bluuebunny/crossref_metadata_2025 huggingface dataset:
Delta‐9‐Tetrahydrocannabinol (THC) Self‐Administration in Male and Female Long Evans Rats
Authors: Wakeford Alison GP , Wetzell Bradley B , Pomfrey Rebecca L , Clasen Matthew , Taylor William , Riley Anthony L
Published: November 2023

Rationale

Rodent preclinical assessments of THC self‐administration have failed to provide a robust and reliable animal model for the widespread use of marijuana in humans. Possibilities for this failure may be related to a) the lack of combining non‐psychoactive cannabinoids when examining THC self‐administration (given that marijuana derivatives abused by clinical populations contain cannabinoids other than THC) and b) the absence of assessments of sex differences in the acquisition of THC self‐administration (given that both clinical and preclinical data suggest that women may be more susceptible to drug use and abuse).

Methods

THC self administration was examined in male and female Long‐Evans rats under an FR1 schedule and across three doses (0.003, 0.01, 0.03 mg/kg/infusion). After each animal received five 2‐hour sessions with each dose (for a maximum of 15 days of intravenous self‐administration), they were then assessed for cocaine self‐administration (0.25 mg/kg/infusion; positive control) for 5 days or for THC self‐administration following pretreatment with cannabidiol at either a 1:1 or 1:10 CBD:THC ratio (all animals received both treatments, but were counterbalanced as to which treatment was received first).

Results

Discrimination between the active and inactive lever was found for almost all animals at the 0.03 mg/kg/infusion dose, but no sex differences appeared between active lever presses or number of infusions. All animals acquired cocaine self‐administration and received more infusions of cocaine than any THC dose examined with females self‐administering more cocaine than males. Overall, cannabidiol failed to impact THC self‐administration, although there were instances in which individual subjects were affected.

Discussion

The failure to find robust THC self‐administration under optimized conditions suggests THC acts as a weak reinforcer in rodent models of self‐administration. These results will be discussed in the context of previous self‐administration research in rodents and primates, along with future research projects that may elucidate mechanisms or other conditions under which rodents might robustly self‐administer THC in an effort to better understand and possibly treat marijuana abuse and dependence.

Support or Funding Information

Funding for this project was provided by American University's Office of Graduate Studies.
Physiological and Pharmacokinetic Effects of E‐cigarette Type Exposure to Δ9‐Tetrahydrocannabinol (THC)
Authors: Nguyen Jacques D. , Aarde Shawn M. , Vandewater Sophia A. , Grant Yanabel , Stouffer David G. , Parsons Loren H. , Cole Maury , Taffe Michael A.
Published: November 2023

The increase in availability of electronic cigarettes (e‐cigarettes) for delivery of nicotine has led to the adaptation of these devices for the delivery of marijuana. Although there have been studies investigating the effects of intrapulmonary delivery of Δ9‐tetrahydrocannabinol (THC), no studies have investigated the effects of e‐cigarette type exposure in rodents. The goal of this study was to determine if vapor delivery of THC using e‐cigarette technology would produce cannabinoid‐typical effects observed in rodents using other routes of administration. Male Wistar rats received an injection of THC (0.3–10 mg/kg,i.p.) or were exposed to vaporized THC or crude marijuana extract in propylene glycol (PG) vehicle (200 mg/mL and 400 mg/mL respectively; 4 puffs per 5 min for 10–40 min). Rats were tested for changes in body temperature and nociception, and plasma THC content was analyzed using fast liquid chromatography/mass spectrometry (LC/MS). Vapor exposure to THC or crude extract significantly reduced body temperature up to 165 min following vape initiation. Tail flick latency was assessed using a hot water bath (52°C), and results showed increased latency up to 60 min post‐vape initiation. Plasma THC levels following a 10 mg/kg intraperitoneal injection were similar to levels produced by 30 min of inhalation exposure to THC in PG. Our results demonstrate that intrapulmonary delivery of THC using‐e‐cigarette type devices produces physiological effects, and this study validates this novel method of intrapulmonary THC delivery in rats.

Support or Funding Information

DA024105 and DA035482
The Effects of Δ9‐Tetrahydrocannabinol (THC) Drug History on THC Taste and Place Conditioning
Authors: Hempel Briana , Wakeford Alison , Clasen Matthew , Friar Mary , Riley Anthony
Published: November 2023

Rationale

Δ9‐Tetrahydrocannabinol (THC) produces both aversive and rewarding effects in animal models. Given that the balance between a drug's affective properties is thought to underlie its abuse potential, it is important to consider these properties of THC and how they (and their balance) are impacted by various factors. In this context, previous studies have examined the effect of THC history on the aversive and rewarding effects of THC. However, such assessments have been made in different animals under different experimental procedures, making it difficult to assess the relative contribution of either affective property to abuse vulnerability.

Methods

In the present experiment, male Sprague‐Dawley rats were administered six pre‐exposure injections (3.2 mg/kg THC) over 12 days. This was followed by a combined taste avoidance/place preference procedure in which a novel saccharin solution and environment were paired with THC (0, 1 or 3.2 mg/kg). Changes in preferences and aversions for the drug‐paired stimuli were evaluated in final place and taste conditioning tests.

Results

Relative to control subjects, THC produced robust taste avoidance, but did not induce place conditioning. Pre‐exposure to THC attenuated THC‐induced taste avoidance and had no effect on place conditioning with THC.

Conclusions

Similar to prior work, THC induced robust and dose‐dependent taste avoidance. The failure to see THC‐induced place preferences is likely due to the aversive effects of THC masking its rewarding properties. Although THC pre‐exposure weakened the aversive effects of THC (as evidenced in the attenuated taste avoidance), some avoidance was still evident (THC‐injected animals drank less than controls). This suggests that the failure to see a change in THC‐induced place preferences following THC pre‐exposure was a function of its residual aversive effects limiting its relatively weak rewarding properties. These findings support the view that the interaction of reward and aversion is important in behavioral displays of the affective properties of abused drugs.

Support or Funding Information

This research was supported by a grant from the Mellon Foundation to Anthony Riley
Characterization of the Behavioral Phenotypes Underlying the Reinforcing Effects of Cannabinoid Receptor Agonists in Rhesus Monkeys
Authors: John William , Nader Michael
Published: November 2023

The lack of a widespread, suitable method to establish robust levels of Δ9‐tetrahydrocannabinol (THC) self‐administration (SA) remains an impediment for developing treatments for human marijuana abuse. Therefore, the major objective of this study was to establish the experimental conditions necessary for demonstrating intravenous SA of THC as well as the cannabinoid receptor (CBR) agonist CP 55,940 in rhesus monkeys. In addition, behavioral phenotypes underlying the individual differences in CBR agonist SA were characterized in order to determine critical variables that could be modified to optimize the procedure. Rhesus monkeys (n=8), implanted with indwelling intravenous catheters, served as subjects and were trained to respond under a fixed‐ratio 10 schedule of food presentation, with a 60‐second timeout after each reinforcer. Once responding was stable, saline was substituted for food pellets for at least 5 consecutive sessions and until responding was extinguished (>80% reduction in responding for 3 consecutive sessions). After re‐establishing food‐maintained responding, CP 55,940 (0.005–1.0 μg/kg) and THC (0.03–10 μg/kg) were substituted for food pellets, with each dose available for at least 5 sessions and until responding was deemed stable. There was a return to food‐maintained responding, for at least 3 sessions, between different CBR agonist doses. CP 55,940 functioned as a reinforcer in three of eight rhesus monkeys at doses lower than previously tested in monkeys. Interestingly, in those three monkeys, non‐contingent administration of CP 55,940 was least potent in decreasing food‐maintained responding indicating an inverse relationship between the rate‐decreasing effects and reinforcing effects (r=0.81, p<0.05). No relationship was found between the reinforcing effects and CP 55,940‐induced hypothermia nor the unconditioned behavioral effects of the dopamine D2/D3 receptor agonist quinpirole. In those monkeys for which CP 55,940 functioned as a reinforcer, THC (0.03–3.0 μg/kg per injection) did not maintain responding higher than vehicle. Therefore, future experiments will determine if tolerance to the rate‐decreasing effects of THC will enhance the reinforcing effects of THC. Such an outcome would suggest that preclinical models require a period of repeated drug treatment before assessing THC SA.

Support or Funding Information

Supported by DA06634 and T32AA007565.
Sex differences in alcohol‐induced HPA activation following systemic vs icv administration in rats: Gene expression studies
Authors: Wakley Alexa , Davis Seth , Yap Parry , Pham Charleen , Selvage Dan John
Published: November 2023

Understanding how alcohol differentially affects the hypothalamic‐pituitary‐adrenal (HPA) axis in males vs. females is important for the development of sex‐specific pharmacological treatments for alcohol abuse. Our lab previously reported that centrally delivered alcohol activated hypothalamic‐pituitary‐adrenal (HPA) axis of rats in a sex‐ and estrous cycle‐dependent manner. This effect was greatest in females in stages of the estrous cycle when the effects of estradiol (E2) are high (proestrus and estrus), as these animals showed greater levels of HPA‐linked hormonal and neuronal activation by alcohol compared to males and to females in stages of the cycle when E2 levels are low (diestrus I and II). In the current study, we used male and normally cycling female rats receiving either intracerebroventricular (icv) or intraperitoneal (i.p.) alcohol injection to determine if there are differences between the ability of central versus systemic alcohol to cause sexually dimorphic changes in the expression of genes coding for neurotransmitters and enzymes implicated in HPA regulation. We also measured levels of the pituitary stress hormone adrenocorticotropin hormone (ACTH) at the time of sampling. Brain areas assessed for changes in gene expression included the paraventricular nucleus of the hypothalamus (PVN), which regulates hormonal and neuronal HPA function, and the locus coeuruleus (LC), which sends noradrenergic (NE) projections to the PVN and has been implicated in sexually dimorphic HPA responses to homeostatic challenges. Both icv and i.p. alcohol injection significantly stimulated ACTH secretion compared to vehicle controls, with this effect being greatest in proestrous rats for both treatment groups. However, neither alcohol administration route significantly altered the expression of corticotropin releasing hormone (CRF), which drives ACTH release, in the PVN or the LC. Icv alcohol stimulated a significant increase in expression of arginine vasopressin (AVP), which is known to contribute to ACTH secretion, in the PVN of proestrous females, but not in other groups. However, i.p alcohol did not alter the expression of this gene. Icv alcohol also significantly stimulated the expression of tyrosine hydroxylase (TH), the rate limiting enzyme for NE synthesis, in the PVN of all female groups and the LC of proestrous females, but not in males. Conversely, i.p. alcohol administration did not significantly alter TH expression in any groups. These results indicate that non‐CNS specific effects of alcohol in the periphery may alter gene responsiveness in areas of the brain that regulate HPA function.

Support or Funding Information

R01 AA016947‐01A1
The Neurotoxic Effects of Manganese on Dopamine Post‐Synaptic Receptors Are Reversed by p‐Aminosalicylic Acid (PAS)
Authors: Dubose Loren , Loney‐Walsh Kurt , Carroll Margaret A , Catapane Edward J
Published: November 2023

Manganese is a neurotoxin that causes the human disease Manganism. It disrupts dopamine neurotransmission in the brain, however the neurotoxic mechanism of action is not fully resolved and there is no effective clinical treatment for it. Reports in the literature postulate that manganese toxicity is more related to dysfunction of the post‐synaptic dopamine D2 receptors (D2DR) rather than degeneration of dopamine neurons. Our previous work showed that gill lateral ciliated cells of Crassostrea virginica have post‐synaptic dopamine receptors that are D2 type and are innervated by cilio‐inhibitory dopamine fibers via the branchial nerve. We also found that manganese treatments block the cilio‐inhibitory effects of dopamine on gill lateral cells and that treatments with manganese in the presence of the drug p‐aminosalicylic acid (PAS) prevented this toxic effect. Our previous immunofluorescence work with 1° antibodies against D2DR and FITC‐linked 2° antibodies showed that treatments with manganese caused reduced fluorescent intensity in the gill lateral cells, suggesting that manganese caused a loss of D2DR number or disruption of their structural integrity. In this study we hypothesized that PAS would reverse the neurotoxic effects of manganese on D2DR when applied after manganese exposure. To test this C. virginia were treated for up to 3 days with manganese (500 μM) followed by 5 more days with PAS (500 μM). Gills were excised, fixed, exposed to 1° antibodies against D2DR and FITC‐linked 2° antibodies, paraffin embedded, sectioned at 10 μm and then visualized on a fluorescence microscope. Fluorescence intensity was quantified using ImageJ software from NSF. The results showed that the fluorescence intensity in animals treated with manganese had a statistically significant progressive decrease in fluorescence of up to 40% less than non‐manganese treated controls. Animals treated with PAS after manganese and animals treated only with PAS did not show reduced fluorescence, indicating PAS reversed the manganese induced loss of post‐synaptic D2DR fluorescence. This immunohistological study shows a positive correlation between the losses of D2DR fluorescence in manganese treated animals vs controls. It also shows PAS effectively reversed the toxic effects of manganese on D2DR after 3 days of manganese treatments. The actions of PAS are likely due to its ability to chelate manganese. The results suggest that manganese chelating agents like PAS that can penetrate the human blood‐brain barrier might be effective therapeutic agents for Manganism.

Support or Funding Information

This work was supported in part by 2R25GM06003 of the Bridge Program of NIGMS and 0516041071 of NYSDOE.
Deorphanizing Renal Olfactory Receptor 1393
Authors: Shepard Blythe D , Peterlin Zita , Firestein Stuart , Pluznick Jennifer L
Published: November 2023

Olfactory receptors (ORs) are G protein‐coupled receptors which serve to detect odorants in the nose. Recently, ORs have been shown to play novel roles beyond odorant detection in a number of tissues, and we previously determined that a subset of ORs are expressed in the kidney. One of these receptors is Olfr1393, an orphan receptor with no known ligand. In order to determine the physiological role of Olfr1393 within the kidney, it is important to understand its ligand profile. Unfortunately, most ORs are orphan receptors, due in large part to the inability to functionally express ORs in heterologous cell systems; ORs are retained in the ER and degraded when heterologously expressed. Luckily, with the assistance of chaperone proteins and a novel cleavable tag that we developed (Shepard, et al PLoS One 2013), we were able to express Olfr1393 on the cell surface of HEK293T cells. Thus, we undertook a large scale ligand screen using a cAMP‐dependent luciferase reporter assay. We began our unbiased screen using different odorant mixtures, each containing a variety of chemicals clustered by functional group such that the mixes, in sum, cover a wide “odorant space”. However, we did not observe any Olfr1393 activation using these libraries. The murine OR gene family (consisting of ~1000 ORs) is subdivided into subfamilies, grouped based on phylogenetic clustering and protein sequence identity, and ORs within a subfamily often share similar ligands. Olfr1393 belongs to the MOR256 subfamily, and several other MOR256 family members have been previously deorphanized. Thus, we screened Olfr1393 against known MOR256 odorants and found that Olfr1393 does respond to one of these chemicals – cyclohexanone. Using cyclohexanone as a guide, we broadened our search by using a multidimensional physiochemical metric for odorant prediction and identified a total of 8 different ligands. All 8 of these ligands are structurally similar, and can be broadly described as pre‐constrained rings containing either ketone or alcohol functional groups. Notably, if the ligand is modified even slightly (with the addition or removal of a functional group) it will no longer activate the receptor. Further unbiased screens ‐including the use of a 1280 compound chemical library ‐ did not reveal any additional ligands for Olfr1393 indicating the narrow range of its ligand profile. Efforts are currently underway to determine the physiological relevance of these 8 ligands using targeted metabolomics of murine urine and plasma; discovering the source of these ligands will contribute to the understanding that Olfr1393 plays in the kidney.

Support or Funding Information

NIDDK
Studies of Ectopically Expressed Olfactory Receptor Olfr558/PSGR2: Gene Expression, Function And Novel Molecular Probes
Authors: Pronin Alexey , Volmar Claude‐Henry , Giulianotti Marc , Brothers Shaun , Slepak Vladlen Z
Published: November 2023

An orphan G protein‐coupled receptor (GPCR), Olfr558 belongs to the class of olfactory receptors. However, in addition to the nasal cavity, it is also expressed in some blood vessels and in the prostate, including prostate cancer cell lines. The human ortholog of Olfr558 (OR51E1 or PSGR2) is dramatically up‐regulated in prostate cancer and is an established biomarker of malignancy. The human and mouse genes encoding this receptor are nearly identical (95%), which is unique for GPCRs in general, and particularly for sensory chemoreceptors. This unusual evolutionary conservation points to indispensability of its physiological function. While the function of this receptor or its native ligand is unknown, in vitro it responds to butyric acid by activating adenylate cyclase with ~25 micromolar EC50. We also found that growth of human prostate cancer cells that express PSGR2 is inhibited by butyric acid. Cells not expressing this gene were unaffected by butyrate. While butyric acid can be used in such experiments, its very low affinity and selectivity complicates interpretation of the data. Thus there is clear need for novel probes that can be used to dissect Olfr558/PSGR2 function. To address this need, we first established an expression system for Olfr558 in HEK293 cells that required addition of three specific tags its cDNA and co‐transfection with three chaperone proteins. We then used this cell system for a high throughput screening of a combinatorial chemical library, which identified chemical scaffolds that have agonist, antagonist and positive allosteric modulator activities. We performed structure‐activity relationship analysis and have begun optimization and subsequent functional analysis of these compounds. Since Olfr558/PSGR2 represents a large class of previously unexplored yet druggable targets, this project facilitates investigation of the entire family of ectopic olfactory receptors.

Support or Funding Information

This work is supported by NIH Grants R01DK105427 (VZS)
GPCRomics of cardiac fibroblasts and cardiac fibrosis
Authors: Sriram Krishna , Michkov Alexander V. , Aroonsakool Nakon , Insel Paul A.
Published: November 2023

G‐protein coupled receptors (GPCRs) are the largest category of cell surface receptors, serving as the most widely used set of targets for pharmacological intervention. Seeking to identify novel targets for cardiac fibrosis, we have used mRNA‐based (GPCRomic) methods to detect and quantify (i.e., profile) non‐chemosensory “endo‐GPCRs” in cardiac fibroblasts (CFs) and, complemented these results by verification of GPCR expression/function using antibody‐based and functional analyses. We used 2D, low passage (passage 1 or 2) cultures of primary isolates of CFs and myofibroblasts (MyoFs) from humans, rats and mice.

RESULTS

We found that CFs express ~120 GPCRs, including a large number (~40–50) of orphan GPCRs, and multiple highly expressed adhesion GPCRs. Biological replicates of human CFs correlate closely in terms of identity of GPCRs expressed and the magnitude of their expression. Further, our data show that MyoFs possess a smaller (number and average expression) of GPCRs but a distinct subset of GPCRs have increased expression in MyoFs compared to CFs. Growth of CFs on soft substrates (which minimize the spontaneous transformation of cultured CFs into MyoFs) produces relatively small effects on the profile of GPCRs expressed by CFs. A subset of GPCRs remains highly and robustly expressed in CFs, regardless of substrate stiffness, pro‐fibrotic stimulation, duration of culture, or growth under hypoxic conditions. The GPCR profiles of mouse and rat CFs closely mimic that of human CFs as do the GPCR profiles of rat and human MyoFs. Further, we find that the GPCR expression profile of rat CFs is very different from that of freshly isolated rat cardiomyocytes, implying that there are numerous potential GPCR targets for cardiac fibrosis, which if used therapeutically, are predicted to have minimal on‐target effects on cardiomyocytes. We conclude that a GPCRomic approach provides novel insights regarding the regulation of CFs and MyoFs and identifies numerous potential targets for cardiac fibrosis.

Support or Funding Information

Supported by Bristol‐Myers Squibb
Establishment of induced Ovarian Cancer Stem Cells by Defined Factors
Authors: Chen Yu‐An , Lu Chen‐Yu , Cheng Wen‐Fang , Chang Nan‐shan , Ho Hong‐Nerng , Chen Hsin‐Fu
Published: November 2023

Drug resistance and metastasis are considered to be the major factors leading to tragic prognosis and high rate of mortality in cancer patients. Recent evidences suggest that cancer stem cells (CSCs) possess the essential property of self‐protection from chemotherapy, exhibit the ability to self‐renew and generate cancer cells by asymmetric cell division. Nevertheless, the currently known mechanisms likely explain only the tip of an iceberg about CSC property, acquisition and maintenance. The information from data mining is still limited due to the rarity of CSCs in clinical samples. Ovarian cancer is the most lethal malignancy of the female reproductive system. CSCs in ovarian cancers are considered to acquire the molecular armaments of stem cells, which carry stem cell property and can determine the level of malignant potential of the cancer cells, similar to the findings in other cancer cell types.

In this study, we introduced a set of defined factors (OCT3/4, SOX2, KLF4 and c‐Myc) into human serous ovarian cancer cells (OVCAR‐3 cell line) through Sendai virus reprogramming, aiming to induce CSC properties in the cancer cells. We observed that the ovarian cancer cells transduced with the four Yamanaka factors showed significantly enhanced pluripotent stem cell properties and drug resistance. We thus named the cells with CSC properties as induced ovarian CSCs (iOCSCs). These iOCSCs could be maintained and expanded up to at least 40 passages. Consistent with the original ovarian cancer cells, iOCSCs could be differentiated into ovarian lineage cells, displaying markers as CA125 and CK‐7. Intriguingly, iOCSCs also acquired the ability to differentiate into cells belonging to the three embryonic germ layers, similar to human iPS cells or ES cells.

This present approach thus makes it feasible to obtain abundant CSCs from ovarian cancer cell line to support our understanding and in‐depth research into CSCs and hopefully can help in developing new therapeutic strategies to treat ovarian cancers.

Support or Funding Information

Research supported by National Science Council of the Republic of China (NSC) and National Taiwan University Hospital (NTUH), Taiwan
Sexual dimorphism in left ventricular and electrocardiographic effects of chronic cyclosporine in rats
Authors: El‐Mas Mahmoud M , El‐Bassossy Hany M. , Banjar Zainy M
Published: November 2023

Although cardiotoxicity has been recognized as one of the adverse effects of the immunosuppressant drug cyclosporine A (CSA), limited or no information is available regarding the sex specificity of CSA cardiotoxicity. Here we tested the hypothesis that the left ventricular (LV) and electrocardiographic effects of chronic CSA and related inflammatory and histopathological derangements are sex‐related. Adult male and female rats were treated with CSA (15 mg/kg/day, sc) or its vehicle for three weeks. Afterwards, studies were undertaken to assess: (i) LV and electrocardiographic characteristics, and (ii) alterations in LV inflammatory (nuclear factor kappa B, NFκB; angiotensin receptors type 1, AT1) and histopathological profiles. In both male and female rats, CSA significantly reduced LV slope of end‐systolic pressure volume relationship (ESPVR), and isovolumic relaxation constant (Tau), suggesting LV systolic and diastolic dysfunction. Histopathologically, the treatment of either rat sex with CSA resulted in vascular congestion, blood extravasation, and pyknotic or even absent nuclei. We also report sex‐dependent electrocardiographic effects for CSA. Whereas CSA increased QTc and T peak trend intervals in male rats, markers of LV arrhythmogenesis, evidence of prolonged AV conduction was noted in female rats as reflected by the prolonged P duration and PR interval. Other sex‐related effects for CSA included (i) increased blood cholesterol, rate of rise (dp/dtmax) and fall (dp/dtmin) in LV pressure and LV content of NFκB and AT1 expression in male rats, and (ii) increased LV content of adiponectin and elevated systolic and diastolic blood pressures in female rats. Although the two rat sexes are prone to the negative LV influences of CSA, the male rat seems to be more vulnerable to inflammatory and LV adverse effects of CSA.

Support or Funding Information

This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. (166 ‐ 1001 ‐D1435). The authors, therefore, acknowledge with thanks DSR technical and financial support
Adipocyte derived factors affect cardioprotective programs initiated by short term high fat feeding
Authors: Haar Lauren , Luther Kristin , Ren Xiaoping , Wang Yang , Jones WK
Published: November 2023

Rationale

High fat mediated cardioprotection is a form of protection that utilizes short‐term high fat diet to decrease infarct size post ischemia/reperfusion injury. This protection is dependent on the increase of autophagy related proteins and the activation of NF‐κB mediated gene transcription. This study addresses the hypothesis that signaling from adipose tissue can remotely influence the cardioprotective markers associated with high fat mediated (HFD) cardioprotection.

Methods

Hormonal activation of cardiac receptors was assessed through treatment with murine recombinant adiponectin and AdipoRon peptide. Isolation of adipocyte derived exosomes was also preformed from 3T3‐L1 (ATCC® CL‐173) cells during pre differentiation, mitotic expansion, and post differentiation states. Viability of cells pretreated with each of the previously described agents was then assessed with Hoescht 33342 and propridium iodide staining before and after the administration of an oxygen glucose assay (OGD) protocol. Autophagy measurements were performed with LC‐3A/B and Beclin‐1 western blot, and the Premo Autohpagy Sensor® transduction for each treatment condition. In vivo studies were preformed in wild type C57/B6J and AdipoQ‐KO murine models, with in vivo LAD occlusion (30m) and reperfusion (24h).

Conclusion

Exosomes isolated from 3 phases during the differentiation of 3T3‐L1 (ATCC® CL‐173) cells into mature adipocytes did not increase the viability of cells after OGD treatment. Post OGD protocol, AdipoRon (25uM) treatment improved viability and cell survival relative to vehicle treated H9c2(2‐1) (ATCC® CRL‐1446) controls. Total serum levels of adiponectin were increased by an average of 15% in 24h HFD wild type animals relative to control fed wild type animals, and AdipoQ‐KO animals lacked cardioprotection at previously reported cardioprotective high fat fed time points. Future studies will focus on the assessment of in vivo hormonal signaling during high fat mediated cardioprotection.
Blocking effect of carvedilol, a beta‐blocker on Na+/Ca2+ exchange current in isolated cardiac ventricular cells of guinea pig
Authors: Watanabe Yasuhide , Kimura Junko
Published: November 2023

Background

Carvedilol has a1, b1, and b2‐adrenergic blocking effects together with the effects such as vasodilating, anti‐ischemic, anti‐oxidant, anti‐apoptotic, anti‐proliferate and anti‐inflammatory activities. Carvedilol inhibited IKr, IKs, ICa‐L and Ito in rabbit cardiac ventricular myocytes. On the other hand, metoprolol, a nonselective b‐blocker also has blocking several channels, including IK1, ICa and Ito in hearts. Murayama et al. (2013) reported that carvedilol and its analogues inhibit delayed after depolarizations (DADs) in Langendorff‐perfused rabbit hearts. It is well known that DADs are caused by the Na+/Ca2+ exchange current (INCX).

Methods

We examined the effects of carvedilol and metoprolol, two b‐blockers, on INCX by using the whole‐cell voltage‐clamp method in isolated guinea pig ventricles and CCL39 fibroblast cells expressing dog cardiac Na+/Ca2+ exchanger (NCX1).

Results

Carvedilol suppressed INCX in a concentration‐dependent manner but metoprolol did not in isolated guinea pig cardiac ventricular myocytes. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 μM and 61.5 μM, respectively. Carvedilol at 100 μM inhibited INCX in CCL39 cells expressing wild type NCX1 similarly to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 μM abolished ouabain‐induced DADs in isolated cardiac ventricular myocytes.

Conclusions

Carvedilol suppressed bi‐directional INCX in a concentration‐dependent manner with the IC50 value of approximately 65 μM. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.
Intralipid Protects against Hypoxia/Reoxygenation Injury in Human Coronary Artery Endothelial Cells and Cardiomyocytes
Authors: Salzman Michele Marie , Warner Edgewood R. , Riess Matthias L.
Published: November 2023

INTRODUCTION

Hypoxic conditions cause extensive cellular damage, particularly to mitochondria and the plasma membrane. Upon reoxygenation, incomplete reduction of oxygen (O2) can produce reactive oxygen species that can cause additional reperfusion injury to damaged and viable cells. Intralipid (IL) has been demonstrated to be cardio‐protective when delivered as a pre‐ or post‐conditioning agent. We hypothesized that IL delivered during reoxygenation will protect human coronary artery endothelial cells (HCAECs) and human cardiomyocytes (HCMs) from reoxygenation (reperfusion) injury.

OBJECTIVES

We compared 1) hypoxic cells vs hypoxic/reoxygenated (H/R) cells vs hypoxic cells reoxygenated with differing concentrations of IL; and 2) cells on the basis of cellular “age” (passage; e.g., P7 vs P15).

METHODS

Cell cultures were placed into complete media/normoxic conditions (FBS & glucose; 95% O2, 5% CO2) or hypoxic media/hypoxic conditions (0% FBS & glucose; 1% O2, 94% N2, 5% CO2) at 37°C for 3 hrs. Samples were then either immediately assayed for cell proliferation, metabolic activity, and cytotoxicity, or they were incubated ± IL (0.1%, 0.5%, 1%, or 2%) in complete media/normoxic conditions for 2 hrs and then assayed for the same parameters.

RESULTS

Younger cells had a greater proliferative capacity than older cells in both cell types. However, there were equivalent decreases in proliferative capacity in both hypoxic and H/R exposed cells, and IL had no effect on cell proliferation, regardless of concentration. Metabolic activity was noted to be higher in older cells than younger cells in both cell types. When exposed to H/R, HCAECs metabolic activity decreased, while HCMs had slightly increased metabolic activity. In both cell types, significant increases in metabolic activity occurred notably with IL concentrations of 1–2%. LDH release was greater in older than in younger cells of both cell types, and was more extensive in hypoxic only cells compared to H/R cells. The dose‐dependent impact of IL on cytotoxicity was indeterminable.

CONCLUSIONS

Cells differ based on their “age” (passage), and also on what cellular function is affected by H/R. IL increased metabolic activity in both cell types, specifically at concentrations of 1–2%; however, there was no determinable effect on cell proliferation or cytotoxicity. Further studies need be conducted to determine which metabolic pathways are being activated, and whether those pathways are related to cell survival during stressful conditions such as H/R.

Support or Funding Information

Supported by the Department of Veterans Affairs (IK2BX001278), NIH (R01 HL095122), and institutional funds.
The Effect of Losartan on Marfan Syndrome Is Angiotensin II Receptor Type 1 (ATR1) Independent
Authors: Sellers Stephanie , Chan Rayleigh , Mielnik Michael , Jermilova Una , Pavlovic Marijana , Hirota Jeremy , Seidman Michael , Hogg James C , Esfrandiarei Mitra , Van Breemen Casey , Bernatchez Pascal
Published: November 2023

Background

Marfan Syndrome (MFS) leads to life threatening cardiovascular and pulmonary complications including aortic root dilation and emphysema‐like lung destruction. The new drug of choice to treat MFS is Losartan as it has been shown to reduce aortic and lung pathology in mouse models of MFS and is the focus of on‐going clinical trials. Losartan selectively blocks angiotensin receptor type I (ATR1), which is a key player in MFS pathogenesis. However, despite its promising therapeutic activity, Losartan has never been evaluated in the absences of its proposed target, and therefore genetic loss of ATR1 should protect against MFS and reveal the full potential of Losartan

Hypothesis

A model of MFS lacking ATR1 will prevent the cardiovascular and pulmonary phenotype of MFS and thereby confirm the mechanism of action of Losartan and reveal the full therapeutic potential of complete ATR1 blockade in MFS.

Methods

A MFS model lacking ATR1 was generated using a commonly used MFS model (Fibrillin C1039G) and ATR1 knockout mice (ATR1−/−). The resulting MFS ATR1−/− mice, both treated and untreated with Losartan, were used to evaluate the impact of ATR1 knockout on MFS. Aortic and lung pathology was evaluated through echocardiogram and histology while MFS skeletal manifestation were analyzed using radiography.

Results

Echocardiograms and histology show that both MFS and MFS ATR1−/− mice have similar aortic root dilation, aortic wall thickening and elastic fibre fragmentation in stark contrast to our hypothesis. Additionally, both MFS and MFS ATR1−/− groups demonstrate emphysema‐like airspace widening and significant changes in skeletal structure. Finally, when treated with Losartan MFS ATR1−/− mice showed similar reduction in aortic root dilation and airspace widening as MFS mice.

Conclusion

Data from our novel model shows that loss of the predominant ATR1 isoform fails to prevent MFS complications. Furthermore, Losartan treatment in the absence of ATR1 is still an effective therapeutic for MFS aortic and lung pathology. This suggests that the therapeutic utility of Losartan in MFS is off‐target and completely independent of ATR1.

Support or Funding Information

This work was supposed by funds from the Canadian Institutes of Health Research (CIHR) and fellowships to SS from CIHR and the University of British Columbia.
Microbiota‐Derived Metabolites Associated with Metabolic Improvements after Gastric Bypass Surgery and their Effects on Intestinal Cells in vitro
Authors: Jennis Matthew , Mabus John R , Cavanaugh Cassandre R. , Leo Gregory , Lenhard James , Hornby Pamela J
Published: November 2023

Gastric Bypass surgery (RYGB) improves glycemic control in obese patients with Type 2 Diabetes (T2DM) by multiple mechanisms, including increased incretins (Reed et al., J Clin Endocrinol Metab 2011). Despite the known RYGB changes in intestinal microbial communities, limited information exists on either the impact of bacterially‐derived metabolites on host metabolism, or their potential mechanisms in T2DM and obesity. To address this, metabolomic data that was generated from the same RYGB study were analyzed by PLS‐DA loadings plot to identify differences in microbial‐related metabolites from lean controls, obese and T2DM obese patients pre‐ and post‐surgery (n ~ 9/group). Next, selected metabolites were incubated with human intestinal T‐84 polarized cell monolayers to determine alterations in permeability/barrier function and gene expression. in addition, monolayers were incubated (24 h) in the presence or absence of stressors to increase permeability (e.g. interferon‐gamma, IFNγ) as an in vitro model of metabolic endotoxemia. Our results demonstrate that bacterially‐derived indoles metabolized from the tryptophan/serotonin pathway (e.g. Indole‐3‐propionic acid, IPA and indoxyl sulphuric acid, ISA) were decreased in obese/T2DM patients, compared to lean, and this was reversed by RYGB. In T‐84 polarized cell monolayers, IFNγ increased intestinal permeability 2–4 fold whereas IPA and serotonin reversed this shift. Serotonin was associated with transcriptional increases (e.g.Muc2, Ocln, Slc10A2, Ccbl1 and glutaminase) and decreases (e.g. Slc2a5, IDO1, and G6PC). In conclusion, microbial‐derived metabolite associations with T2DM and their reversal by RYGB have been identified. Furthermore, these metabolites affect intestinal permeability and gene expression for junctional, glucogenic, glutaminolytic or lipogenic proteins. The results are being confirmed in 3D intestinal organoids for further translation and will identify novel targets within the tryptophan/serotonin pathway for therapeutic assessment in in vivo animal models of diabetes and obesity.
Bone Marrow‐Derived Stromal Cells (BMSCs) Improve Microvascular Perfusion in Skin Flaps Subjected to Ischemia Reperfusion Injury (IRI)
Authors: Tang Ya Hui
Published: November 2023

Skin flaps are frequently used for the functional and cosmetic repair of wounds and defects left behind after trauma, burns, and tumors. Flap survival mainly depends on the blood supply provided through re‐established vessels of the dermal and subdermal plexus. We have previously demonstrated the efficacy of BMSCs in the enhancement of wound healing and in improving flap survival.

Objective

To determine if the intravascular delivery of BMSCs improve blood perfusion in the skin flaps following IRI.

Methods

A cutaneous flap model perfused via the inferior epigastric artery was created in wild type (C57Bl/6), which underwent 3.5 hours of ischemia, followed by reperfusion and BMSCs transplantation. Blood perfusion in the flap was monitored using a noncontact, real‐time and noninvasive Laser Speckle Contrast Analysis (LSCA) system. Flap survival and BMSCs recruitment were also assessed.

Results

The numbers of trans‐migrated BMSCs appearing in the flap interstitial spaces were increased, compared to control (sham) mice, within 4h after IRI. Blood flow, both proximal and distal to the vascular pedicle was marked increased (p<0.01) in postischemic flaps treated with BMSCs treatment, when compared to IRI controls at 3 days of reperfusion.

Conclusion

The results of this study indicate that the enhanced wound healing and improved flap survival observed following BMSC treatment may be related to an improvement in tissue blood flow.

Support or Funding Information

Supported by a Malcolm Feist Postdoctoral Fellowship
Attenuation of EPA and DHA‐Mediated Platelet Inhibition Effects Following Heterogeneous Agonist Treatment
Authors: Bormann Jordan L. , Meyer Evan S. , Voigt Ellen M. , Whiteheart Sidney W. , Larson Mark K.
Published: November 2023

Agonist‐induced platelet activation following vascular damage is a critical event for the propagation of thrombi formed as a part of either hemostasis or thrombosis. Several studies have indicated this activation can be impaired after platelet membrane enrichment with the polyunsaturated omega‐3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This attenuated platelet function represents a potential mechanism behind the anti‐thrombotic benefits of EPA and DHA observed in many human trials. However, nearly all platelet studies with EPA and DHA examined the alterations to platelet activation in response to a single agonist, rather than with multiple agonists that mimic the more complex heterogeneous environment of a developing arterial clot. We therefore asked if high levels of EPA and DHA could still inhibit platelet activation if platelets were stimulated with a combination of agonists designed to simulate the activating conditions found in the thrombus inner core (using thrombin and collagen) or outer shell (using ADP and thromboxane A2). To test this question, we treated platelets with EPA and DHA concentrations in vitro that raise the membrane content of both fatty acids to levels analogous to subjects taking EPA and DHA supplements. After this modification, the platelets were stimulated with the agonist combinations and measured for an activation response. Surprisingly, inherent measures of platelet activation using these agonist combinations, including adhesion, aggregation, granule release, and integrin activation were either only mildly affected or non‐significantly altered after EPA and DHA membrane enrichment. We also tested the ability of the core agonist combination to stimulate platelet‐derived thrombin generation, a catalytic process thought to be necessary for rapid thrombus core development. Contrary to the other measures of platelet function, thrombin generation was still consistently impaired by elevated EPA and DHA levels. We conclude that elevated levels of EPA and DHA in the developing clot environment do not readily inhibit most events of platelet activation. Instead, the impairment of amplified thrombin generation in the platelet‐rich arterial clot may provide a more likely candidate for how EPA and DHA prevent detrimental cardiovascular events.

Support or Funding Information

Research reported in this abstract was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443, a South Dakota/National Science Foundation EPSCoR RII Track 1 grant, the Sanford Program for Undergraduate Research, and an American Physiological Society Undergraduate Summer Research Fellowship.
Effects of SIRT3 deficiency on vasomotor function and atherosclerotic plaque composition in mice
Authors: Roos Carolyn M , Hagler Michael A , Zhang Bin , Miller Jordan D
Published: November 2023

Excess mitochondrial reactive oxygen species can drive endothelial dysfunction and vascular calcification in atherosclerosis. Recently, SIRT3 has been linked to the activity of the ROS‐degrading enzyme MnSOD through regulation of protein acetylation levels. It remains unclear, however, whether SIRT3 protects against endothelial dysfunction and osteogenic signaling in hypercholesterolemia. We hypothesized, that reduction of SIRT3 will impair endothelial function, decrease mitochondrial antioxidant capacity, and increase osteogenic signaling in hypercholesterolemic mice. We used Ldlr−/−/ApoB100/100 (LA) mice that were either wild‐type (LA‐SIRT3+/+) or null for SIRT3 (LA‐SIRT3−/−). After 12 months of western diet feeding, we assessed endothelial function in aorta using isolated organ chamber baths and measured changes in gene expression using qRT‐PCR. As expected, SIRT3 gene expression was significantly reduced in LA‐SIRT3+/− mice compared to LA‐SIRT3+/+ mice, and undetectable in LA‐SIRT3−/−. Surprisingly, endothelial‐dependent relaxation to acetylcholine (MRAch) was unchanged between LA‐SIRT3+/+ and LA‐SIRT3−/− (37.0±4.4%, 38.2±4.4%). Endothelial‐independent relaxation (sodium nitroprusside) was also unchanged. MnSOD expression was reduced in LA‐SIRT3−/− compared to LA‐SIRT3+/+ (0.80 ± 0.02 vs 1.0±0.08, p < 0.05). In contrast, the late‐stage osteoblast marker, osterix, tended to increase in LA‐SIRT3−/− (1.3 fold) but failed to reach statistical significance. Collectively, these results suggest that losses of SIRT3 do not impact endothelial function in advanced atherosclerosis, but may contribute to augmentation of osteogenic signaling and accelerated progression of vascular calcification.

Support or Funding Information

NHLBI