bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining the Role of ARRDC3 in Regulation of PAR1 Trafficking and Signaling in Invasive Breast Carcinoma
Authors: Arakaki Aleena
Published: November 2023

The G protein‐coupled receptor (GPCR) protease‐activated receptor‐1 (PAR1) is activated by proteases secreted in the extracellular environment. Overexpression of PAR1 in breast cancer correlates with poor patient prognosis and increased breast cancer invasion and metastasis. However, the mechanism(s) by which PAR1 promotes breast cancer invasion and metastasis are not known. Activation of PAR1 occurs through an irreversible proteolytic cleavage event and thus, the precise regulation of receptor signaling is critical for proper cellular responses. The arrestin‐domain containing ARRDC3 protein functions as a tumor suppressor and mediates activated PAR1 lysosomal degradation in HeLa cells. We have previously shown that PAR1 trafficking is dysregulated in invasive breast carcinoma cells, in which ARRDC3 expression is low or absent, resulting in persistent signaling and invasion. We hypothesize that loss of ARRDC3 expression in invasive breast carcinoma results in the dysregulated PAR1 trafficking, persistent signaling and breast cancer invasion we observe. We used a tetracycline‐inducible lentiviral vector to re‐express ARRDC3 in the highly invasive breast carcinoma cell line MDA‐MB‐231. Using biochemical‐ and microscopy‐based approaches, we found that re‐expression of ARRDC3 was sufficient to rescue defective PAR1 trafficking and restored agonist‐induced receptor lysosomal degradation. In addition, activation of PAR1 resulted in a marked increase in Hippo‐YAP signaling in MDA‐MB‐231 cells that was attenuated upon ARRDC3 re‐expression. These findings suggest that ARRDC3 is a critical regulator of PAR1 signaling in invasive breast cancer. We will further investigate whether loss of ARRDC3 affects PAR1‐mediated Hippo‐YAP pathway activity and modulation of breast cancer migration, invasion and tumor growth and metastasis. These studies will be the first to link ARRDC3 to GPCR‐stimulated Hippo‐YAP signaling and will reveal an important pathway for dysregulation of Hippo‐YAP signaling induced by GPCRs in breast cancer.

Support or Funding Information

Funding for this project was provided by the UCSD Tribal Membership Initiative Graduate Fellowship and UCSD Graduate Training Program in Cellular and Molecular Pharmacology through NIH General Medical Sciences, T32 GM007752 (Aleena Arakaki) and by the National Institute of Health NIGMS R01 GM090689 (JoAnn Trejo).
Muscarinic cholinergic receptors regulate oscillation of intracellular Ca2+ in steroid‐producing adrenocortical carcinoma cells
Authors: Sharthiya Harsh , Surachaicharn Nuntida , Shams Yara , Arshad Mohammad , Kopf Phillip G. , Malaiyandi Latha M. , Dineley Kirk E.
Published: November 2023

Elevated circulating aldosterone levels are associated with hypertension, thrombosis formation, cardiac hypertrophy, and congestive heart failure. Aldosterone secretion is regulated by angiotensin II (Ang II), potassium, and adrenocorticotropic hormone (ACTH). Calcium is required for the stimulation of aldosterone secretion by these steroidogenic stimuli. Despite the importance of calcium in steroidogenesis, the relationship between intracellular Ca2+ homeostasis and steroid production by the adrenal cortex remains incompletely understood. We used the Ca2+‐sensitive fluorophore Fluo‐4 to study whole‐cell calcium in human adrenocortical carcinoma (HAC15) cells. We found that oscillation in intracellular Ca2+ could be triggered in a subset of cells by the cholinergic agonist carbachol. The oscillations were characterized by a relatively rapid periodicity, and often returned completely to baseline before spiking again. Removal of calcium from the extracellular perfusate suggested that oscillations resulted from repetitive release and recapture of calcium stored internally in the endoplasmic reticulum. Carbachol‐triggered oscillations were inhibited by muscarinic antagonists including atropine, pirenzepine, and darifenacin. Because darifenacin nearly completely abolished the carbachol effect, we concluded that regulation by M3 receptors is most important for the oscillations. Nicotinic cholinergic receptors were ruled out because nicotine did not alter intracellular calcium levels. Angiotensin II increased intracellular Ca2+, however these changes were relatively large and sustained, and not oscillatory in nature. Further experiments will investigate the relationship between muscarinic receptor activity and aldosterone production stimulated by angiotensin II.

Support or Funding Information

Supported by funds from Midwestern University
Altered spinophilin interactions in the pancreas of a leptin receptor mutant (db/db) model of obesity
Authors: Stickel Kaitlyn C. , Watkins Darryl S. , Edler Michael C. , Dharmarajan Subramanian , Belecky‐Adams Teri L. , Berbari Nicolas F. , Mastracci Teresa L. , Baucum Anthony J
Published: November 2023

Spinophilin is a multi‐domain scaffolding protein known to bind with multiple neuronal proteins, such as F‐actin and protein phosphatase 1 (PP1). Spinophilin modulation of glucose and M3 muscarinic receptor‐dependent insulin secretion has been recently evaluated. These studies established that spinophilin KO mice have decreased fat mass and increased lean mass. Yet spinophilin specific expression and the physiological and pathological functions of spinophilin within pancreatic beta cells have not been analyzed. Here we report that spinophilin is expressed in a rat INS‐1 pancreatic beta cell line and also human pancreatic islets. In isolated pancreatic mouse islets, spinophilin was detected in both pancreatic beta cells and non‐beta cell populations. Using proteomics‐based approaches we identified multiple putative spinophilin interacting proteins isolated from intact pancreas, including PP1, neurabin and myosin‐9. We are continuing to assess the interaction of spinophilin with these proteins in pancreata from mice that have a mutation in the leptin receptor (db/db), a well‐characterized mouse model of type 2 diabetes and obesity. We have found that the associations of PP1 and neurabin with spinophilin were decreased in db/db mice as early as 6 weeks. The association of myosin‐9 with spinophilin was substantially increased in this same population. We are currently working with a transgenic mouse population that expresses a tagged form of human spinophilin in pancreatic beta cells to evaluate if changes in the spinophilin interactions are occurring within the pancreatic beta cells. Ultimately, Our data demonstrate that spinophilin is expressed in pancreatic beta cells and that spinophilin protein interactions in the pancreas are modulated under pathological conditions such as obesity. Future studies will examine how these changes in the spinophilin interactome may contribute to pathological sequelae associated with obesity and diabetes.

Support or Funding Information

Support and funding provided by NIH ‐ K01NS073700, R21DA041876 and by Indiana University School of Medicine Center for Diabetes and Metabolic Diseases Pilot and Feasibility Program and by IUPUI Department of Biology.
The Aortic function of Male UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) Rats: Possible Involvement of Potassium Channels
Authors: Akther Farjana , Shaligram Sonali , Karimi Mujibullah D , Graham James L. , Stanhope Kimber L. , Havel Peter J. , Rahimian Roshanak
Published: November 2023

UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) rat is a validated model of type 2 diabetes mellitus. UCD‐T2DM is characterized by polygenic, adult‐onset obesity and spontaneous β‐cell failure and, as a result, more closely models the pathophysiology of type 2 diabetes in humans than other rodent models. The objectives of this study were to determine whether the aortic function is altered in male UCD‐T2DM rats, and to test the hypothesis that potassium (K+) channels contribute in modulating vascular reactivity of aorta in this model. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10‐8 to 10‐5M) was measured in intact aortic rings pre‐contracted with phenylephrine (PE, 2μM) before and after pretreatment with tetraethylammonium (TEA, 2 mM), a non‐selective K+ channel blocker. Endothelium‐independent vasodilation to sodium nitroprusside (SNP, 10‐9 to 10‐5 M) was assessed in endothelium‐denuded rings pre‐contracted with PE (2μM). Furthermore, constrictor response curves to PE (10‐8 to 10‐5 M) were generated before and after incubation with L‐NAME (200μM), an endothelial nitric oxide synthase (eNOS) inhibitor.

ACh responses were significantly potentiated in aortic rings of male UCD‐T2DM rats. Both maximal relaxation and sensitivity to ACh were significantly enhanced compared to those in controls. Incubation of aortic rings with TEA blunted the relaxation responses to ACh in both groups. However, the inhibitory effect of TEA on the ACh‐induced relaxation in UCD‐T2DM was greater than that in control rats. The smooth muscle sensitivity to NO as measured by SNP‐induced relaxation was not altered in aorta of UCD‐T2DM group. The responsiveness to PE was significantly enhanced in UCD‐T2DM rats. Accordingly, the basal nitric oxide (NO), as indicated by the potentiation of the response to PE after L‐NAME, was reduced in aorta of this group.

These data, for the first time, show that the aortic function of male UCD‐T2DM rats is altered and that K+ channels may be involved. Further studies is required to document the underlying mechanisms of the altered vascular reactivity and to identify the contribution of specific subtypes of K+ channels in the potentiation of aortic endothelial function in this model.

Support or Funding Information

Supported by NIHLBI, R15HL128988
Targeting Cathepsin K to Promoted Diabetic Wound Healing
Authors: Nair Sreejayan , Guo Rui , Nair Rama , Zgheib Carlos , Xu Junwang , Liechty Kenneth W.
Published: November 2023

Impaired wound healing is serious complication of diabetes and is responsible for over 70,000 amputations annually in the United States. Increased degradation of extracellular matrix (ECM) proteins by elevated levels of proteases has been established as a key pathogenic factor in diabetic wounds. Collagen is the most abundant ECM in the wound, which aids healing by facilitating migration of dermal fibroblasts and keratinocytes and stimulating endothelial cell angiogenesis. The most potent collagenolytic and elastolytic protease in the mammalian species is the cysteine protease cathepsin K. This study was undertaken to explore the hypothesis that cathepsin K is elevated in diabetic wound and pharmacological inhibition of cathepsin K improves wound healing. To this end, 8mm excision skin wounds were made in wild type and db/db mice, and cathepsin K mRNA and protein levels were determined in the wound tissue by real‐time PCR and immunohistochemistry. In vitro monolayer wound closure assay was used to determine the effect of cathepsin K inhibitor‐II on migration of human fibroblasts under high glucose (20mM) conditions. Cathepsin K puncta and mRNA levels were significantly elevated in the wound tissue of db/db mice compared to that in the wild‐type mice. Migration of fibroblasts was significantly impaired when subjected to high‐glucose treatment, was reversed by cathepsin K inhibitor‐II. Even under basal (5mM glucose) conditions, treatment with cathepsin K inhibitor‐II augmented fibroblast migration. Collectively, these data demonstrate that cathepsin K is upregulated in the diabetic wound and inhibition of cathepsin K accelerates migration of fibroblasts, suggesting that pharmacological or genetic targeting of cathepsin K may represent a viable strategy to facilitate diabetic wound healing.
Critical role for adenylyl cyclase 5 on L‐type CaV1.2 channel potentiation and vasoconstriction during diabetic hyperglycemia
Authors: Syed Arsalan U , Nystoriak Matthew A , Prada Maria Paz , Nieves‐Cintrón Madeline , Navedo Manuel F
Published: November 2023

Diabetes mellitus is a devastating metabolic disease affecting millions worldwide that carries with it a major risk factor of cardiovascular disease. Foremost among the symptoms of uncontrolled diabetes is elevated blood glucose (i.e. hyperglycemia). The correlation between elevated glucose and hypertension has emerged as an important factor for cardiovascular disease. Chronic increase in blood pressure is a consequence of increased vasoconstriction (myogenic tone) in resistant arteries, resulting in impaired organ perfusion. Our lab has recently demonstrated that this pathophysiological increase in vasoconstriction as a result of elevated glucose is due to dysfunction of L‐type Ca2+ channels (LTCC) activity due to phosphorylation of CaV1.2 at S1928 by cAMP‐dependent protein kinase (PKA) in vascular smooth muscle (VSM). This novel result challenged long‐held views that associate PKA activity with VSM relaxation and vasodilation. In the present study we test the hypothesis that local PKA signaling associated with CaV1.2 phosphorylation and activation of LTCC in response to elevated glucose is regulated by adenylyl cyclase isoform‐5 (AC5) via A‐kinase anchor protein 150 (AKAP‐150). Proximity ligation assay (PLA) and ground state depletion (GSD) super‐resolution imaging suggest that AC5 co‐localizes and exit in a signaling complex along with AKAP150 and LTCC. Using patch clamp electrophysiology and pressurized arteriography, we observed decrease potentiation of LTCC and attenuated vasoconstriction of cerebral arteries in response to elevated glucose in vascular smooth muscle cells and arteries from wild type mice pre‐incubated with the general AC inhibitor 2,5‐DDA or from AC5 knockout mice. Taken together, these results suggest that AC5 is a critical component in the signaling cascade required to activate LTCC and induce vasoconstriction in response to elevated glucose.

Support or Funding Information

T32 HL086350

NIH RO1 HL098200

NIH RO1 HL121059
Structure based drug design with Diabetic Retinopathy and Rage
Authors: Sriramoju Swetha , Evangelista Diana Evangelista , Goetz Kerry Goetz
Published: November 2023

Objective

Diabetic Retinopathy is the leading cause of blindness, stimulating the rise in 20,000 cases in the United States per year, and leaving over 3 million individuals vulnerable at risk. During the early stages of Diabetic Retinopathy, inflammatory and innate immune responses are derivative from pattern‐recognition receptors (PRRs) expressed as Advanced Glycation End‐products (AGEs). It has been observed that AGEs increases the progression of retinopathy in patients enduring the early stages of retinopathy. The reason behind the complication progression induced by AGEs is the ligand ‐ receptor binding with the Receptor Advanced Glycation End‐products. Therefore, by instituting a drug to bind with RAGE, may prevent the ligand‐receptor binding between RAGEs and AGEs. The following study analyzed drug targets based on their hydrophobicity, as to whether they will be able to bind with RAGE.

Methods

A docking program, AutoDock Tools and AutoDock Vina, were both used to determine the binding affinity, rmsd values, and Log P values of the drug targets.

Results

It was found that Statin drugs and Fibric Acid drugs are the types of drugs that were able to bind with RAGE with a high hydrophobicity and affinity, while a very popularly marketed drug, Metformin, wasn't able to bind with RAGE.

Conclusion

RAGE may be of importance in the prevention and management of diabetes complications.

Support or Funding Information

none
Mid‐Term Exposure to Prednisone is Less Detrimental than Short‐Term Dexamethasone on Glucose Homeostasis in Rats
Authors: Ribeiro Nathalee Xavier Ribeiro , de Souza Melo Danylo Noleto , Sanches Jonas Rodrigues , França Lucas Martins , Cappelli Ana Paula Gameiro , Rafacho Alex , Abrau Iracelle Carvalho , de Andrade Paes Antonio Marcus
Published: November 2023

Synthetic glucocorticoids (GCs) are pontent and widely used anti‐inflammatory and immunosuppressant agents. However, prolonged or high‐dose usage of GCs promote several side effects, especially on glucose and lipid metabolism. In spite of the abovementioned factors, there is scanty data on differential impact of distinct synthetic GC on metabolic targets, particularly glycolipidic homeostasis. Thus, this work aimed to assess the metabolic alterations caused by subchronic treatment with prednisone in comparison to acute dexamethasone. For this, 90‐days old Wistar rats were treated with dexamethasone (1 mg/kg/day, i.p., D5 group, n=8) for 5 consecutive days and compared to saline‐treated control rats (C5, n=9). Additionally, 60‐days old rats were treated with prednisone (80 mg/kg/day, orally) for 15 days (P15, n=7) and 30 days (P30, n=12) and compared to their respective controls (C15, n=7 and C30, n=12), which received vehicle solution. D5 rats showed decreased body weight (−12.3%) and lower retroperitoneal fat accumulation (−38%), increased fasting (+12%) and fed (+30%) glycemia, higher insulinemia (+80%), higher free fatty acids (+114%) and hypertriglyceridemia (+339%). Total fat and triglycerides content in the liver were 29% and 52% higher in D5 than in C5 rats, respectively. P15 rats weight gain was 61% lower, with reduction of retroperitoneal fat (−29%) and increased plasma triglyceride levels (+60%), as compared to C15 rats. 30 days treatment promoted similar but deeper effects on the same parameters. In vivo protocols revealed impaired glucose tolerance (oGTT) in D5 and P30 rats. However, P30 rats did not show reduced insulin sensitivity (ipITT, HOMA, TyG), as observed in D5 animals. Ex vivo assessment of glucose‐stimulated insulin secretion showed dysfunctional response in D5 but not P30 rats. In conclusion, our data suggest that mid‐term administration of prednisone promoted lesser metabolic side effects on glucose homeostasis, as compared to short‐term administration of dexamethasone, supporting its preferential use in order to minimize adverse metabolic effects associated to GCs treatment.

Support or Funding Information

FAPEMA, UFMA and CAPES
Novel antiplatelet role for a protein disulfide isomerase‐targeted peptide: Evidence of covalent binding to C‐terminal CGHC redox motif
Authors: Gaspar Renato Simões , Sousa Hiran Reis , Sena Elyjany , Da Silva Samira Abdalla , de Lima Fontelles João Lucas , Araujo Thais , Fries Diana , Azevedo‐Santos Ana Paula , Laurindo Francisco , Trostchansky Andrés , de Andrade Paes Antonio Marcus
Published: November 2023

Protein disulfide isomerase (PDI) plays a major role in platelet aggregation and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI‐modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized this peptide would also inhibit platelet aggregation by association to surface PDI. Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. We showed that CxxC decreased platelet aggregation in a dose‐dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. In thrombin‐activated platelets, surface expression of activated αIIbβ3 integrin, P‐selectin and granulophysin were analyzed by flow cytometry. CxxC decreased αIIbβ3 activation, but had no effect on the other markers. In addition, CxxC was the only peptide able to decrease cell‐surface PDI pull down, with no detectable unspecific binding to total free thiols. Finally, the capacity of CxxC to covalently bind to PDI active site was analyzed by ESI LC‐MS/MS. We detected the addition of one CxxC molecule to PDI through binding to Cys400, an important site for PDI prothrombotic activity. In summary, our data support an antiplatelet activity for CxxC through binding to Cys400 in PDI a′ domain, which can be further exploited as a model for site‐driven antithrombotic agents development.

Support or Funding Information

Foundation for Research and Scientific Development of Maranhão ‐ FAPEMA, Nacional Counsel for Scientific Development and Technology ‐ CNPq, Coordination of Improvement of Higher Level Personnel ‐ CAPES and CSIC grupos I+D 2014.
RGS10 serves as a braking system for platelet hyperactivity
Authors: Hensch Nicole R , Karim Zubair A , Qasim Hanan , Khasawneh Fadi T
Published: November 2023

Regulators of G protein signaling (RGS) proteins act as GTPase activating proteins to negatively regulate G protein‐coupled receptor (GPCR) signaling. Although several RGS proteins including RGS2, RGS16, RGS10, and RGS18 are expressed in human and mouse platelets, the respective unique function(s) of each have not been fully delineated. RGS10 is a member of the D/R12 subfamily of RGS proteins and is expressed in microglia, macrophages, megakaryocytes, and platelets. We used a genetic approach to examine the role(s) of RGS10 in platelet activation in vitro and hemostasis and thrombosis in vivo. GPCR‐induced aggregation, α‐granule secretion, and integrin activation were much more pronounced in platelets from Rgs10−/− mice relative to wild type (WT). Accordingly, these mice had markedly reduced bleeding times and were more susceptible to vascular injury‐associated thrombus formation than control mice. These findings suggest a unique, non‐redundant role of RGS10 in modulating the hemostatic and thrombotic functions of platelets in mice. RGS10 thus represents a potential therapeutic target to control platelet activity and/or hypercoagulable states.

Support or Funding Information

This research was supported by funds provided by the College of Pharmacy at Western University of Health Sciences (to F.T.K). This work has been approved by Institutional Review Board and the Institutional Animal Care and Use Committee at Western University of Health Sciences, as applicable.
A Prospective, Open Label Study to Evaluate the Pharmacokinetics of Dabigatran in Patients With Non‐Valvular Atrial Fibrillation and Severe Renal Impairment Who Are Receiving Dabigatran Etexilate (75 mg twice daily)
Authors: Martin Jack , Esmaeili Habib , Manuel Raymond C. , Petrini Michaela , Wiebe Sabrina , Maas Hugo
Published: November 2023

Introduction

Dabigatran etexilate (DE) is a direct oral thrombin inhibitor. Dabigatran excretion is 80% renal, and exposure increases with the severity of renal failure. The FDA approved DE 75 mg BID for non‐valvular atrial fibrillation (NVAF) patients with severely impaired renal function (SIRF), defined as a CrCl of 15–30mL/min, based on post‐hoc pharmacokinetic (PK) modelling.

Objectives

To assess dabigatran exposure at both trough and peak levels in patients with NVAF and SIRF, who were receiving DE 75 mg BID.

Methods

Patients received DE (75 mg BID) for at least 7 days before 2 blood samples were obtained; the first at 10–16 hrs post‐dose (immediately prior to next dose) for Cpre,ss (steady state predose; trough), and the second 2 hrs (±30 min) post‐dose for C2,ss (steady state 2 hrs post‐dose; expected peak). Pharmacodynamic effect parameters at Ebase (pre‐DE; baseline), Epre,ss (steady state predose; trough), and E2,ss (steady state 2 hrs post‐dose; expected peak) were assessed by established coagulation assays (aPTT, dTT, ECT and TT).

Results

Of 150 patients screened, 60 were treated; 40% were male, 78.3% white, and their median age was 84. Reported Cpre,ss values (N=51) and variability were mostly in the predicted ranges of the PK model, with a gMean of 155 ng/mL, a gCV of 76.9%, and a range of 15.6 to 498 ng/mL. The C2,ss gMean value (N=59) was 202 ng/mL, with a gCV of 70.6%, and a range of 42.0 to 680 ng/mL. The pharmacodynamic effects of dabigatran on coagulation assays are presented in Table 1. Five patients reported bleeding events during the trial, all of which were considered to be minor bleeds by the investigators.

Conclusions

The levels of dabigatran exposure reported here in patients with NVAF and SIRF largely confirmed earlier PK predictions (Lehr et al. J Clin Pharmacol. 2012 Sep;52(9):1373–8), supporting the use of 75 mg DE BID in this patient group. Relationships between plasma dabigatran levels and coagulation assay results were also in line with earlier studies.

Support or Funding Information

Boehringer Ingelheim Pharmaceuticals, Inc. funded the study, and provided support for medical writing and editorial support. The authors did not receive any direct payment for the writing of this abstract.

aPTT
ECT
dTT
TT

N*

60
59
60
31

Ebase
s†(SD)
43.1(12.5)
67.7(36.1)
43.9(14.7)
49.4(73.3)

Epre,ss
s(SD)
52.3(10.9)
96.0(37.6)
54.2(12.9)
154.0(66.0)

E2,ss
s(SD)
61.3(35.6)
117.0(50.8)
60.0(16.6)
153.0(51.8)

aPTT, activated prothrombin time; dTT, dilute thrombin time; ECT, ecarin clotting time; s, seconds; SD, standard deviation; TT, thrombin time

N displayed is maximal N; E, effect; Epre,ss and E2,ss were calculated with slightly smaller Ns. All values include samples from both treatment naїve and pre‐treated patients.

values presented are arithmetic means.
12‐HETrE Exerts its Antithrombotic Effects Through the Prostacyclin Receptor
Authors: Tourdot Benjamin , Isingizwe Zitha , Adili Reheman , Holman Ted , Holinstat Michael
Published: November 2023

The antithrombotic effect of dihomo‐γ‐linolenic acid (DGLA), an ω‐6 fatty acid, was recently shown to be dependent upon 12‐lipoxygenase (12‐LOX) and its DGLA‐derived metabolite, 12‐hydroxy‐eicosatrienoic acid (12‐HETrE). 12‐HETrE inhibits platelet activation via the Gαs signaling pathway; however, the Gαs‐coupled receptor by which 12‐HETrE mediates its antiplatelet effects has yet to be identified. We hypothesized that 12‐HETrE exerts its antiplatelet effects through a known lipid binding Gαs‐coupled prostanoid receptor expressed by platelets: IP, EP2, EP4, or DP1. In this study, we found that pharmacological inhibition of the IP receptor reduced 12‐HETrE's antiplatelet effects in humans, while genetic ablation of the IP receptor in murine platelets completely abolished the inhibitory effects of 12‐HETrE. Consistent with our in vitro studies, 12‐HETrE's antithrombotic effects were abolished in IP receptor deficient mice. Further, ectopic expression of the IP receptor in Jurkat cells was sufficient for 12‐HETrE to induced Gαs signaling. Together this data demonstrates that 12‐HETrE's antiplatelet effects are at least partially dependent on IP receptor signaling. Importantly, this work identified a new class of endogenous IP receptor agonist that will aid in the rationale design of novel IP receptor therapeutics. Additionally, this study provides further knowledge of the mechanism by which DGLA supplementation inhibits platelets function.

Support or Funding Information

This work was funded in part through the National Institutes of Health grants R01 GM105671 (MH & TRH), R01 HL114405 (MH & TRH), R01 MD 007880 (MH), R01 AG047986 (MH & TRH), and F32 HL129491 (BET), as well as the University of Michigan Frankel Cardiovascular Center Summer Fellowship Program (ZRI).
Effect of Sevoflurane plus Ondansetron and Ranitidine vs. Propofol and Remifentanil for the Prevention of Post‐operative Nausea and Vomiting
Authors: Lynch Sharlette M , Tulp Orien L , Einstein George P
Published: November 2023

Postoperative nausea and vomiting (PONV) are frequent and distressing complications following anesthesia [AN] and general surgery [GS]. The purpose of this study was to compare total intravenous AN (TIVA) using propofol and remifentanil [TIVA‐PR] vs. sevoflurane with prophylactic ondansetron and ranitidine [TIVA‐SF group] for the prevention of PONV. Hemodynamic changes [heart rate [HR] blood pressure [BP], and the quality of recovery such as post‐operative pain [POP] were compared between the two groups. Patients [n=60] undergoing AN and GS were randomly assigned to one of two prophylactic interventions [n=30/group]: (1) ondansetron 4 mg and ranitidine 50 mg before the induction of sevoflurane anesthesia [TIVA‐SF] group); and (2) propofol at 50–150 mcg/kg/minute + remifentanil 0.1 mcg/kg/min; (TIVA‐PR group). PONV incidence and severity was monitored immediately after GS for 24 hours. Recovery was assessed with a simulated wake up test and a simple behavioral score to assess overall recovery. The POP was assessed in each group via the visual analogue scale (VAS). The incidence of PONV was similar in the TIVA‐SF and TIVA‐PR groups during the 24 hours post GS [pGS], [TIVA‐SF=30.0%; TIVA‐PR 33.0%]. pGS HR was similar in both groups, but the GS mean arterial pressure (MAP) was significantly lower after induction and higher during surgical incision in the TIVA group [TIVA‐PR > TIVA‐SF, P<0.05]. The MAP in the TIVA‐PR was better controlled throughout GS than in TIVA‐ SF [MAP TIVA –PR < TIVA‐ SF, p<0.05]. The mean pain rating [MPR] was 3.5±3.4 in the TIVA‐PR group and 5.4±2.9 in TIVA‐ SF, while MPR was 3.1±3.1 in the TIVA‐PR group and 5.6±1.6 in the TIVA‐SF group. In the TIVA‐PR group, 8 out of 30 patients reported a pain score [PS] > 4; In the TIVA‐SF group, 18 out of 30 patients reported a PS > 4. In conclusion, IV ranitidine and ondanestron immediately before induction of TIVA using SF resulted in a similar reduction in PONV in comparison to TIVA‐PR. The overall PS recovery and HR did not differ significantly in both groups. The MAP demonstrated a significant increase after intubation in both the TIVA‐SF and TIVA‐PR groups, but was lower in TIVA‐PR group throughout the remainder of surgery. Upon emergence from anesthesia, the POP, behavioral scores, and the pGS recovery time were similar in both groups, but indicate that MAP was better controlled during GS with the TIVA‐PR than the TIVA‐ SF regimen.

Support or Funding Information

Supported by Institutional Resources of USAT Colleges of Medicine and Graduate Studies and the Einstein Institute
Influences of Experimental Air Pollution on Human Sympathetic Nerve Traffic and Norepinephrine Metabolism: A Double Blind, Randomized, Twofold Crossover Study
Authors: Tank Jens , Heusser Karsten , Holz Olaf , May Marcus , Brinkmann Julia , Diedrich André , Framke Theodor , Koch Armin , Grosshennig Anika , Sweep Fred C.G.J. , Koch Wolfgang , Krug Norbert , Jordan Jens , Hohlfeld Jens
Published: November 2023

Background

Much of the evidence linking air pollutant exposure with changes in human cardiovascular autonomic regulation relied on epidemiological studies, exposure estimates, and indirect autonomic nervous system measurements. We tested the hypothesis that in healthy older individuals, experimental exposure to fine particles increases sympathetic nervous system activity and more so with addition of ozone.

Material and Methods

Eighteen healthy participants (age >50 years) completed all study visits and were included in the final analysis. Participants were exposed to clean air (‘placebo’), ultrafine particles (UFP, 50μg/m3), and combination of UFP+ozone (250 ppb) for 3 hours combined with intermittent bicycle ergometer training in a randomized, three‐period, cross‐over, double‐blind fashion. Three hours following exposure, respiration, ECG, blood pressure, and muscle sympathetic nerve activity (MSNA) were continuously recorded at supine rest, during deep breathing, and during a Valsalva maneuver. Venous blood samples for plasma catecholamine measurements were taken at baseline. Induced sputum was obtained at the end of each study day.

Results

Combined exposure to ozone and UFP but not UFP alone caused a significant increase in sputum neutrophils and circulating leucocytes. We did not detect significant effects on blood pressure or heart rate. Resting MSNA was 47±12 with clean air, 47±14 with UFP, and 45±14 bursts/min with UFP+ozone. Maximum MSNA during Valsalva phase IIb was similar. Yet, with UFP+ozone plasma norepinephrine concentrations were significantly increased with concomitant reduction in the dihydroxyphenylglycol (DHPG)/norepinephrine ratio. Respiratory sinus arrhythmia and Valsalva ratio were unaffected by experimental air pollution.

Conclusion

Exposure to ultrafine particles with or without ozone does not elicit clinically relevant changes in central sympathetic or parasympathetic activity in healthy older subjects. The paradoxical norepinephrine increase with UFP+ozone may be explained by reduced peripheral norepinephrine uptake and metabolism.

Support or Funding Information

MM was funded by a grant from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, awarded to JJ: JO 284/8‐1). The German Aerospace Center (DLR) supported JT, KH, and MM (50 WB1117, 50WB1517).
Clinical Pharmacokinetics and Preclinical Pharmacodynamics of (+)‐Epicatechin on Cardiometabolic Endpoints
Authors: Villarreal Francisco , Moreno‐Ulloa Aldo , Ciaraldi Theodore , Best Brookie , Ramirez‐Sanchez Israel , Ceballos Guillermo , Dugar Sundeep , Schreiner George , Henry Robert
Published: November 2023

We have previously reported on the capacity of the cacao flavanol (−)‐epicatechin to vigorously stimulate mitochondria biogenesis and beneficially impact metabolism. However, preliminary studies indicate that the (+) epimer form may be more potent. In the present study, we report on the pharmacokinetics (PK), partial pharmacodynamics (PD) and initial safety analysis of (+)‐epicatechin (Epi) using a Phase I study performed in pre‐diabetic subjects. Using a mouse model of diet‐induced obesity and insulin resistance we also provide PD evidence of the metabolic effects of (+)‐Epi vs. those of (+)‐catechin to determine if the effects observed correspond to class actions. For the Phase I PK study, subjects were provided a single incremental dose of (+)‐Epi using either 10, 30 or 100 mg in capsules. For the PD study, subjects were provided a single 30 mg dose/day for 7 days. In all subjects blood samples were collected and an assessment of standard safety measures were performed. Results indicate that incremental doses of (+)‐Epi increase the half life of blood metabolites ranging from ~2–6 h. Seven day dosing of prediabetic subjects leads to a trend in reducing blood glucose levels (p=0.053) and markers of inflammation. No adverse effects were reported and the compound was well tolerated. In obese‐insulin resistant animals, 2 weeks of dosing (0.003, 0.01, 0.03, 0.1 and 0.3 mg/kg/day by oral gavage) led to a dose dependent significant improvement in markers of metabolism (weight gain, glucose, cholesterol, triglycerides with doses as low as 0.01 mg/kg/day). (+)‐Catechin yielded no effects at 0.1 mg/kg/day. Overall results indicate that (+)‐Epi evidences a favorable PK and safety profile. The compound exerts a positive impact on metabolism which may link to enhanced mitochondrial bioenergetics. The effects noted are not due to antioxidant actions as (+)‐catechin yielded no effects.

Support or Funding Information

NIH AT8310, DK9871, Cardero Therapeutics, Inc.
Pretreatment With Probenecid Downregulates Kidney Pendrin Expression and Potentiates Hydrochlorothiazde Diuresis in Rat
Authors: Barone Sharon , Xu Jie , Zahedi Kamyar , Soleimani Manoocher
Published: November 2023

Background

Concomitant deficiency of the Na‐Cl co‐transporter (NCC) and pendrin leads to significant salt wasting. Probenecid is an inhibitor of the organic anion transporters in the kidney proximal tubule and enhances uric acid excretion. Probenecid is also an inhibitor of pendrin in mammary cells (AJP Endo/Metab, 2003). The diuretic effect of Hydrochlorothiazide (HCTZ), which is a specific inhibitor of NCC, is significantly blunted consequent to compensatory salt absorption by pendrin. The downregulation or inactivation of pendrin enhances the diuretic effect of HCTZ.

Hypothesis

Pre‐treatment with probenecid will downregulate pendrin, therefore leaving NCC as the main salt absorbing transporter in the distal nephron. These changes enhance the diuretic effect of HCTZ.

Results

Male Sprague Dawley rats (200–225 gm BW) were pre‐treated with probenecid, at 250 mg/kg/day IP for 6 days. Urine output increased from 9.5 ml to 13.8 ml /24 hr on day 6 of probenecid treatment (p<0.05, n=5). Treatment of rats with HCTZ alone (40 mg/kg) for 4 days caused a mild diuresis which did not achieve statistical significance. However, treatment of rats that were pretreated with Probenecid for 6 days with a combination of Probenecid plus HCTZ for 4 additional days increased the urine output by greater than 300% to 42.1 ml/day (p<0.001, n = 5) compared to Probenecid‐treated or HCTZ‐treated animals. The animals treated with Probenecid plus HCTZ developed significant volume depletion as judged by >3 fold increase in the expression of renin in their kidneys. Immunofluorscence studies demonstrated an ~80% reduction in pendrin expression in kidneys of rats treated with Probenecid. Probenecid treatment reduced HCTZ excretion by ~40% suggesting competition between probenecid and HCTZ for secretion via organic anion transporters in the proximal tubule. The impaired excretion of HCTZ was unlikely to be responsible for the profound diuresis by probenecid plus HCTZ.

Conclusion

Despite being considered a mild agent, we propose that HCTZ could be a potent diuretic when administered in individuals pre‐treated with probenecid. The treatment with probenecid should also prevent the generation of hyperuricemia by HCTZ.

Support or Funding Information

Merit Review Award by Veterans Administration.

Center on Genetics of Transport, University of Cincinnati
YiqiHuoxue decoction and its constituents inhibit the activation of platelets and thrombosis for the prevention and treatment of cardiovascular diseases
Authors: Wu Hong , Lei Zhen , Gao Shuibo , Han Lihua
Published: November 2023

YiqiHuoxue decoction (YHD) is frequently used for the prevention and treatment of cardiovascular diseases. Previous studies have demonstrated that YHD can promote angiogenesis and reduce left ventricular remodeling. The present study further investigated the effects of both YHD and ethanol‐precipitated YHD (EYHD) on the activation of platelets and thromboxane B2 (TXB2) in vitro and thrombosis in vivo. The results showed that 20 mg/mL of YHD and 2–20 mg/mL of 75% EYHD inhibited adenosine diphosphate (ADP)‐induced platelet aggregation. Moreover, collagen‐induced platelet aggregation was significantly suppressed by 20 mg/mL YHD, 20 mg/mL of 50, 50–75, and 75% EYHD, and 2 mg/mL of 50–75 and 75% EYHD. Furthermore, rats administered with 4 mg/mL 75% EYHD displayed a marked reduction in collagen‐induced platelet aggregation at 2 h post‐administration. Meanwhile, we observed the effects of YHD and EYHD on bleeding and clotting time in rats, and on mesenteric arterial thrombosis in mice. Our data indicates that YHD and EYHD markedly prolonged the onset of thrombosis and led to a loose attachment of the thrombus with the vascular endothelium, albeit with no significant influence on their bleeding or clotting times. Finally, we found that YHD and EYHD markedly reduced the level of thromboxane B2 (TXB2) using washed platelets. In conclusion, YHD and EYHD can effectively inhibit arterial thrombosis in vivo due to antiplatelet activity, which may exert beneficial effects on the prevention and treatment of cardiovascular diseases.

Support or Funding Information

This work was financially supported by grants from the National Natural Science Foundation of China (Grant No. 81072968) and Collaborative Innovation Center of Henan University of Traditional Chinese Medicine
Effects of GLA Precursors Added to Atherogenic Diet on Platelet Insulin Sensitivity, Plasminogen Activator Activity and Atherogenic Plaque Development in Rabbits
Authors: Beard Elizabeth Letitia , Edupuganti Srilatha
Published: November 2023

Maintaining rabbits on a diet containing 2%Cholesterol (Ch) for 4–6 weeks usually promotes development of hypercholesterolemia, diminished plasma plasminogen activator activity(PPA) and atherosclerotic plaque formation. Also tissue insulin sensitivity and tissue delta‐6‐desatuase (D‐6‐D) levels needed to convert cis cis linoleic acid(LA) to gamma linoleic acid (GLA) are lowered. GLA is a precursor for the anti‐atherogenic prostaglandin, Prostacyclin (PGE2) production in normal tissues.

In this study, three groups of 5 New Zealand strain male rabbits were fed AD (2% Ch, 6%Corn Oil, 92% Purina Rabbit Chow) for an 8 week period.. In Group I – the AD Control animals‐ the rabbits were fed AD alone. Group II rabbits were fed AD supplemented with 5% LA and in Group III, the animals received AD with 5% PRO added. In blood plasma assays for plasminogen activator activity (PAA), mean PAA values found in Group I AD Control animals was 11.7 %. In the (LA+ AD) Group II samples, it was 15.7% and 16.6% in (PRO+AD)‐Group III assays. The mean for blood cholesterol levels was 689.9mg% in the Group I AD Controls and lowered to 612.4 mg% in the (LA+AD) Group II and 595.1 mg% in the (PRO+AD) Group III samples. Tissue insulin sensitivity was measured by assaying blood platelet glucose content after incubation with exogenous glucose and insulin. Group I AD Control platelets had a mean value of 219.2 mg% Glucose. In (LA+AD) Group II, it increased to 304.4 mg% and in (PRO+AD) Group III‐ platelets, the mean was 308.7mg%. Gas chromatographic analyses of the aortae assayed for Linoleic Acid levels found a mean of 2645 nmol/mg dry wt. in Group I AD controls, 4310nmol/mg for LA+AD Group II aortae and 4290nmol/mg in the PRO+AD Group III aortae. Assays for GLA levels in aortae from AD Group I gave 10.1 nmol/mg dry wt., for LA+AD Group II, the value was 172nmol/gm and for PRO+AD Group III‐ it was 540nmol/mg.

Atherosclerotic plaque present in the wall of the aortae, estimated on a scale of 0–5, indicated the maximum aortal plaque development was 4.5 in AD Control Group I rabbits, 3.5 in LA+AD Group II and 2.5 in P RO+AD Group III aortae.

Addition of linoleic acid and primrose oil to AD, appeared to reduce atherogenesis that follows feeding rabbits AD alone. Addition of LA and PRO to AD increased blood levels of fibrinolytic activity and tissue sensitivity to insulin. These supplements lowered blood cholesterol and increased tissue levels of LA and GLA opposing the effects of AD dieting lacking them. Addition of LA and PRO to AD dieted rabbits resulted in increased tissue GLA levels which serve as a precursor for synthesis of the antiatherogenic Prostacylin (PGE2) in tissues.

Support or Funding Information

Funded by a grant from the American Heart Association
Activator of G‐protein signaling 8 is involved in VEGF‐induced choroidal angiogenesis
Authors: Hayashi Hisaki , Al Mamun Abdullah , Sakima Miho , Takeyama Masayuki , Yagasaki Rina , Zako Masahiro , Nakahara Tsutomu , Sato Motohiko
Published: November 2023

Choroidal neovascularization is associated with an age‐related macular degeneration (AMD), a major cause of vision loss among aged people. It is known that targeting of vascular endothelial cell growth factor (VEGF) signaling molecules are effective for the AMD treatment. From the ischemic cardiac tissue with highly developed collaterals, we have identified activator of G‐protein signaling 8 (AGS8) as a regulatory protein for heterotrimeric G‐proteins that binds to Gbg subunit. We recently reported that AGS8 bound to VEGF receptor type 2 (VEGFR‐2) and was involved in VEGF mediated angiogenesis in cultured human umbilical endothelial cells; however, role of AGS8 in vivo was not understood yet. This study aimed to explore the role of AGS8 in laser‐induced choroidal neovascularization, a murine model of AMD. Real‐time PCR demonstrated that AGS8 mRNA was significantly upregulated in laser‐induced choroidal lesion [238.3 ± 24.2%], and immunostaining indicated that AGS8 was only expressed in newly formed vessels originated from choroidal vasculature. For knockdown AGS8 expression in choroidal lesion, AGS8 siRNA was introduced into murine vitreous, which significantly reduced AGS8 expression [54.0 ± 18.2%], and also area of choroidal neovascularization. Furthermore, knockdown of AGS8 in RF/6A choroidal endothelial cells inhibited VEGF‐induced tube formation [22.7 ± 2.1%]. These results indicated that AGS8 play critical roles on VEGF‐induced choroidal angiogenesis in vivo as well as in vitro, and suggested AGS8 as an alternative target for AMD treatment.
Interleukin‐10 (IL‐10) Enhances Wound Healing of Cutaneous Flaps Following Ischemia Reperfusion Injury (IRI)
Authors: Tang Ya Hui , Lian Timothy
Published: November 2023

Cutaneous flaps are routinely used by reconstructive surgeons for the functional and cosmetic repair of wounds and defects that are the result of burn injuries, trauma, or tumor ablation. Successful reconstructions rely on the survival of these transferred tissues subjected to IRI. We found that IL‐10 is highly and persistently expressed in bone marrow stromal cells (BMSCs) and demonstrated that the introduction of BMSCs into IRI cutaneous flaps has a protective effect.

Objective

To determine whether IL‐10 treatment alone can protect cutaneous flaps from IRI or IL‐10 mediates the protective effect of BMSCs therapy in an animal model of cutaneous flaps following IRI.

Methods

A 1 × 2 cm cutaneous flap perfused by the inferior epigastric vessels were raised on C57BL/6 wild type and IL‐10−/− mice and were subjected to 3.5 hours of ischemia and then reperfused. Animals were divided into Sham, Ischemia, Ischemia + IL‐10 and Ischemia + BMSCs. IL‐10 and BMSCs were administered intravenously. Both blood perfusion images and perfusion units were recorded by a Laser Speckle Contrast Analysis System at day 1, 3, 5, 7, 9, 14 post ischemia and reperfusion. Digital photography of the flap was performed on the daily basis for analysis of flap size and area of necrosis. Surface area measurements of necrosis was determined with digital image software analysis (Image J; NIH). Biomarkers of endothelial cells (CD31) and immune cells (CD3 and F4/80) were also analysed in IRI flaps using Immunohistochemistry.

Results

The percent necrosis decreased significantly in mice with either IL‐10 or BMSCs treatment comparison with control on day 3, 5 and 7 post IRI. There was a significant increase in perfusion in all proximal areas at day 5 and distal areas at day 9 compared with immediate postoperative values (p<0.05). The perfusion was significantly decreased in proximal areas at day 1, and in distal areas at day 1 to 7 in both ischemia control and IL‐10 and BMSCs groups compared with Sham (p<0.05). There was a modest increment in blood perfusion in both proximal and distal areas of IL‐10 and BMSCs groups compared to the Ischemia control.

Conclusion

IL‐10 participated and contributed the protective effect to the animal model of cutaneous flap subjected to IRI.

Support or Funding Information

Supported by a Malcolm Feist Postdoctoral Fellowship