United States

Background. In addition to providing the energy for cardiac muscle contraction and building blocks for cellular homeostasis, nutrient metabolism also generates signaling molecules for the regulation of cardiac functional and structural remodeling under various conditions. While the alteration of these metabolic signals has been linked to the development of heart failure, the molecular mechanisms involved remain incompletely understood. Olfactory receptors (ORs) are G-protein-coupled receptors which play a pivotal role in the body’s response to chemical stimuli from the external environment. Although initially believed to be exclusively located at the olfactory epithelium, ORs have been detected in many other tissue and organs where they allow for cellular adaptation to changes in metabolite levels in the bloodstream. Recent work from our lab and others has identified OR51E2 as a cardiac OR. 
Hypothesis. We hypothesize that cardiac expression of OR51E2 is altered by the development of metabolic diseases and heart failure. We further anticipate that that the dysregulation of OR51E2 expression plays a role in cardiac adaptation to stress. 
Methods. In order to determine the impact of metabolic derangements on cardiac OR51E2 expression, OR51E2 mRNA levels were quantified by real-time PCR in the normal mouse heart (n=4) and compared to that of hearts from mouse models of obesity and type 2 diabetes (ob/ob mice, n=4; db/db mice, n=4). The same method was used to quantify cardiac OR51E2 mRNA levels in normal rats (n=8) and rats rendered diabetic via intraperitoneal injection of streptozotocin (STZ; n=10). Western blotting was used to correlate mRNA data with OR51E2 protein expression levels in the mouse hearts. Next, OR51E2 mRNA levels were quantified in hearts from mice subjected to transverse aortic constriction (TAC)-induced pressure overload (n=10) in order to determine whether the development of heart failure affects OR51E2 expression. 
Results. The expression of OR51E2 increased by 36-43% in hearts of ob/ob mice (p=0.010) and STZ-treated rats (p=0.024) when compared to hearts from non-obese and non-diabetic animals. While no changes were detected at the mRNA level for hearts of db/db mice, OR51E2 protein levels were found to increase by 83% (p=0.001). Lastly, the failing heart of mice subjected to TAC-induced pressure overload also exhibited higher OR51E2 mRNA levels when compared to sham-operated mice (p=0.001). 
Conclusions. Our results demonstrate that cardiac OR51E2 is upregulated in states of metabolic dysregulation and hypertrophic heart failure. Future investigations will aim to identify the nature of the signals transduced by OR51E2 in the mammalian heart, and the consequences of their dysregulations for cardiac structure and function during disease states.

AMA Research Challenge 2023

A Potential Role for the Olfactory Receptor OR51E2 in Cardiac Adaptation to 
Metabolic and Hemodynamic Stress

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Background
Atherosclerosis is a chronic inflammatory disease that contributes to over 50% of deaths in western societies. Adult men are at a unique risk because estrogen provides a well-documented atheroprotective effect for premenopausal women. Therefore, this study focuses on development of lesions in adult male mice. Atherosclerosis is characterized by the formation of plaques, lipid-filled deposits on arterial walls. Late stage plaques are susceptible to rupture, leading to life-threatening events like stroke and myocardial infarction. Plaques are primarily composed of foam cells, or macrophages laden with cholesterol. The formation of foam cells is mediated by various receptors to engage in endocytosis. One key enzyme in this process is Protein Kinase C epsilon (PKCε), which catalyzes the addition of membrane lipids to a forming phagosome. In vitro, PKCε deficient macrophages are unable to efficiently phagocytose and thus PKCε has a demonstrated potential to be atheroprotective. To study this potential in vivo, C57Bl/6 mice underwent Cre-Lox recombination to create a mεKO strain, which lacks PKCε in myeloid cells. We predict that the mεKO mice, which are PKCε deficient, will accumulate more plaque burden than WT mice.

Methods
At 8 weeks, mice were injected with the PCSK-AAV8 virus to induce hypercholesterolemia by inhibiting LDL receptors in the liver. This state was further reinforced by a high fat (“Western”) diet. Serum was collected during this time to confirm hypercholesterolemia. 16 weeks post injection, the aortic arch and descending thoracic aortas of WT and mεKO mice were collected. These samples were pinned flat, stained with Oil Red O, a fat soluble dye, to mark areas of plaque development, and photographed. Using ImageJ, sample area was standardized across all aortas and a color threshold was determined as a double-blinded method to calculate percent lesion coverage or “plaque burden.”

Results
WT mice (n = 8) had an average plaque burden of 1.393% (+/- 1.141%) while mεKO mice (n=7) had a significantly higher average plaque burden of 5.820% (+/- 2.411%). The p-value of a Student’s t-test is <0.001 (***).

Conclusions
The results confirm our hypothesis and demonstrate that PKCε may play a role in preventing lesion development in atherosclerosis. Additionally, current research suggests that plaque composition may be as important as plaque area in determining prognosis; mεKO mice developed visibility more opaque plaques than WT mice, and analyzing differences in composition may be productive.

Deletion of myeloid protein kinase C epsilon exacerbates atherosclerosis

Pregnancy requires significantly increased cardiac output (~30%) achieved through peripheral vasodilation and increased stroke volume, due to increased end-diastolic volume. Most women resolve these physiological changes within 6 months of delivery; however, some women progress to heart failure late in pregnancy or in the immediate post-partum period. Peri-partum cardiomyopathy (PPCM) is a rare condition of largely unknown etiology which, at its most-severe and life-threatening, requires cardiac transplantation. Pre-eclampsia occurs at much higher rates in the PPCM population (~40%) compared with the generally pregnant population (~2-5%). We have previously shown that transgenic mice expressing the human-specific isoform of the TBXA2R gene (TPβ) spontaneously develop pre-eclampsia accompanied by all the associated hallmarks expected from the human disease. We now show TPβ transgenic (TPβ-Tg) mice are a model of PPCM. Using trans-thoracic echocardiographic imaging, TPβ-Tg mice had no basal phenotype; however, unlike C57/Bl6J (WT) mice that resolved the pregnancy-induced cardiac remodeling, TPβ-Tg mice had exaggerated cardiac dilatation at term (E18.5; P<0.01) which increased in severity over the subsequent 6 months (P<0.005). In addition, TP-Tg mice exhibited reduced ejection fraction (22±13% vs 67% in WT), significant LV wall thinning (1.12±0.16 mm vs 1.6±0.17 mm WT), and loss of LV mass (66.55±9 mg vs 95±11mg WT). Analysis of myocardium of human PPCM patients undergoing heart transplant (n=12) compared to healthy myocardium from non-diseased donors (n=42) by RT-PCR and IHC showed highly expressed TPβ in PPCM patients. In human PPCM samples, there was a significant (p<0.01) dysregulation of genes associated with circadian rhythm as determined by RNAseq analysis. Quantitative PCR analysis of hearts from non-pregnant (WT=8; TPβ-Tg=4) and pregnant (WT=10; TPβ-Tg=13) mice found similar dysregulation of multiple genes associated with circadian rhythm compared to WT mice (p<0.01). This indicates that PPCM may be associated with failure to terminate CLOCK signaling as a result of TPβ activation. Our results support dysregulation of TBXA2R splicing as part of the pathogenesis of PPCM and identify new therapeutic targets for this condition.

Characterization of a Novel Clinically Relevant Mouse Model of Postpartum Cardiomyopathy

Background: Cardiomyocytes are the contractile muscle cells of the heart. A key developmental feature of mammalian cardiomyocytes is their terminal differentiation early in life and inability to proliferate following myocardial injury. As cardiomyocyte function is directly correlated to cellular metabolism and mitochondrial respiration, we aim to explore their respiration early in life when the cells have proliferative capabilities. Based on previous work in the lab, we hypothesize that our dual cellular ATP indicator and continuous oxygen monitoring sensor will be useful in analyzing the mitochondrial respiration changes in early cardiomyocytes.
Methods: To test the hypothesis, this study used isolated neonatal mouse cardiomyocytes which are useful cell models as the cells undergo proliferation for 7 days following their isolation. To culture these cells, hearts from mouse pups were harvested at an age of 1 to 3 days. The tissue was lysed and enzymatically digested to isolate the cardiomyocytes. Following isolation, the cells were cultured for around 7 days. A novel genetically encoded fluorescent ATP indicator of cytosolic and mitochondrial ATP (smacATPi) was transduced in the cultured cardiomyocytes to monitor the dynamic changes of ATP in response to metabolic inhibitors. Continuous recording of oxygen consumption of the cardiomyocytes was achieved using a Recipher cultured cell oxygen sensing system. The drug 2-deoxy-D-glucose (2-DG), a glycolysis inhibitor, was applied to the cells.
Results: Evaluating changes in mitochondrial respiration and ATP production was performed by live cell imaging using a Cytation 5 imaging system. Cardiomyocytes with green and red fluorescence were identified, indicating the feasibility of the adenovirus-mediated smacATPi expression in cultured cardiomyocytes. Furthermore, the respective changes in green (cytosolic ATP) and red (mitochondrial ATP) fluorescence in response to 2-DG were observed. The oxygen consumption rate of the cultured cardiomyocytes was continuously monitored over a 7-day period using the online oxygen sensing device. Analysis of the data collected by the above methods demonstrates the predicted changes in mitochondrial respiration following drug treatment in that mitochondrial respiration decreases in response to the metabolic inhibitor, 2-DG.
Conclusion: Experimentation demonstrates that the methods employed in this study provide insight into mitochondrial respiration changes in early cardiomyocytes and how cellular respiration responds to different metabolic inhibitors. For future investigations, evaluating real-time spatiotemporal cellular ATP dynamics and cellular respiration may help identify novel insights into previously unknown biochemical mechanisms in cardiomyocytes for the development of effective treatments of cardiac disorders.

Oxygen Consumption and ATP Production in Neonatal Mouse Cardiomyocytes

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules derived from molecular oxygen (O2), which can cause DNA damage and lead to skin cancer. NADPH oxidase 1 (Nox1) is a major producer of ROS in the skin upon exposure to ultraviolet light. Functionally, Nox1 forms a holoenzyme complex that generates two superoxide molecules and reduces NADPH. The signaling activation occurs when the organizer subunit Noxo1 translocates to the plasma membrane bringing a cytochrome p450 through interaction with Cyba. We propose to design inhibitors that prevent Cyba-Noxo1 binding as a topical application to reduce UV-generated ROS in human skin cells. 

Inhibitor design started from an apocynin backbone structure to generate a small molecule to serve as an anchor point, inhibitor 1, followed by adding a polyethylene glycol linked biotin in inhibitor 2. Further in vitro experiments using isothermal calorimetric (ITC) were used to quantify the binding affinity (KD). ITC and MALDI (TOF) MS was also used to show if binding of a Cyba peptide to Noxo1 was blocked. In vivo experiments were performed using donated skin explants with topical application of the two inhibitors while exposing the samples to UV. 

Inhibitors 1 and 2 were nontoxic in human cells, and successfully increased cell viability after UV exposure. Biophysical using ITC and MALDI revealed biotin as not necessary for inhibition while confirming affinity between inhibitor 2 and Noxo1. Further in vitro experiments using isothermal calorimetric binding quantification showed that inhibitor 2 bound a Noxo1 peptide with a KD of 2nM. Both using isothermal calorimetric binding and MALDI (TOF) MS showed that the binding of a Cyba peptide to Noxo1 was blocked. Experiments show that in primary skin explant models with ultraviolet light exposure, inhibitor 2 reduced the amount of cyclobutene pyrimidine dimers in DNA, a molecule known to lead to carcinogenesis. 

These findings demonstrate a novel inhibition of Nox1, which serves as a basis for further research to determine if this inhibitor can be added to topical sunscreens for public use in hopes of decreasing UVR-induced DNA damage and subsequent carcinogenesis. In addition, this novel sunscreen formulation could be a non-toxic alternative to chemical based barrier sunscreens that are currently on the market. 


Serendipitous Identification of a NOXO1 Inhibitor By Addition of a Polyethylene Glycol Chain

Background
Ceramides play an essential role in the formation of the stratum corneum, and include a long-chain sphingoid base in an amide linkage with one or more fatty acids (FAs). Skin contains many unique FAs, particularly esterified omega (EO)-hydroxy(OH)ceramides with ultra-long FA chains, which are critical to the CLE.
Atopic Dermatitis, a common pediatric skin disorder with cutaneous inflammation and barrier impairment, is recognized to have a deficiency of ceramides, including EO-OH ceramides, based on targeted lipidomic analyses. Untargeted lipidomics (Q Exactive HF OrbiTrap) is a new technology that, as RNA-Seq or proteomics, allows comprehensive comparison of diseased vs healthy tissues but has yet to be described for skin.

Methods
We extracted lipids from tape strips of lesional atopic dermatitis (AD-L) and nonlesional (NL) skin from 31 AD patients (0.2-17.3 yrs old, mean 7 yrs; 51% female; 58% White) and 14 age- and site-matched healthy controls (C) for untargeted lipidomic analysis.

Results
PCA plots showed distinct separation between AD-L and C skin, regardless of sex or age. 637 distinct ceramide species were detected in AD and 616 in C skin, with 291 decreased in AD-L vs. C and 242 reduced in AD-L vs NL skin. Total normalized signal response of ceramides decreased 84% in AD-L and 51% in NL skin vs C (p < 0.001). Most ceramide classes decreased by >80% in AD-L vs C skin (EO by 88-93%; ODS/ADS 91%; NP 90%; NH 87%; NSD 83%; OH/AH 83%; and OSD/ASD 82%).
We found a new ceramide class (m), overall decreased in AD-L by 81%. A few species were increased in EOSD, OH/AH, OSD/ASD, NDS, NH, NP, and NS classes. Ceramide changes in NL skin were all in AD-L. Decreased AD-L ceramides correlated with eczema severity indices: SCORAD and EASI scores (Spearman r= –0.4 to -0.57, p < 0.001); m-ceramides with EASI and Pruritus ADQ scores (r= -0.43 and r= -0.44, p < 0.01). Increases of NDS in AD-L correlated with EASI (r=0.48, p < 0.01); and NH, NP, and NS with Itch VAS (r= 0.73 to 0.93, all p < 0.05).

Conclusion
The discovery of a new ceramide class (m), which is found to be decreased in AD-L highlights the value of untargeted lipidomic analyses for understanding skin disorders and in clinical trials that can target specific ceramides in the metabolic pathway.


Untargeted Lipidomic Analysis from Atopic Dermatitis Stratum Corneum Reveals Novel Ceramide Defects

INTRODUCTION: Snail, also known as the “land helix,” has been used in traditional and contemporary medicine to treat various ailments and has provided dermatological benefits in human history. Snail secretions have been shown to improve wound healing, relieve pain, and prevent associated complications due to their antimicrobial, anti-inflammatory, and anti-aging properties. We performed a literature review on topical snail derivatives and their potential to treat different dermatological conditions and the limitations of each publication. 

METHODS: A literature search was performed using 324 search terms with combinations using Boolean operators. Inclusion and Exclusion criteria were used to review the relevance of each paper. Literature analysis included two randomized controlled trials that fit both inclusion and exclusion criteria. 

RESULTS: Two studies illustrated the use of snail derivatives on dermatological conditions in humans and mice, respectively. A double-blinded randomized controlled trial showed significant improvement in periocular and perioral rhytides when using topical combination serum of Cryptomphalus aspersa after 12 weeks of treatment. Similarly, another randomized controlled trial showed significant improvement in wound healing when using topical snail secretion from Helix aspersa after 14 days of treatment. 

DISCUSSION: Literature has shown that snail mucus has properties that allow improvement of skin appearance thanks to its use as emollient with moisturizing and lubricating properties. Growth factors present in snail mucus have can reduce the presence of wrinkles, leading to anti-aging properties. Additionally, snail mucus aids in collagen production, a key factor in wound healing, and decreases inflammatory cells and cytokine release, which are usually seen in chronic inflammation. 

CONCLUSION: Results from this literature review show that topical application of snail mucus can have beneficial dermatological outcomes, such as anti-aging properties and improved wound healing. More research is needed to investigate additional therapeutics and cosmetic effects of snail secretion on the skin.


A Systematic Review of the Dermatological Benefits of Topical Snail Derivatives

• Silicone and other Polymers are used to create simulated skin for task trainers and other products in health care simulations.
• Silicones are created and sold in varying firmness termed durometer.
• Platinum cured Silicones are fixed after cure and cannot be melted or
reformed after they are cut or punctured.
• A newer Polymer that is thermoplastic (able to be reshaped and formed by melting) - is called synthetic ballistics gelatin or Styrene ethylene butylene styrene (SEBS).

Adjusting durometer to create more realistic soft- tissue in synthetic ballistics gelatin

Pathologic white adipose tissue (WAT) expansion is known to occur under conditions of metabolically “unhealthy” obesity. Such obesity is marked by physiologic abnormalities and depends on the location of WAT depots, as well as an individual’s comorbidities. Hypoxic conditions, in particular, can shift the direction of metabolic pathways, with the signaling molecule HIF1α playing a large role in driving a cascade resulting in adipose tissue expansion. This study aimed to identify the specific mitochondrial metabolic changes that arise in WAT precursor cells in response to HIF1α induction. WAT was extracted from mice inguinal fat depots and the stromal vascular fraction, containing adipose precursor cells, was isolated and plated. They were treated with either of two HIF1α inducers, COCl2 or DMOG, or left without treatment as controls. Targeted metabolomics through gas chromatography-mass spectroscopy (GC-MS) was utilized to assess levels of both glycolysis-related metabolites and tricarboxylic acid (TCA) cycle intermediates produced within the cells. Compared to the control group, COCl2 significantly increased lactate levels (t(10) = 5.93, p < .001) and α-ketoglutarate levels (t(10) = 6.75, p < .001) while significantly decreasing malate levels (t(10) = -5.03, p < .001). Both COCl2 (t(10) = -8.12, p < .001) and DMOG (t(10) = -2.69, p < .05) significantly decreased citrate levels compared to the control group. All other metabolites assessed showed no significant difference between treatment and control groups. The metabolomics data revealed dysfunctions of glycolysis and the TCA cycle upon treatment of adipose precursor cells with inducers of HIF1α. A potential cause of this may have been a reliance on reductive glutamine metabolism to drive α-ketoglutarate into fatty acids as an alternate energy source under hypoxic conditions. Understanding the molecular controls at play in this process is critical to preventing the adverse effects of metabolically unhealthy obesity, such as fibrosis and organ damage. Previous literature has shown that there is the potential to modify the fate of adipose precursor cells to prevent them from committing to a fibrotic phenotype. By incorporating metabolic data similar to that produced in this study on HIF1α with findings on modifying adipose precursor cell fate, it may be possible to prevent or treat unhealthy obesity from a clinical standpoint.

HIF1α Signaling to Target Mitochondrial Metabolism for Regulation of Adipose Precursor Cell Fate

Introduction 
Pre-eclampsia is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality, complicating about 4% of pregnancies. Recent work has identified PERIOD3 (PER3), a gene regulating 24-hour circadian rhythms, to be downregulated in the preeclamptic placenta. The role of circadian rhythms in the placenta largely remains unknown. Our goal is to establish an organotypic placental preparation to allow screening of drugs that regulate circadian rhythms. These studies are a first step towards understanding how circadian rhythm changes are caused in the placenta and will be the basis for future studies to understand how circadian rhythm disruption in the placenta cause pregnancy complications, such as preeclampsia.

Methods 
We established a novel organotypic placenta preparation allowing to study circadian rhythms in a LumiCycle using gestation day (GD) 14 and GD18 placental explants from the validated circadian reporter mouse Per2::Luciferase. Using H&E we validated the explants to be >80% enriched in either decidua (DC) which is strictly of maternal origin, labyrinth (LB) which is strictly of fetal origin, and the junctional zone (JZ) which consists of binucleated cells fused of maternal and fetal cells.

Results 
Through H&E staining, we validated by distinct morphology that the DC is enriched with neutrophils; the JZ contains spongiotrophoblasts and glycogen trophoblasts; whereas the LB has anucleated red blood cells. We next established a protocol to record Per2::Luciferase circadian rhythms for <6 days. We found that the Per2::Luciferase circadian rhythm shortens with increased gestational age, where the period in DC, LZ and LB in the GD14 placenta is ~4h longer than in the GD18 placenta, showing an unexpected plasticity in circadian timekeeping in the placenta. Because PER3 is downregulated in the pre-eclamptic placenta, we asked if the explants would be useful for pharmacologically screenings. We applied PF670462 (1μM), which stabilizes PER1/2/3, or epidermal growth factor (EGF, 30ng/mL), a growth factor important in placental function and a PER2 enhancer. PF670462 lengthened Per2::Luciferase period in the DC and JZ, confirming the expected circadian rhythm stabilizing effect of PF670462. Surprisingly, EGF only modulated circadian rhythms in the GD14 placental, where EGF shortened Per2::Luciferase period in DC, and lengthen the period in the JZ and LB zones in the GD14 placenta. This data suggests EGF has gestational age and placenta zone specific effects.

Conclusion 
We validated a novel ex vivo placental preparation to study circadian rhythms at GD14 and GD18. We identified a placenta zone and gestation age specific effect of EGF, an effect future studies will further expand on. This model will be useful to complete pharmacological screenings of drugs that can restore circadian rhythms in the preeclamptic placenta.

Validation of a Novel Placenta Explant Protocol Allowing
Screening of Drugs Modulating Circadian Rhythms in the Mouse Placenta

Objective / Hypothesis: Cell replacement therapy is rapidly emerging as a viable cure for Type 1 Diabetes Mellitus; however, the supply of insulin-producing cells for transplantation is severely limited due to a shortage of pancreatic islet donors. Current strategies include generating stem-cell derived pancreatic islets (SC-islets) for eventual implantation, but current methods generate functionally immature cells (mostly due to lack of vascularization and the importance of endothelial cell proximity), which complicates clinical translation. Our goal with this project is to create functionally competent insulin-secreting cells with a built-in vascular network. We hypothesize that the orthogonal differentiation of human induced pluripotent stem cells (h-iPSCs) into beta cells and vascular cells will enable the development of SC-islets with a built-in vasculature.

Approach: We developed an orthogonal differentiation strategy using doxycycline-inducible transgenes encoding key transcription factors to induce differentiation of h-iPSCs into endothelial cells (iECs) and mural cells (iMCs). We are using our differentiation protocol that generates SC-islets from human embryonic stem cells (hESCs) through a multi-stage procedure reminiscent of pancreatic development to generate islet endocrine cells. In the initial phases of the experiment, we used INS1 insulin-secreting rat insulinoma cells in place of SC-islet cells due to their ease of use (no need for >31-day differentiation protocol) and direct translatability to the behavior of SC-islet cells in vitro. These cells were co-cultured and studied by functional assays, imaging, and flow cytometry.

Results: Both transcription factor-based differentiation methods enabled highly efficient differentiation of human iPSCs into competent vascular cells (iECs and iMCs) – h-iPSCs were rapidly and robustly differentiated by simple exposure to doxycycline (4-day process, >95% efficiency). These cells were phenotypically and functionally validated, with the iECs staining positive for CD31, CD144, and VE-Cadherin, and iMCs staining positive for Calponin1, Vimentin, and SM22. These results were confirmed with flow cytometry. Using the INS1 cell line, co-culture of these cells in different ratios of INS1:iEC:iMC did show integrated tube formation reminiscent of vasculature at day 8 (80:18:2 and 91:9:1). In contrast, other ratios (such as 50:50 and 66:33 INS1 to iEC) did not show tube formation, and instead formed segregated cell clusters in which iECs, iMCs, and INS1 cells did not integrate.

Summary / Conclusions: This data shows promising results and suggests an in vitro model for creating an intra-islet vasculature may be achievable, as we did see endothelial/mural cell tube formation in our experiments when using INS1 cells, iECs, and iMCs. Future studies may include additional functional testing and analysis, such as using GSIS to quantify insulin secretion and sc-RNA-seq to study maturation markers pre- and post-differentiation. Once the orthogonal differentiation protocol shows significant success, transplantation studies into animal models could demonstrate how these islets function in vivo.


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