats of both sexes have rhythmic BP, but lower than Bmal1+/+ controls. Given the importance of the renin‐angiotensin system in BP control, we hypothesized that loss of Bmal1 may reduce plasma renin activity in the Bmal1‐/‐ rat. For this experiment, male rats were housed in a 12:12h light/dark cycle with constant humidity and temperature. Water and food (0.49% NaCl) were available ad libitum. BP was measured by a telemetry transmitter implanted in the abdominal aorta of 10‐week‐old rats. After a 10‐day surgical recovery period, BP was recorded for 2 minutes every 10 minutes at 1000 samples/second. Mean BP, assessed by cosinor analysis, was lower in male Bmal1‐/‐ (MBP MESOR: 109±2 mmHg, n=9) animals compared to littermate Bmal1+/+ controls (MBP MESOR: 117±3 mmHg, n=8, p<0.05, ANOVA). In a separate cohort of animals, plasma was collected from male Bmal1‐/‐ rats and littermate Bmal1+/+ controls across the circadian day every four hours in groups of 2‐6 at each time point starting at zeitgeber time (ZT) 0 (07:00) and ending at ZT 20. Plasma renin activity (PRA) and total prorenin/renin were measured by ELISA (name the kits used). PRA was similar between male Bmal1‐/‐ and Bmal1+/+ rats during the light (Bmal1+/+: 1.4±0.5 vs Bmal1‐/‐: 2.5±0.6 ng/mL, p=0.21, Student’s t‐test) and dark (Bmal1+/+:2.4±0.7 vs Bmal1‐/‐: 2.4±0.5 ng/mL, p=0.97, Student’s t‐test) periods. Plasma total prorenin/renin was similar between Bmal1‐/‐ and Bmal1+/+ rats during the light (Bmal1+/+: 8.8±1.7 vs Bmal1‐/‐: 6.9±0.7 ng/mL, p=0.36, Student’s t‐test) and dark (Bmal1+/+: 2.7±1.3 vs Bmal1‐/‐: 5.2±1.7 ng/mL, p=0.27, Student’s t‐test) periods. These data suggest that loss of Bmal1 does not directly affect systemic plasma renin activity or total prorenin/renin concentrations in the rat. Furthermore, the lowered blood pressure observed in rats lacking Bmal1 does not appear to be a result of systemic changes in circulating renin activity or concentration.
Cardiac Fibroblasts Secrete Galectin‐1 After Myocardial Infarction to Communicate With Macrophages
Authors: Chalise Upendra , Lindsey Merry
Published: May 2022

The cardiac fibroblast is the key cell type regulating scar formation after myocardial infarction (MI). Whether the fibroblast also serves a role in the early pro‐inflammatory phase has not been evaluated. For this study we evaluated secretome from no MI day (D) 0 and MI D1 cardiac fibroblast to understand the roles of D1 fibroblast in cellular communication during the pro‐inflammatory phase of MI wound healing. Cardiac fibroblasts were isolated from day (D) 0 (n=3) left ventricle and MI D1 (n=3) infarct region, and the 24 h secretome was collected from passage 2 cells. We used mass spectrometry to catalogue and quantify the secretome. We identified galectin‐1 as a highly upregulated D1 MI secreted factor. Because galectin‐1 is known in other systems to be anti‐inflammatory, we stimulated bone marrow derived macrophages (1.5x106 cells/ well; n=8 4M/4F paired sets) with galectin‐1 (500 ng/ml) in the presence of interleukin (IL)‐1b (200 ng/ml) or IL‐10 (50 ng/ml). The conditioned media was assayed using proteome profiler mouse XL cytokine array (RnD) and immunoblotting validation. We also used a human glycosylation array 1000 (RayBio) to evaluate plasma from healthy controls (n=18) and patients 48h after MI (n=41). Galectin‐1 was 3‐fold elevated in the MI D1 secretome (p=0.007). Galectin‐1 increased IL‐10 release (p=0.02) in the macrophages treated wtih IL‐10, a finding validated by two methods. In humans, Galectin‐1 was 2.2‐fold increased at 48h after MI compared to healthy controls (p=0.013). In conclusion, the fibroblast secretes galectin‐1 to modulate the macrophage response after MI by stimulating anti‐inflammatory macrophage polarization and potentially improve MI wound healing.
Exposure to Doxorubicin Modulates the Cardiac Response to Isoproterenol in a Sex‐Dependent Manner
Authors: Agostinucci Kevin , Grant Marianne K. O. , Melaku Wongel , Abdelgawad Ibrahim Y. , Zordoky Beshay N.
Published: May 2022

Introduction

Subclinical cardiotoxicity occurs in cancer patients who receive low doses of anthracyclines such as doxorubicin (DOX), which can develop into overt cardiomyopathy when triggered by other cardiovascular stressors. Female rodents are protected from DOX‐induced cardiac dysfunction in experimental studies. However, the sex‐related differences in DOX‐induced increased susceptibility to later‐onset cardiovascular stressors are yet to be determined. Therefore, in the present study, we determined the effects of a low dose of DOX on the cardiac response to catecholamine stress induced by isoproterenol (ISO) in male and female mice. Since sex hormones are believed to mediate the sexually dimorphic response to DOX, we determined whether gonadectomy is able to reverse the observed response.

Methods

Five‐week old intact and gonadectomized male and female C57Bl/6 mice were administered saline or DOX (4 mg/kg/week for 5 weeks by intraperitoneal injections). After a 5‐week recovery period, mice were administered daily subcutaneous injections of ISO (10 mg/kg) or saline for 14 days. Cardiac function was measured by echocardiography before, immediately after the first dose of ISO (stress echo), and following 14 days of daily ISO injections. Hearts were harvested and weighed at the end of the experiment.

Results

Stress echo shows that the positive chronotropic effect of ISO was blunted in DOX‐treated male and female mice, while the positive inotropic effect is maintained. Following 14 days of daily ISO injections, saline‐treated male mice exhibited a hypertrophic response to ISO, indicated by an increase in the heart weight‐to‐tibia length, while DOX‐treated male mice did not. In addition, ISO caused more reduction of cardiac output in DOX‐treated male mice than that in saline‐treated male mice. On the other hand, both saline‐treated and DOX‐treated female groups exhibited a hypertrophic response to ISO. ISO treatment for 14 days did not cause significant reduction in cardiac output in either saline‐treated or DOX‐treated female mice. Similar experiments were performed in a cohort of gonadectomized male and female mice. Intriguingly, sexually dimorphic responses are observed in gonadectomized mice similar to those observed in intact mice.

Conclusion

Exposure to DOX modulates the cardiac response to isoproterenol in a sex‐dependent manner. DOX treatment abrogates the adaptive hypertrophic response to ISO in male mice, making them more susceptible to declining cardiac output. Deprivation of sex hormones by gonadectomy did not reverse this sexual dimorphism, suggesting that other mechanisms are implicated. Further research is warranted to determine the mechanisms of this sexual dimorphism.
Characterization of molecular basis of anti‐platelet effect of daphnetin
Authors: Chaudhary Preeti K. , Kim Sanggu , Kim Soochong
Published: May 2022

Coumarin derivates have been known for their antithrombotic, anti‐inflammatory, and antioxidant activities and daphnetin is one of the natural coumarin derivatives isolated from Daphne Korean Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect is not studied till date.

We investigated the functional role of daphnetin and its molecular basis on the regulation of platelet activation using murine platelets.

In order to check the effect of daphnetin on GPVI‐mediated platelet response, we first measured the effect of daphnetin on collagen‐induced platelet aggregation and secretion. Collagen‐induced platelet aggregation and dense granule secretion were partially inhibited in the presence of daphnetin. Interestingly, 2‐MeSADP‐induced secondary wave of aggregation and secretion were completely inhibited in the presence of daphnetin. It has been known that 2‐MeSADP‐induced secretion and the resultant secondary wave of aggregation are mediated by positive‐feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2‐MeSADP‐induced platelet aggregation in aspirin‐treated platelets where the contribution of TxA2 generation is blocked. Additionally, AYPGKF‐induced platelet aggregation and secretion were inhibited in the presence of daphnetin by blocking the positive‐feedback effect of TxA2 generation. Moreover, 2‐MeSADP‐induced TxA2 generation was completely inhibited in the presence of daphnetin confirming the role of daphnetin on TxA2 generation. Finally, 2‐MeSADP‐induced ERK phosphorylation was inhibited in the presence of daphnetin.

We conclude that daphnetin plays a role in platelet function by inhibiting the TxA2 generation through the regulation of ERK phosphorylation.
Development of Myotropic Extracellular Vesicles for Targeted Delivery of Therapeutics to Skeletal Muscle
Authors: Shuler Kyle T. , Mitchell Andrew D. , Muñoz Eric R. , Sperduto John L. , Balascio Gabriel M. , Wilson Brittany E. , Hudson Matthew B.
Published: May 2022

Muscular pathologies comprise a family of diseases and disorders resulting in the degeneration of skeletal muscle tissue. Current therapeutics are non‐specific to skeletal muscle, which often reduces efficacy and causes harmful off‐target effects in patients. Thus, a myotropic (muscle‐targeted) drug delivery system is needed to shuttle therapeutics to muscle cells to improve efficacy and limit off‐target effects. The objective of this investigation was to develop biological lipid nanoparticles, extracellular vesicles (EVs), with enhanced myotropism. We hypothesized that displaying myotropic transmembrane proteins at the surface of EVs would improve delivery of molecular cargo into muscle cells. To this end, we displayed the myotropic transmembrane proteins MyoMaker (MYMK), MyoMixer (MYMX) and M‐Cadherin (M‐CAD) in the membranes of EVs from Human Embryonic Kidney (HEK293) cells. Using flow cytometry, we measured the incorporation of each protein candidate into the EVs via endogenous green fluorescent protein (GFP) tags included on the cytosolic c‐terminus of each protein. We then measured the delivery of fluorescently‐labeled protein by each myotropic EV candidate in mouse (C2C12) myotubes. In addition to using EVs as delivery vehicles for exogenously loaded therapeutics, the native EV cargo may also be of therapeutic relevance. Given this, we examined the endogenous protein cargo of non‐engineered HEK293‐EVs via mass spectrometry. MYMK displayed the highest degree of incorporation into EVs relative to control HEK293‐EVs (p = 0.006). The MYMK‐EV formulation displayed the greatest increase in protein cargo delivered into myotubes relative to control HEK293‐EVs (~125%, p = 0.0005). Proteomic analysis revealed the top biological processes and molecular functions of HEK293‐EV protein cargo to be ribonucleic acid binding, processing, and metabolism, potentially altering translation in recipient cells. The in vitro data in the present study establish the feasibility of using MYMK‐EVs as a myotropic delivery system. Future investigations are needed to fully elucidate the cell/tissue tropism and physiological effects of MYMK‐EVs in vivo.
Epigenetic regulation of Anoctamin1 in pulmonary arterial hypertension
Authors: D'Silva Natalie M. , Wang Eric R. , Fernandez‐Nicolas Ana , Vang Alexander , Yano Naohiro , Zimmer Alexsandra , Fedulov Alexey V. , Zhang Peng , Choudhary Gaurav
Published: May 2022

Introduction/Hypothesis

Pulmonary arterial hypertension (PAH) is associated with substantial pulmonary vascular remodeling wherein endothelial cells (ECs) within the lumen hyper‐proliferate leading to occlusive plexiform lesions. We previously reported that Anoctamin‐1 (ANO1) expression is increased in lung ECs from patients with PAH and is associated with increased EC proliferation. However, the mechanism of ANO1 expression regulation in PAH remains unclear. DNA methylation has been recognized as an important mechanism in gene regulation. In this study, we test the hypothesis that ANO1 expression in PAH is epigenetically mediated via DNA methylation.

Methods/Results

DNA was extracted from lung ECs of patients with idiopathic PAH (n=7) and controls (n=6), followed by Bisulfite Next‐Generation Sequencing (EpigenDx, MA) to determine DNA methylation profile in human ANO1 gene (Ensembl Gene ID: ENSG00000131620). We used 19 assays to cover 97 CpG sites in the human ANO1 gene, including regions of 5’‐upstream, 5’‐UTR, and different introns. Our data show that the methylation of 29 of 97 (30%) CpG sites in human ANO1 gene is significantly altered in lung ECs from PAH patients than that from control subjects. Among the significantly altered 29 CpG sites, the methylation at 22 CpG sites (76%) is significantly decreased. To determine whether decreased DNA methylation changes human ANO1 gene expression, we then exposed normal human pulmonary artery ECs to RG108, which is a DNA methyltransferase inhibitor that broadly reduces DNA methylation. Our data show that cells treated with RG108 for 72 hours have a significant 2‐fold increase in ANO‐1 mRNA expression in comparison to control cells, suggesting that decreased DNA methylation is sufficient to upregulate human ANO1 expression. We are developing CRISPR‐based approaches to target these identified CpG sites to determine the specific contribution of their methylation in regulation of human ANO1 gene.

Conclusions

Our results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH.
Difelikefalin, a peripherally restricted kappa opioid receptor agonist, evokes diuresis through a central kappa opioid receptor pathway
Authors: Meariman Jacob , Sutphen Jane , Gao Juan , Kapusta Daniel
Published: May 2022

Kappa opioid receptor (KOR) agonists produce a variety of beneficial effects such as nonaddictive analgesia and diuresis, but their translation into the clinic has been hindered by central adverse effects (dysphoria). In clinical studies, difelikefalin, a KOR agonist recently FDA approved for treatment of pruritus in hemodialysis patients, was shown to be devoid of producing adverse CNS effects suggesting that this compound is peripherally restricted. This is of interest from a renal excretory perspective, since it would be predicted that difelikefalin would not produce diuresis, which is a classical centrally mediated response produced by KOR agonists. However, in preliminary studies in rats we have shown that IV administration of difelikefalin markedly increased urine output and decreased urinary sodium and potassium excretion similar to that produced by other KOR agonists. Therefore, we hypothesized that difelikefalin activates a central KOR pathway to cause diuresis. To test this hypothesis, Sprague‐Dawley rats were implanted with bladder, femoral artery, and femoral vein catheters and administered an isotonic saline infusion. To delineate the central and/or peripheral site of action of difelikefalin, renal excretory function was measured in rats pretreated centrally or peripherally with norBNI (KOR antagonist) or vehicle via an intracerebroventricular (ICV) cannula or IV catheter. Following stabilization, conscious rats were administered an IV bolus of difelikefalin (10 µg/kg) and urine output, mean arterial pressure (MAP), and heart rate (HR) were recorded for 90 minutes. The results demonstrated that ICV norBNI pretreatment significantly decreased the diuretic response to IV difelikefalin but had no effect on urinary sodium or potassium concentration (Table 1). In contrast, IV norBNI pretreatment prevented the diuresis to IV difelikefalin and increased urinary sodium/potassium concentration (Table 1). Notably, ICV and IV norBNI pretreatment abolished the difelikefalin‐induced decrease in MAP that was also seen in the vehicle pretreated groups. Together, these findings suggest that difelikefalin produces its diuretic and blood pressure effects through a central KOR pathway, while the sodium/potassium retaining effects of difelikefalin are mediated through peripheral KORs.