bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluating Pharmacokinetic Drug‐Drug Interactions Due to Time Dependent Inhibition
Authors: Yadav Jaydeep D , Pham Chuong , Korzekwa Kenneth , Nagar Swati
Published: November 2023

Time dependent inhibition (TDI) represents a more severe form of inhibition for cytochrome P450 (CYP) enzymes relative to reversible inhibition because inactivated enzymes have to be regenerated to restore their function by de novo synthesis. Moreover, it has been observed that many in vitro TDI exhibiting drugs do not exhibit significant TDI in vivo. The overall goal of this project is to improve TDI predictions with in vitro data. To this end, in vivo TDI data were generated in rats for future in vitro‐in vivo correlation analysis.

Drugs exhibiting TDI in vitro (podophyllotoxin (PPT), verapamil, paroxetine) and probe substrate for specific rat cyps (midazolam for cyp3a and desipramine for cyp2d1) were selected for pharmacokinetic (PK) studies. PK studies were conducted in singular jugular cannulated Sprague Dawley (SD) rats with selected CYP substrates. Rats were divided in two groups, control and inactivator treated group. Treated group was initially administered the inactivator and subsequently administered the probe substrate. Blood samples were withdrawn and analyzed for substrate concentration by LC‐MS/MS. Area under the curve (AUC(0‐∞)) ratios were compared between the two groups. Equilibrium dialysis was conducted to find the fraction unbound in plasma (fu) of substrate and the inactivator in five different matrices. PK studies were additionally conducted in SD rats for inactivator alone.

The AUC(0‐∞) ratios of two groups were calculated by both non‐compartmental analysis and compartmental analysis. The midazolam AUC ratio for PPT treated vs control groups was found to be 1.18 ± 0.24. The midazolam AUC ratio for verapamil treated vs control groups was 1.26 ± 0.43. The fu for midazolam was 0.04, 0.016, 0.78, 0.43, 0.76 in rat plasma, human plasma, human liver microsome (HLM), rat liver microsome (RLM) and rat hepatocytes respectively.

The AUC ratios indicate that the inactivators might not have a significant effect in vivo in rats. However an AUC ratio higher than 1 indicates some enzyme inactivation. In vitro studies are currently underway to conduct in vitro‐in vivo correlation of TDI.

Support or Funding Information

This work was supported by NIH grant GM1R01GM114369 to Ken Korzekwa and Swati Nagar.
Drug Interaction of Pirfenidone with Commonly Prescribed Pharmaceutical Drugs
Authors: Kale Vijay , Manoi Tharit , Porter Caleb , Atkinson Sabrina
Published: November 2023

Purpose

Pirfenidone was initially developed as an antihelminthic, antipyretic agent now approved by US FDA as antifibrotic drug for the treatment of idiopathic pulmonary fibrosis. In vitro experiments with cell fractions from human liver and recombinant cytochrome P450 enzymes indicate that pirfenidone is primarily metabolized by CYP1A2, although multiple other CYP isoforms contribute to metabolism. Numbers of clinical drugs are substrates to these isoforms and thus it will have significant effects on pirfenidone metabolism. Thus in the present study, we sought to investigate effects of ethynyl estradiol, zileuton, itraconazole, diazepam, omeprazole and naproxen on pirfenidone metabolism using human liver microsomes.

Methods

We ordered human liver microsomes from Life Technologies, Grand Island, NY (Pooled human liver chromosome from 50 donors, CYP450 levels 0.286 nmol/mg; CYP1A2 activity Vmax 0.73 nmol/min/mg of protein, Km 78μM). Pirfenidone, ethynyl estradiol, itraconazole, zileuton, diazepam, omeprazole, naproxen, and DMSO were from Sigma Aldrich, St. Louis MO. Ethyl acetate and 100mM phosphate buffer were from VWR, Randor, PA, C18 (5μm × 4.6mm × 150mm) and guard column were from Waters, Milford, MA. Waters HPLC equipped with photodiode array detector was used for separation and quantification. All other reagents were of analytical and HPLC grade. Briefly the microsomes were incubated with or without drugs (DMSO) at 37°C for 1h in phosphate buffer (total reaction volume was 200uL). Reaction was initiated by adding 20mM NADPH and terminated with equal volume of ethyl acetate. The reaction tubes were vortexed for few seconds to mix all contents and then centrifuged at 7,000g for 10 minutes at 4°C. Supernatant was isolated and stored until further processing at −80°C. 20uL of samples were eluted on HPLC using acetonitrile:water (65:35) as mobile phase at the rate of 0.7 mL/min. Detection wavelength was 317nm. Standard curve was run with same condition using denatured human liver microsomes.

Results

Our microsomal incubation studies show that pirfenidone concentration was significantly increased (126%) when used together with omeprazole as compared to pirfenidone alone. No significant increase in pirfenidone concentration was noted with other drugs (estradiol, itraconazole, zileuton, or naproxen) when used together with pirfenidone.

Conclusion

Our study suggests that there is a potential drug interaction of pirfenidone with omeprazole when used simultaneously.

Support or Funding Information

Roseman University of Health Sciences
Mechanism‐based Inhibition of CYP3A4 by Podophyllotoxin: Integrating Biophysical Measurements into Predictive Analysis
Authors: Barnaba Carlo , Nagar Swati , Korzekwa Ken R , Jones Jeffrey P
Published: November 2023

Avoiding drug‐drug interactions (DDIs) is a critical effort during drug discovery and development. One of the most difficult DDIs to predict with preclinical data is the time‐dependent inhibition (TDI) of the cytochrome P450 enzymes. Since TDIs result in a loss of enzyme over time, the in vivo impact of TDI is more difficult to assess than competitive inhibitors. Current methods to determine TDI parameters have provided limited success in predicting clinical outcomes. A probably more significant complexity in TDI kinetics is the mechanism of metabolic intermediate complex (MIC) formation. Podophyllotoxin is a methylenedioxyphenyl (MDP) compounds known to cause time‐dependent inhibition of CYP3A4, through a carbene intermediate that can form a pseudo‐irreversible complex with the heme of P450. The goal of the present study is to characterize the time‐course of MIC formation using biophysical techniques and relate these rate constants to observed time‐dependent inactivation kinetics. Here we report novel evidence for the existence of a reversible intermediate that plays a key role in the kinetics of MIC formation. This finding is then used with in vitro TDI kinetics, to greatly increase the accuracy of in vivo DDI predictions relative to existing methodologies.

Support or Funding Information

NIH
INTRINSIC INCREASED ACE2 EXPRESSION PROTECTS SPINY MOUSE ACOMYS CAHIRINUS AGAINST ISCHEMIC‐INDUCED CARDIAC DYSFUNCTION
Authors: Qi Yanfei , Zhang Juan , Wang Lei , Kumar Ashok , Vohra Ravneet , Walter Glenn A , Maden Malcolm , Katovich Michael J , Raizada Mohan , Pepine Carl J
Published: November 2023

Angiotensin converting enzyme 2 (ACE2), a key component of the renin angiotensin system (RAS), is a potential therapeutic target for ischemic heart disease as it protects cardiovascular function. Recently, extensive evidence points to a significant role of ACE2 in regulation of hematopoiesis and vasculogenesis via correction of endothelial progenitor cell dysfunction and increases in the number of cardiac and endothelial progenitor cells after ischemic injury. Spiny mice (Acomys cahirinus, SM) possess lizard‐like abilities when a large portion of skin is “torn away” the deep tissue injury heals rapidly by regenerating complete hair follicles, dermis, epidermis, sebaceous glands, adipose tissue, microvessels, smooth muscle and skeletal muscle of the panniculus carnosus without scaring (Nature 2013). This skin auto to my provides an effective defense against predators. We tested the cardiac regenerative capacity of SM using a myocardial infarction (MI) model. Following ligation of left anterior descending coronary artery, the SM exhibited smaller infarct size (SM‐MI 20% of left ventricular free wall vs CD1‐MI 50%, p<0.05) with preserved ejection fraction vs CD1 mice (SM‐MI 62 ± 2% vs CD1‐MI 32± 7%, p<0.05). At baseline ACE2 activity in SM sham was elevated 12% in myocardial tissue and 23% in plasma vs CDl sham (both, p<0.05). MI resulted in a 37% decrease in ACE2 activity in CD1 mice (p<0.05 vs baseline and vs SM‐MI). Interestingly, SM exhibited further elevation of ACE2 activity (7%) in myocardial tissue following MI, while plasma ACE2 activity remained unchanged after MI. Collectively, our findings suggest that ACE2 is likely to be modulating, at least in part, the myocardial preservation/regeneration process in the SM.

Support or Funding Information

2 UM1 HL087366‐06, R01 HL056921, HL033610, and UL1 TR000064 from the National Institutes of Health
Amiloride Enhances Arterial Flow‐Induced Dilation in Female Mice Fed a Western Diet
Authors: Martinez‐Lemus Luis A. , Ramirez‐Perez Francisco I. , Jia Guanghong , Habibi Javad , Aroor Annayya , DeMarco Vincent , Sun Zhe , Meininger Gerald , Jaffe Iris , Sowers James
Published: November 2023

We have previously shown that consumption of a western diet (WD) high in fat and sugars induces obesity, insulin resistance, cardiac diastolic dysfunction and vascular endothelial dysfunction. These deleterious effects of WD‐consumption appear earlier and are greater in female than in male mice. We hypothesize that the WD‐induced endothelial dysfunction is caused in part by overexpression and increased activity of epithelial Na channels (ENaC) in the vascular endothelium, that the endothelial cell (EC) ENaC overexpression is driven by an augmented EC mineralocorticoid receptor (MR) activity, and that the increased ENaC activity in the endothelium would be associated with increased EC stiffness and reduced flow‐induced dilatory capacity. To test these hypotheses, 4‐week old ECMR+/+ and ECMR−/− female mice were fed a WD (46% fat, 17.5% sucrose, 17.5% high‐fructose corn syrup) for 16 weeks. At 20 weeks of age mice were sacrificed and their aortae and mesenteric resistance arteries excised for ex vivo analyses. Aortic endothelial ENaC content was greater (P<0.05) and the aortic endothelium was stiffer (P<0.05) in ECMR+/+ than ECMR−/− mice as determined by immunohistochemistry and atomic force microscopy, respectively. Ex vivo exposure to the ENaC inhibitor, Amiloride (1mM), reduced endothelial stiffness in aortae. Accordingly, flow‐induced vasodilatory responses were greater (P<0.05) in mesenteric resistance arteries from ECMR−/− than in those from ECMR+/+ and Amiloride (1mM) improved flow‐induced dilations only in arteries from ECMR+/+ mice. These results suggest that endothelial dysfunction in WD‐fed female mice is associated with vascular endothelial stiffness resulting from endothelial MR‐dependent overexpression and increased activity of ENaC in the vascular endothelium. Moreover, our results indicate that ENaC inhibition with Amiloride may represent a novel strategy to prevent and treat the endothelial dysfunction associated with consumption of a WD in females.
VEGF/eNOS‐mediated Cardiomyocyte Proliferation: a Novel Mechanism to Regulate Cardiovascular Regeneration in Mice and Humans
Authors: GENTILE CARMINE , Chuang Christine Y , Drake Christopher J , Davies Michael J
Published: November 2023

An absence of cardiomyocyte proliferation in adult hearts limits their regeneration potential following tissue damage arising from adverse cardiac events such as a myocardial infarction or heart failure. There is considerable interest, and therapeutic potential, in unveiling mechanisms that enhance heart regeneration using stem cells and progenitor cells. Our studies have previously demonstrated that vascular endothelial growth factor (VEGF) regulates proliferation of cardiomyocyte progenitor cells via endothelial nitric oxide synthase (eNOS) in murine embryos. We have now investigated the role played by the VEGF/eNOS signalling pathway by evaluating cardiomyocyte proliferation in both WT and eNOS KO mouse embryos, and in human heart samples. Confocal analysis of WT and KO embryonic hearts showed no difference at embryonic stage E8.0 by using antibodies against markers of progenitor cells in the heart (VEGFR2/Flk1), cardiomyocytes (smooth muscle α‐actin), endothelial cells (CD31), and cell proliferation (phospho‐histone H3). In contrast, cardiomyocyte proliferation was reduced in E8.5 and E9.5 NOS3 KO embryos when compared to WT controls. Heart tube formation was also impaired. Consequently, E17 NOS3 KO embryos had smaller hearts (approximately 20% decrease of the volume) compared to WT controls. To translate our findings to humans, we analysed human heart biopsies from young and older donors using similar antibodies. Confocal analysis showed, for the first time, a correlation between eNOS expression and cardiomyocyte proliferation in a young human heart. To further investigate the role played by the VEGF/eNOS signaling pathway in human hearts, we developed an in vitro 3D co‐culture model of human endothelial cells, cardiomyocytes and fibroblasts, the human cardiac spheroid (hCS). A higher level of extracellular matrix (ECM) synthesis was observed in hCSs generated by young cells compared to hCSs generated by older cells. Interestingly, VEGF‐treatment of older hCSs increased ECM synthesis compared to untreated hCSs controls, demonstrating therapeutic potential in enhancing the activity of the VEGF/eNOS signaling pathway in hCSs. In conclusion, our data suggest that a similar role is played by the VEGF/eNOS signaling pathway in mice and human cardiomyocyte proliferation. Current studies are focusing on identifying molecular targets of the VEGF/eNOS signaling pathway in the regulation of human cardiomyocyte proliferation as novel therapeutics to induce tissue regeneration in diseased hearts.

Support or Funding Information

This study was supported by AHA 11PRE7530048 and Marcus Blackmore Fellowship to CG
Inhibition of the Sodium‐Glucose Co‐Transporter Protein Type 2 Decreases Oxidative Stress and Improves Cardiac Remodeling in Rats with Diabetes Mellitus
Authors: Rosa Camila Moreno , Campos Dijon Henrique Salome , Reyes David Rafael , Pagan Luana Urbano , Gomes Mariana Janini , Damatto Felipe , Fernandes Ana Angélica Henrique , Okoshi Marina Politi , Okoshi Katashi
Published: November 2023

Purpose

A new class of antidiabetic drugs aims to inhibit the sodium‐glucose co‐transporter type 2 (SGLT2). Thus, SGLT2 inhibition results in a reduction of hyperglycemia by decreasing reabsorption of glucose and increasing urinary excretion. Therefore, this study aimed to analyze the effects of SGLT2 inhibition by dapagliflozin (DAPA) on cardiac remodeling in rats with diabetes mellitus (DM).

Methods

Male Wistar rats with 450 g were divided into four groups: control (CTL, n=15); CTL+DAPA (n=15); DM (n=15); DM+DAPA (n=15). Diabetes was induced by streptozotocin (40 mg/kg, i.p., single dose). CTL+DAPA and DM+DAPA groups received DAPA (5 mg/kg/day, added to the rat chow) for 8 weeks. In vivo cardiac structures and functions were assessed by echocardiogram. Oxidative stress was evaluated in left ventricular myocardial tissue by measuring the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), catalase, and lipid hydroperoxide (LPO).

Statistics

Comparisons were performed by ANOVA ‐ 2×2 factorial (p<0.05).

Results

Compared to the DM group, DM+DAPA group presented higher body weight (DM: 343±52; DM+DAPA: 389±46 g; p<0.05), lower blood glucose (DM: 487±80; DM+DAPA: 172±89 mg/dL; p<0.05), and lower systolic blood pressure (DM: 140±7; DM+DAPA: 133±7 mmHg; p<0.05). Echocardiography showed lower left ventricular (LV) and left atrial diameter, and improved LV systolic and diastolic function in DM+DAPA compared to the DM group. Activities of SOD (DM: 13.7±3.90; DM+DAPA: 23.5±4.12 nmol/mg protein; p<0.05), GSH‐Px (DM: 19.7±5.07; DM+DAPA: 33.5±6.84 nmol/mg tissue; p<0.05), and catalase (DM: 85.4±23.0; DM+DAPA: 117±20.1 μmol/g tissue; p<0.05) were increased, and LPO levels (DM: 385±53.8; DM+DAPA: 304±39.8 nmol/g tissue; p<0.05) decreased in the DM+DAPA, compared to the DM group.

Conclusion

Inhibition of the sodium‐glucose co‐transporter type 2 by dapagliflozin decreases oxidative stress and improves cardiac remodeling in rats with diabetes mellitus.

Support or Funding Information

FAPESP, and CNPq.
Acute GPER1 Activation During Reperfusion Protects the Myocardium Against Ischemia/Reperfusion Injury by inhibiting of the mPTP Opening and Mitophagy
Authors: Feng Yansheng , Kabir E. Mohammad , Madungwe Ngonidzashe Benson , Bopassa Jean C.
Published: November 2023

Introduction

We recently found that acute pre‐ischemic estrogen (17 b‐estradiol) treatment induces cardioprotection against ischemia/reperfusion (I/R) injury mainly via G protein‐coupled estrogen receptor1 (GPER1) activation. We, furthermore, showed that pre‐ischemic estrogen‐GPER1 effects preserve mitochondrial integrity and function via MEK/ERK/GSK‐3b pathway.

Hypothesis

We investigated whether acute post‐ischemic estrogen (PI‐E2) treatment can also induce cardio‐protective effects via GPER1 activation and determine whether PI‐E2 action involves the reduction of mitophagy.

Methods

Male and ovarectomized female Sprague‐Dawley rats were anesthetized with ketamine (80 mg/kg i.p.) and xylazine (8 mg/kg i.p.). Hearts were subjected to 35 min of the left anterior descending (LAD) artery occlusion, followed by 180 min reperfusion. An estrogen bolus (50 mg/kg body weight) or PBS (same volume) was applied via the femoral vein 5 min before reperfusion, and GPER1 antagonist, G15, was given 10 min before estrogen. Area at risk (AAR) was identified using Evans Blue dye and myocardial infarct size assessed by TTC staining method. Mitochondrial calcium retention capacity (CRC) required to induce mitochondrial permeability transition pore (mPTP) opening was assessed after 20 min reperfusion. Expression levels of LC3I LC3II; p62; PINK1; and pERK were detected by western blot in whole cell lysates. Parkin and ubiquitinated protein were measured by Western blot in cytosolic and mitochondrial fractions.

Results

We found that PI‐E2 treatment reduced myocardial infarct size normalized to the AAR or to the whole LV and increased mitochondrial CRC compared to untreated. We also found that PI‐E2 treatment decreased the level of LC3II, increased the level of p62 and pERK1/2, inhibited the translocation of Parkin to mitochondria and decreased ubiquitinated proteins in mitochondria compared to control. Interestingly, all these PI‐E2 effects were abolished by addition of G15.

Conclusion

Acute post‐ischemic GPER1 activation by estrogen treatment induces cardioprotective effects against I/R injury. PI‐E2 effects through GPER1 involve the inhibition of mitophagy and reduction of mitochondrial proteins ubiquitination. Together, these post‐ischemic estrogen effects via GPER1 lead to inhibition of the mPTP opening, and subsequently cardioprotection.

Support or Funding Information

Voelcker Fund
Impact of Static and Pulsatile Blood Pressure on Cerebral Blood Flow Regulation
Authors: Tarumi Takashi , Zhang Li , Liu Jie , Xing Changyang , Turner Marcel , Repshas Justin , Lewis Tammy , Zhang Rong
Published: November 2023

Background

Elevated blood pressure (BP) is a major risk factor for stroke and cognitive impairment in older adults. BP consists of static and pulsatile components that may have differential impacts on cerebral hemodynamics. The objective of this study is to investigate the main and interaction effects of static and pulsatile BP components on cerebral blood flow (CBF), perfusion, and pressure‐flow relations.

Methods

We tested 126 subjects aged 55–80 years (mean age = 65±6 SD). Static and pulsatile BP components were defined as diastolic BP and pulse pressure respectively, as measured by arterial tonometry from the carotid artery. Global CBF, regional brain perfusion, and vascular resistance were measured by phase‐contrast MRI and arterial spin labeling. Transfer function metrics of cerebral dynamic pressure‐flow relations were calculated from the cardiac and lower frequencies to quantify cerebrovascular impedance and autoregulation.

Results

CBF pulsatility increased dose‐dependently with pulse pressure while global CBF and regional perfusion remained at the similar levels. Figure shows the main and interaction effects of static and pulsatile BP components on cerebral pressure‐flow relations. Cerebrovascular resistance increased with diastolic BP whereas the modulus of impedance increased with both diastolic BP and pulse pressure. There were significant main and interaction effects of diastolic BP and pulse pressure on transfer function phase of cerebral autoregulation. These findings remained statistically significant after adjusting for covariates (e.g. age).

Conclusions

Elevations of static and pulsatile BP components increase cerebrovascular resistance/impedance and alter dynamic cerebral autoregulation in middle‐aged and older adults, which potentially may have impact on brain structure and function.

Support or Funding Information

This study was supported by the National Institute of Health (R01AG033106 and R01HL102457) and the American Heart Association (14POST20140013).
The Role of the Endothelium in Altering Vascular Reactivity Following Acute Hyperbaric Oxygen Treatment
Authors: Janardan Veena , Bontekoe Jack , Ophoff Brad , Horr Kyle , Sylvester Francis
Published: November 2023

Acute hyperbaric oxygen treatment (HBOT) alters vascular reactivity, yet the underlying mechanism requires further clarification. As Furchgott and Zawadzki previously demonstrated the obligatory role of the endothelium in regulating blood vessel tone, our lab seeks to determine if the simple squamous epithelium of the tunica intima mediates the observed changes in vascular reactivity following HBOT. Intact or chemically denuded porcine, mesenteric arterial rings were exposed to one of three treatments: 100% oxygen at 1.75 atm, room air at 1.75 atm, and room air at 1.00 atm. Additionally, a separate series of experiments utilized a Clark type electrode to measure the oxygen concentrations observed in the hyperbaric chamber. These recordings demonstrated significant differences among the three treatment groups. Following two hours of exposure to hyperbaric treatment, vessels were mounted in isolated organ baths and coupled to force transducers so that changes in arterial force were recorded in response to multiple agonists. The vasoconstrictors, potassium chloride (6×10−2 M) and phenylephrine (10−7 ‐3×10−6 M), were used to record increases in tension. The vasodilators, acetylcholine (10−7 ‐ 10−4 M) and sodium nitroprusside (10−7 ‐ 10−4 M), were used to record decreases in tension. Lack of a dilator response to acetylcholine, an endothelial‐dependent vasodilator, was used as validation for denudation. Potassium chloride and phenylephrine induced significant increases in tension in all treatments. Nitroprusside induced significant decreases in tension in all treatments. Comparison of the intact arteries to the denuded arteries following HBOT indicated that the endothelium did not account for the changes in vascular reactivity. Further investigation of the acute effects of HBOT on vascular reactivity is needed to better understand the potential benefits of hyperbaric oxygen therapy for treating cardiovascular disease.
Induction of Heme Oxygenase with Hemin Improves Impaired Insulin Signaling and Compromised Cardiac Structure and Function in Obese Zucker Rats
Authors: Tiwari Shuchita , Ndisang Joseph Fomusi
Published: November 2023

Background

Diabetic cardiomyopathy is characterized by cardiac dysfunction and impaired insulin signaling. The mechanisms that drive obesity and insulin‐resistant mediated cardiomyopathy are not completely elucidated. Accordingly, the aim of this study was to examine the effects of upregulating the heme‐oxygenase (HO) on obesity related cardiac dysfunction and impaired insulin signaling in obese Zucker rats, a model characterized by insulin resistance and cardiomyopathy.

Methods

Immunohistochemistry, spectrophotometry, enzyme‐linked immunosorbent assay (ELISA) and Western blotting were used. In addition non‐invasive echocardiography and invasive left ventricular (LV) cannulation was employed to evaluate the hemodynamic, echocardiographic and electrophysiological parameters of the myocardium. HO was enhanced with hemin or blocked with stannous mesoporphyrin.

Results

Our data demonstrated that administering hemin to obese Zucker rats; (i) enhanced important proteins of insulin signaling (IRS‐1, PI3K and GLUT4), (ii) reduced glucose intolerance, (iii) suppressed HOMA‐index, but (iv) increased adiponectin and atrial‐natriuretic‐peptide (ANP). Interestingly, the improvement of insulin sensitivity and glucose metabolism was accompanied by attenuation of cardiomyocytes longitudinal muscle‐fiber thickness, the reduction of diastolic/systolic free‐wall thickness and the suppression of LV hypertrophy. Furthermore, hemin attenuated inflammation/oxidative stress, suppressed fibrosis, extracellular matrix/pro‐fibrotic proteins (collagen‐IV, fibronectin, TGF‐β1) as well as proteins of heart failure such as osteopontin and osteoprotegerin. These were associated with improved cardiac function as evidenced by improved hemodynamics such as mean‐arterial pressure, arterial‐systolic pressure, +dP/dt, arterial‐diastolic pressure and improved electrocardiographic parameters associated with cardiac conduction system including PR‐interval, QRS‐duration, ST‐segment and QT interval. However, the HO blocker, stannous‐mesoporphyrin reversed the cardio‐protective effects of hemin.

Conclusion

Our findings demonstrates that hemin improved compromised cardiac structure and function in obese Zucker rats. Interestingly, attenuation of inflammation, oxidative stress, extracellular‐matrix/profibrotic proteins, cardiac hypertrophy and fibrosis alongside the potentiation of adiponectin, ANP and proteins of insulin signaling are among the multifaceted mechanisms through which an upregulated HO‐system improves insulin sensitivity, glucose metabolism and diabetic cardiomyopathy. Thus, HO‐inducers may be explored as a novel therapy for diabetic cardiomyopathy.

Support or Funding Information

Heart & Stroke Foundation of Saskatchewan, Canada
Long QT‐syndrome Associated Caveolin‐3 Mutations F97C and S141R Alter the Cardiac Transient Outward Kv Current and the L‐type Ca2+ Currents
Authors: Balijepalli Ravi , Tyan Leonid , Keefe Alexis , Foell Jason D , Kamp Timothy J
Published: November 2023

Caveolae are discrete microdomains in cardiomyocytes that localize many ion channels and signaling proteins. Mutations in the CAV3 gene, encoding caveolin‐3 (Cav‐3), a muscle specific scaffolding protein integral to caveolae in the cardiomyocytes, have been associated with a congenital Long QT syndrome (LQTS9). Previously we have shown that the LQTS associated Cav‐3 mutations, F97C and S141R; increase late sodium channel current in cardiomyocytes contributing to the prolonged action potential duration (APD), but co‐immunoprecipitation using lysates from mouse ventricular myocytes and western blot analysis revealed that Kv4.2, Kv4.3 and Cav1.2 channels also co‐IP with Cav‐3. Thus we evaluated whether disease the LQT associated mutations impacted heterologously expressed Kv4.2, Kv4.3, and Cav1.2 channels in HEK293 cells using whole cell patch clamp electrophysiology. Co‐expression of F97C (40.7 ± 5.9 pA/pF) or S141R (58.8 ± 7.7 pA/pF) significantly (p<0.001) reduced the peak IKv4.2 densities compared to cells co‐expressing either Cav‐3 WT (80 ± 9.1 pA/pF) or pcDNA3.1 (88.6 ± 12.7 pA/pF). The co‐expression of F97C significantly reduced IKv4.3 (213.8 ± 35 pA/pF) compared to Cav‐3 WT (336.7 ± 48.2 pA/pF). F97C also caused a reduction in the activation time constants of Ikv4.2 and Ikv4.3, but did not impact the inactivation. Co‐expression of F97C and S141R significantly (p<0.05) slowed the recovery from inactivation of Kv4.2. On the other hand, co‐expression of S141R significantly increased the peak ICa,L (‐14.8 ± 2.5 pA/pF, p<0.05) compared to Cav‐3 WT (−9.5 ± 2.4 pA/pF). Co‐expression of F97C (−7.6 ± 1.2 pA/pF) did not alter the peak ICa,L density but significantly slowed the calcium dependent inactivation (CDI) of the ICa,L. We conclude that the LQTS linked Cav‐3 mutations F97C and S141R, impart loss‐of‐function effects on Ito but cause a gain‐of function effect on the ICa,L and together these may contribute to the delay in ventricular repolarization and arrhythmogenesis in a setting of LQTS.

Support or Funding Information

R01 HL078878
Fetal Hypoxia Programs High Blood Pressure And Reduced Adaptation To Repeat Psychological Stress
Authors: Prestipino Louise , Ritchie Helen , Polson Jaimie W
Published: November 2023

Numerous studies in both humans and animal models highlight intrauterine hypoxia as a potential stressor capable of programing hypertension in the offspring. The underlying causative mechanisms are unclear, but may be due to altered cardiovascular responses to chronic stress. In this study in the rat, we used dofetilide (dof), a class III antiarrhythmic agent reported to reduce fetal heart rate, without affecting the dam, and thereby induce hypoxia specific to the fetus. Blood pressure (BP), heart rate (HR), HR variability (HRV) and BP variability (BPV) were compared in dof programed and control offspring at 3–6 months of age. Pregnant rats were dosed daily with dof (2.5 mg/kg orally) or saline from gestational days 11–14. Embryonic HR was determined using an ex vivo embryonic culture preparation, which confirmed a period of ~6h embryonic bradycardia after dosing. The absence of an effect of dof on adult HR was confirmed in a separate cohort of control adult rats following surgical implantation of radiotelemetry BP transmitters. Dof and control offspring were telemetered and cardiovascular variables were measured at rest and following an air jet stress (AJS) protocol, repeated twice daily for 7 days. Dof rats were hypertensive at rest before AJS (day1 mean BP: dof, 92±2.3 mmHg; control, 83±1.9 mmHg, P=0.03). The increase in BP in response to the first AJS was similar between groups (dof, 23.8 mmHg; control 22.8 mmHg), as were the increases in HRV and BPV. However, after 7 days, there was a significant attenuation of the AJS pressor response in control (51.2%, P=0.04), but not dof (28.9%, P=0.48) rats. This pattern was also reflected in the total power component of BPV, with control rats displaying significantly greater attenuation (72%, P=0.02) than dof (41%, P= 0.06). The 7 days of repeated AJS did not affect resting blood pressure in either group (day 7 mean BP: dof, 95±2.2 mmHg, P=0.31 compared to day 1; control, 85±2.3 mmHg, P=0.46). The results indicate that fetal hypoxia programs for hypertension and altered responses to repeated psychological stress. Although repeated stress showed reduced adaptation of the pressor response, more than 7 days of chronic stress may be required before an effect on resting BP may be observed.
Cytotoxic CD8+ Interleukin (IL)‐17+ T‐cells Play a Role in Hypertension‐Specific Inflammatory Responses Associated with Ovariectomy in Dahl Rats
Authors: Pai Amrita V , West Crystal A , Souza Aline A , Ji Hong , Wu Xie A , Sandberg Kathryn L
Published: November 2023

Background and Objective

Oophorectomy is associated with increased body weight (BW) gain and incidence of hypertension (HT) compared with age‐matched women. Increased BW gain and HT coincide with increased inflammatory biomarkers. To separate immune responses due to HT from those due to BW gain, we compared T‐cell subpopulations in normotensive Dahl (DR) and hypertensive Dahl (DS) rats after ovariectomy.

Methods

Six week old DS and DR rats were ovariectomized (Ovx) or had sham surgery (Sham). Mean arterial pressure (MAP) was measured in anesthetized rats after 4 and 10 months (mo) acutely by indwelling vascular catheter. Splenic T‐cells were assessed by flow cytometry at 10 mo.

Results

Ovariectomy increased the MAP in DS but not DR rats at 4 mo [MAP (mmHg): DS‐Ovx, 143±1.0 vs DS‐Sham, 129±2.9; p=0.0002 & DR‐Ovx, 123±4.4 vs DR‐Sham, 126±1.3; ns n=10–12/group]. At 10 mo, the MAP remained higher in the ovariectomized DS compared to DR rats [MAP (mmHg): DS‐Ovx, 190±12 vs DR‐Ovx, 114±1.2; n=5–8/group; p=0.002]. At 10 mo, ovariectomy increased BW to a similar extent in DS and DR rats [BW gain (g): DS, 179±13 vs DR, 202±14; ns]. The frequency of CD8+IL17a+ T‐cells increased in the ovariectomized DS but not DR rats [Frequency (%) at 10 mo: DS‐Ovx, 9.6±2 vs DR‐Ovx, 1.3±0.4; n=5–8/group; p<0.0001].

Conclusion

Cytotoxic CD8+ IL‐17a+ T‐cells may play a role in the HT‐specific inflammatory response in ovariectomized DS rats.

Support or Funding Information

Georgetown University Medical Center, and Translational Biomedical Science Predoctoral Training Program Georgetown‐Howard Universities Center for Clinical and Translational Science
Pathogen‐derived biosensors for detection of phosphoinositides in mammalian cells
Authors: Pike Colleen
Published: November 2023

Phosphoinositide lipids (PIPs) have a major role in defining organelle identity in eukaryotic cells by coordinating recruitment of protein effectors to distinct membranes in a time‐ and organelle‐dependent manner. Bacterial pathogens are emerging as a valuable resource for the development of high affinity PIP biosensors which can be used to monitor membrane trafficking events in live cells. Legionella pneumophila is an opportunistic bacterial pathogen that infects alveolar macrophages and causes severe pneumonia known as Legionnaires’ disease. The success of infection is attributed to L. pneumophila's ability to evade lysosomal degradation and establish a plasma membrane‐derived compartment called the Legionella containing vacuole (LCV). Throughout the course of infection L. pneumophila translocates over 300 effector proteins into the host cytosol, many of which bind and/or modify distinct species of PIPs. We hypothesized that L. pneumophila has many more PIP binding effector proteins that have not yet been identified. We performed in vitro binding studies with purified L. pneumophila effector proteins and identified a set of fourteen novel PIP‐binding L. pneumophila effector proteins with varying PIP substrates and binding affinities. These effectors predominantly bind PI3P and PI4P and they may be important for targeting vesicular trafficking to establish the LCV and evade degradation. We expect that the PIP binding candidates identified in our screen have distinct PIP binding domains that can be utilized as novel PIP biosensors.
Pioglitazone Inhibits Pyruvate Metabolism in Hepatocytes
Authors: Shannon Christopher , Daniele Giuseppe , Galindo Cynthia , DeFronzo Ralph , Abdul‐Ghani Mohammed , Norton Luke
Published: November 2023

The thiazoldinedione compound pioglitazone (PIO) is an effective hypoglycemic agent commonly prescribed for the treatment of type 2 diabetes mellitus (T2DM), the actions of which have typically been assigned to transcriptional regulation through the peroxisome proliferator‐activated receptor gamma (PPAR‐γ). Growing evidence supports the presence of non‐PPAR‐γ related targets of PIO. For example, modulation of glucose metabolism has been demonstrated in human skeletal muscle myotubes through PIO‐mediated inhibition of the newly identified mitochondrial pyruvate carriers MPC1 and MPC2 (Divakuruni et al,. 2013). However, the existence of PPAR‐γ sparing mechanisms of PIO in hepatocytes has not been established. Therefore, we explored the effects of pharmacologically‐relevant concentrations of PIO in cultured hepatocytes (H4IIE) on whole‐cell substrate oxidation (14CO2 capture), mitochondrial ATP production (luciferin‐luciferase assay) and pyruvate/lactate‐driven glucose production, as well as any impact upon the expression of key regulatory gluconeogenic genes. We also assessed the dependence of these effects, if any, on MPC1 and MPC2 using siRNA transfection by electroporation.

Four hour PIO treatment (10 μM) suppressed pyruvate oxidation by 29% (P<0.01), but had no impact upon palmitate oxidation. Consistent with this, a dose‐dependent inhibition of mitochondrial ATP production from 500 μM pyruvate/2 mM malate was observed with PIO at 1 μM (27%; p=0.07), 5 μM (36%; P<0.05) and 10 μM (61%; P<0.01) concentrations, but did not influence ATP production from glutamate, succinate or palmitoylcarnitine substrates. Glucose production from pyruvate/lactate was 53% lower following overnight treatment with 10 μM PIO (P<0.01) and this occurred in the absence of any changes in the expression of phosphoenolpyruvate carboxykinase, fructose 1,6‐bisphosphatase, glucose 6‐phosphatase or pyruvate carboxylase. Treatment with the specific inhibitor of mitochondrial pyruvate import UK50995 (5 – 10 μM) similarly inhibited pyruvate oxidation (−85%), pyruvate‐driven mitochondrial ATP production (−66%) and glucose production (−71%). Silencing of MPC1 or MPC2 led to a ~20% reduction in pyruvate oxidation, but only MPC1 silencing significantly impaired glucose production (−48%). The inhibition of pyruvate oxidation by PIO was preserved in cells silenced for MPC1 (P<0.001) and MPC2 (P<0.05), as was the suppression of glucose production in cells silenced for MPC1 (P<0.05).

Together, these data demonstrate a role for PIO in the downregulation of liver mitochondrial metabolism that is specific to pyruvate and not dependent on the expression of MPC1 or MPC2. Moreover, these experiments indicate the existence of a novel PPAR‐γ‐independent mechanism through which PIO may suppress hepatic glucose production and hence ameliorate hyperglycemia in T2D patients.
CHANGE IN ENVIRONMENTAL GLUCOSE CONCENTRATION ALTERS ASTROCYTIC INSULIN AND INSULIN RECEPTOR LEVELS
Authors: Cohen Susan , Liu Qiuli , Ryan Robert , Gebremedhin Debebe , Harder David , Rarick Kevin
Published: November 2023

BACKGROUND

The actions of insulin in the brain include effects on glucose homeostasis, cell proliferation, cell differentiation, cognition, and memory. Cellular constituents of the neurovascular unit, including the astrocyte, contribute to insulin responses in the brain. Astrocytes are an ideal target cell type for investigation because of their dynamic character, multitude of functions, and their important role in brain homeostasis. We hypothesize that astrocytes respond to environmental glucose changes and could be a source of insulin supply to the microenvironment.

OBJECTIVE

To investigate astrocyte insulin and insulin receptor response to environmental glucose changes and to determine insulin secretion levels.

METHODS

Neonatal rat astrocytes in culture were exposed to changes in environmental glucose levels (LOW‐ 0mM, NORMAL‐5.5mM, and HIGH‐25mM) over three passages. Astrocyte whole cell lysates were used to determine message (qPCR) and protein levels (Western blot) for insulin and insulin receptor. Insulin Ultrasensitive ELISA was used to measure NORMAL astrocytic insulin secretion in response to glucose (25mM) and potassium (30mM) stimuli.

RESULTS

Astrocytes grown in LOW, NORMAL, and HIGH glucose levels resulted in equal cell counts and morphological appearances. Increasing glucose level from LOW, to NORMAL, or to HIGH in astrocytic culture media significantly increased insulin‐1 (exon 1–2) and insulin‐2 (exon 1–2) mRNA levels (One‐way ANOVA, n=4, p<0.05). Increased glucose level also resulted in a positive correlation with increased protein levels of insulin and insulin receptor in astrocytic cell lysate (Pearson Correlation, r=>0.85, p<0.01, n=3, R2=>0.73). Astrocytes secreted measurable levels of insulin (0.02–0.05μg/L) but did not increase significantly with glucose or potassium stimuli.

CONCLUSIONS

Astrocytes respond to changes in environmental glucose levels with increased insulin and insulin receptor message and protein levels. Astrocytes secrete measurable amounts of insulin but do not respond to stimuli as other insulin secreting cells. We speculate that such insulin responses to glucose levels from the astrocyte may be adaptive for its own homeostasis rather than to affect the microenvironment.

Support or Funding Information

Funding from NIH (NHLBI) R01 HL033833 and HL105997 and the Veterans Administration Research Career Scientist Award
VTA Lamotrigine microinfusions accelerate the development of cocaine sensitisation
Authors: Vazquez Rafael , Santos‐Vera Bermary , Arencibia‐Albite Francisco , Vaquer‐Alicea Ana , Maria‐Rios Cristina , Montiel‐Ramos Alan , Devarie‐Hornedo Marcos , Jimenez‐Rivera Carlos
Published: November 2023

Support or Funding Information

2SC1GM084854‐05A1

5R25GM061838‐15

2G12‐RR003051

8G12‐MD007600

Dopaminergic projections arising from ventral tegmental area (VTA) play a key role on drug induced effects in the mesocorticolimbic system. Chronic cocaine administration produces an increased VTA dopamine (DA) cell excitability. VTA DA neurons express a large hyperpolarization activated cation current, known as Ih, that contributes to the electrophysiological properties of neurons in the CNS. We hypothesized that Ih ‐enhances VTA DA cell activity during the development of cocaine sensitization. Cocaine sensitization is the progressive escalation of psychomotor responses that results with repeated cocaine administration. We performed intra VTA microinfusions of Lamotrigine (LTG), an Ih current enhancer. Male Sprague‐Dawley rats (250–300g) were anesthetized and stereotaxically implanted with bilateral 26‐gauge cannulas in the VTA. Animals were divided as following: Vehicle/Saline, LTG/Saline, Vehicle/Cocaine, LTG/Cocaine (microinjections/i.p. injections, respectively). Daily LTG microinjections (0.1ug bilateral) were administered for 7 days. Cocaine treated rats (15 mg/kg, i.p.) were injected 10–15 minutes after the LTG microinfusion. Locomotor activity was recorded for 1 hour every day. On day 8, rats received a first cocaine challenge in the absence of LTG. After a 7 days withdrawal period, a second cocaine challenge was performed. Total ambulatory and stereotype activities were analyzed using One‐Way ANOVA followed by Newman‐Keuls Test. LTG/Cocaine rats showed a significant increase in the acute response to cocaine (p< 0.01). Moreover, these animals developed sensitization two days before the control group. Their locomotor response to cocaine during days 1–3 was significantly higher (p< 0.01) compared to Vehicle/Cocaine rats. However, during cocaine challenge, when no LTG was administered the LTG/Cocaine group's locomotor activity was not significantly different from the Vehicle/Cocaine group. We postulate that LTG in the VTA, acting through the Ih current, enhances neural excitability caused by chronic cocaine administration. Taken together, our data suggest that an enhanced Ih current activity in the VTA may contribute to a greater susceptibility to cocaine neurobiological effect.
Renal Nerve Denervation Suppresses the Progression of End Organ Damage via Modulating the GABAergic Mechanisms in the Brain in Hypertensive Mice with Chronic Kidney Disease
Authors: Nishihara Masaaki , Hirooka Yoshitaka
Published: November 2023

Background

Sympathoexcitation is strongly involved in the pathogenesis of hypertension accompanied with chronic kidney disease (CKD). The paraventricular nucleus of the hypothalamus (PVN) in the brain controls the sympathetic outflow through GABA‐ergic mechanisms in hypertension. The renal nerve denervation (RDN) exerts a certain long term antihypertensive effect; however the precise mechanism is not known. In the present study, we examined whether RDN reduces sympathetic outflow through GABA in the PVN and improves the end organ damage in mice with CKD.

Methods and Results

In 5/6‐nephrectomized ICR‐mice (Nx) at 4‐weeks after nephrectomy, systolic BP (SBP) was significantly increased (vs. Sham, 143±2 vs. 109±2mmHg, n=12–24, p<0.01), accompanied by sympathoexcitation (urinary norepinephrine (uNE): vs. Sham, 424±26 vs. 226±17μg/24hrs, n=12–24, p<0.01). Nx‐mice developed albuminuria (vs. Sham, 510±97 vs. 102±17mg/g creatinine, n=6–16, p<0.01), and echocardiographic left‐ventricular hypertrophy (LVH) (vs. Sham, 1.18±0.01 vs. 0.98±0.03mm, n=6–24, p<0.01) compared with sham‐mice. We performed RDN or sham operation and divided into 3 groups (Sham‐sham, Nx‐sham, and Nx‐RDN, n=12 for each). At 2‐weeks after RDN, SBP was significantly decreased (123±2 vs. 136±2 mmHg, n=12 for each, p<0.01), and urinary sodium excretions were increased (0.64±0.04 vs. 0.47±0.03 mmol/24hrs, n=8 for each, p<0.01) in Nx‐RDN compared with those in Nx‐sham. The uNE levels were not different between two groups. At 6‐weeks after RDN, SBP was kept decreasing (125±1 vs. 140±3 mmHg, n=12 for each, p<0.01) and uNE levels were also decreased (386±32 vs. 541±35 μg/24hrs, n=12 for each, p<0.01) in Nx‐RDN compared with those in Nx‐sham. The urinary sodium excretions were not different between two groups. Bicuculline (GABA‐A receptor antagonist, 50pmol) microinjection into PVN increased mean arterial pressure (MAP) and lumbar sympathetic nerve activity (LSNA) in all groups. The pressor responses and the change in LSNA were significantly attenuated in Nx‐sham (vs. Sham‐sham, ΔMAP/baseline MAP [%], 33±4 vs. 66±6 %; ΔLSNA (%baseline), 57±7 vs. 112±8 %, p<0.01, n=6 for each), but were enhanced in Nx‐RDN at 6‐weeks after RDN (vs. Nx‐sham, ΔMAP/baseline MAP [%], 60±6 vs. 33±4 %; ΔLSNA (%baseline), 94±5 vs. 57±7 %, p<0.05, n=6 for each). The albuminuria was suppressed (533±121 vs. 1001±192 mg/g creatinine, n=8, p<0.01), and LVH was regressed (1.08±0.02 vs. 1.24±0.03mm, n=12, p<0.05) in Nx‐RDN compared with those in Nx‐sham at 6‐weeks after RDN. The plasma aldosterone concentration (PAC) level was significantly decreased in Nx‐RDN compared with Nx‐sham (365±38 vs. 633±42pg/ml, n=12, p<0.01).

Conclusion

These data indicate that long term antihypertensive effect exerted by renal nerve denervation suppresses the end organ damage, at least in part, by reducing sympathetic outflow through GABA in the PVN in hypertensive mice with CKD.
IRF6 is critical mediator of alternative macrophage activation
Authors: Tseng Alexander Mike , Cheng Chang‐An , Wang Haiqing , Lin Yu‐Lieh , Zhou Beiyan , Brehm Tyler
Published: November 2023

Macrophages display a spectrum of activated phenotypes in response to microenvironmental signals. Pro‐inflammatory and anti‐inflammatory macrophages have important roles in promoting or resolving disease states and also regulate homeostatic conditions of the tissues they reside in, as is the case with adipose tissue macrophages (ATMs). Indeed, under obese stress ATMs expand in number and undergo predominantly pro‐inflammatory activation which propagates dysregulated metabolic and immunologic adipose tissue homeostasis. In this study, we report a new regulator, IRF6, in controlling macrophage alternative activation (M2).

IRF6 is a member of the interferon regulatory transcription factors (IRFs) which share a high degree of conservation in their DNA binding domain but less similarity within their protein interaction domain. Knowledge of IRF6 is currently limited to its role in mediating epithelial cell differentiation and the epithelial innate immune response to pathogen challenge. We observed that expression of IRF6 was dramatically suppressed in M2 macrophages. Using both gain and loss of function strategies, we further confirmed that IRF6 specific shRNA enhanced M2 activation as evidenced by elevated levels of activation‐related cell surface markers CD69 and CD86 and the expression of M2‐related genes including IRF4, PPARγ, Arginase1, and IL10. Conversely, IRF6 overexpression dramatically attenuated M2 activation. Furthermore, chromatin immunoprecipitation revealed IRF6 regulates anti‐inflammatory macrophage activation by repressing the expression of PPARy, a critical mediator of macrophage polarization, through binding two IRF recognition sites located upstream of the PPARγ coding region. Our investigation also indicated IRF6 expression is significantly increased in obese ATMS, suggesting that a dysregulated IRF6/PPARγ regulatory axis suppresses anti‐inflammatory ATM responses and ultimately contributes to dysregulated metabolic and immunologic homeostasis.

Support or Funding Information

This work was supported by the Texas A&M Graduate Merit Fellowship (to A. Tseng), American Diabetes Association (1‐13‐JF‐59 to B. Zhou) and National Institute of Health/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NIDDK 1R01DK098662 to B. Zhou).