bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cardiomyocytes Release IFN‐β in Response to Hypoxia: A Previously Unrecognized Cardiomyocyte to Fibroblast Signaling Pathway
Authors: Bangeranye Catherine , Wang Ping , Chatterjeee Prodyot K , Predmore Jamie , McNeil Franklin , Metz Christine , Miller Edmund , Day Sharlene , Powell Saul R 
Published: November 2023

We have observed a persistent increase in the expression and activity of the immunoproteasome (iProt) in post‐ischemic heart (post‐MI). The objective of this study was to identify the pathway and mediator(s) responsible for iProt upregulation. Using RT‐PCR, ifng, tnfa, and ifnb1 mRNAs were assayed. There were no changes in either ifng or tnfa but ifnb1 was elevated ~ 17‐fold (P<0.01) in the heart 4 days post‐MI. Exposure of a mixture of rat neonatal cardiomyocytes/fibroblasts (CM/CFs) to IFN‐β from 1U/ml up to100u/ml for 24h resulted in a variable response with an 85% (P<0.05) increase in iProt at 100 u/ml as measured using the ProCISE assay. Since the CM/CF preparation typically contains about 20% cardiac fibroblasts, (confirmed by vimentin staining), isolated CFs were exposed to IFN‐β for 24hs which resulted in a robust concentration‐dependent increase in iProt that was almost 3‐fold (P<0.01) at 100 u/ml. An enriched CM (eCM) preparation in which CFs were limited to ≤5% was prepared and these had the lowest iProt response to 10u/ml IFN‐β (eCM, +27% ± 5%; CF, +104 ± 15%; CM/CF, +52 ± 18%). To determine a potential source of IFN‐β in the post‐MI heart, CFs, CM/CFs and eCMs were exposed to 1% O2 for 22h to simulate ischemia. This resulted in +2 ±1%, +31 ± 8% (P<0.05), −16 ± 5% changes in iProt, respectively, suggesting that the CMs were the source of the IFN‐β. RT‐PCR analysis revealed that ifnb1 transcription in the control and in hypoxic eCMs was increased 2.75‐fold (P<0.01) and 3.3‐fold (P<0.01) respectively when compared to hypoxic CM/CFs. To probe a potential function of IFN‐β, CM/CFs were pretreated with 100 u/ml IFN‐β 24h prior to exposure to hypoxia (1% O2) for 22h. Cardiomyocyte injury was determined using protein array to measure release of cardiac injury markers, cTNI, cTNT and FABP3, into the media. All of the markers were significantly increased at the end of the hypoxic challenge in cells that were not pre‐treated with IFN‐β as compared to controls. In the absence of hypoxia, IFN‐β had no effect on the levels of these markers. However, when IFN‐β pre‐treated CM/CFs were exposed to hypoxia there was a significant reduction (P<0.05) in all three markers as compared to hypoxic CM/CFs alone. These results suggest that in response to hypoxia CMs release IFN‐β which then stimulates CF to upregulate an iProt that serves to protect CMs. Overall, these studies suggest the existence of a previously unrecognized signaling pathway in the heart involving CM IFN‐β signaling to the cardiac fibroblast.Support or Funding InformationThis study was supported in part by an AHA ‐ Founders Affiliate Grant in Aid and a grant from Onyx Pharmaceuticals, Inc., an Amgen subsidiary.
Interleukin‐19 Can Simultaneously Reduce Atherosclerosis and Increase Perfusion in Hind Limb Ligated LDLr−/− Mice
Authors: Ray Mitali , Gabunia Khatuna , Kelemen Sheri , Kako Farah , Richards James , Autieri Michael V. 
Published: November 2023

Peripheral arterial disease is often a co‐morbidity with hypercholesterolemia and atherosclerosis. We have previously characterized Interleukin‐19 (IL‐19) as an anti‐inflammatory, Th2 cytokine. Past studies in our lab have determined that IL‐19 can decrease atherosclerosis in LDLr−/− mice and increase angiogenesis in the hind limb ischemia (HLI) model, suggesting that IL‐19 has both anti‐atherosclerotic and pro‐angiogenic effects.ObjectiveThe purpose of this study was to determine whether IL‐19 can induce angiogenesis within the background of hyperlipidemia and atherosclerosis and potentially reduce atherosclerosis and induce angiogenesis simultaneously.Methods and ResultsWe utilized three different, yet complementary platforms to test this hypothesis. We fed LDLr−/− mice high fat diet (HFD) for 12 weeks, then performed HLI surgery and continued HFD and treatment with rIL‐19 (10ng/g/day) or PBS intraperitoneal (IP) injections for an additional 4 weeks. Doppler imaging revealed increased perfusion in mice treated with IL‐19 when compared to PBS controls. Additional en face evaluation of the aortic arches of these mice demonstrated a trend toward decreased plaque burden in the IL‐19 treated mice, though it was not statistically significant. These data suggest that IL‐19 is capable of promoting angiogenesis, even in a hyperlipidemic environment. We used this same experimental model, but instead treated LDLr−/− mice with IL‐19 (10ng/g/day) or PBS by daily IP injection throughout the initial 12 weeks of HFD, in addition to four weeks following HLI surgery. Doppler imaging demonstrated increased perfusion in IL‐19 treated mice when compared to PBS controls, and en face evaluation showed decreased plaque burden in the IL‐19 treated mice, supporting our hypothesis that IL‐19 is capable of simultaneously decreasing atherosclerosis and promoting angiogenesis in the background of hyperlipidemia. Finally, we hypothesized that lack of IL‐19 would both reduce perfusion and exacerbate atherosclerosis. We generated a double knockout mouse model (dKO) by crossing the LDLr−/− with an IL‐19−/− mouse. We placed dKO and LDLr−/− mice on HFD for 12 weeks, completed HLI and performed Doppler imaging for 4 weeks. Preliminary results suggest dKO mice demonstrate both decreased perfusion and increased plaque burden.ConclusionsThe current dogma referred to as the Janus phenomenon suggests that pro‐angiogenic cytokines are also pro‐atherosclerotic. Collectively, these data suggest IL‐19 is the only cytokine to date that challenges this theory, demonstrating both pro‐angiogenic and anti‐atherosclerotic effects.Support or Funding InformationThis work was supported by grants HL115575 and HL117724 from the National Heart Lung, and Blood Institute of the National Institutes of Health, and Grant 13GRNT1685003 from the American Heart Association to MVA.
Stimulation of Mitochondrial Fragmentation and Mitophagy by TNFα in Microvascular Endothelial Cells
Authors: Kalogeris Theodore , Korthuis Ronald 
Published: November 2023

The inflammatory cytokine, TNFα, decreases mitochondrial mass in cultured microvascular endothelial cells. We previously showed that this effect might be explained by an inhibitory effect of TNFα on eNOS‐dependent expression of PGC‐1α. However, other investigators have reported that TNFα stimulates mitophagy, and a possible mitophagic effect of TNFα was not ruled out in our own studies. In order to more fully understand how TNFα affects endothelial mitochondrial function, we have begun to examine the effect of this cytokine on measures of mitochondrial network integrity and mitophagy in human dermal microvascular endothelial cells (HMEC‐1). Since fragmentation of mitochondrial networks via mitochondrial fission has been shown to be necessary for subsequent mitophagy, we examined the time‐dependent effect of TNFα on mitochondrial network integrity using fluorescence microscopy of cells loaded with the mitochondrial‐specific dye, mitotracker green (MTG). Compared with control cells, TNF treatment increased mitochondrial fragmentation by 12% and 20% at 24 and 48 h, respectively. This was associated with a similar time‐dependent increase in mitochondrial PINK‐1, and recruitment of both drp‐1 and parkin, suggesting increases in both mitochondrial fission and mitophagy progression. We also observed an increase in the cellular ratio of LC3II/LC3I, a marker of autophagasome formation. Finally, TNFα‐induced decrease in mitochondrial mass was significantly attenuated in cells co‐incubated with mitophagy inhibitors, 3‐methyladenine or m‐divi. Taken together with our previous results, these findings suggest that TNFα may exert negative effects on endothelial mitochondrial capacity by simultaneously blocking organellar biogenesis and stimulating mitophagic destruction.Support or Funding InformationSupported by AA014945 & HL095486.
Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase and Endoplasmic Reticulum Stress Protects Myocardial from Ischemia ‐ Reperfusion Injury in Type 2 Diabetic Mice
Authors: Mali Vishal R. , Ali Maha , Haddox Samuel , Matrougui Khalid , Belmadani Souad 
Published: November 2023

BackgroundWe previously reported that epidermal growth factor receptor tyrosine kinase (EGFRtk) activity and endoplasmic reticulum (ER) stress are enhanced in type 2 diabetic mice and cause vascular dysfunction. In the present study, we determined the in vivo contribution of EGFRtk and ER stress in acute myocardial infarction in type 2 diabetic (db−/db−) mice.Methods and ResultsWe treated type 2 diabetic mice (db−/db−) mice with EGFRtk inhibitor (AG1478, 10 mg/kg/day) or ER stress inhibitor (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) for two weeks. After the treatment, mice were subjected to myocardial ischemia for 40 minutes and followed by reperfusion for 24 hours. The treatment with AG1478 and TUDCA reduced infarct size illustrating a cardioprotection effect of inhibiting EGFRtk signaling or ER stress against ischemia‐reperfusion injury in type 2 diabetic mice. To determine the signaling mechanism responsible for the observed cardioprotection, we determined the level of ER stress proteins Chop and BIP expression. The AG1478 or TUDCA significantly decreased the expression of BIP and Chop in diabetic mice compared to untreated db−/db− mice. Also, AG1478 and TUDCA reduced cardiomyocyte apoptosis, p38, and ERK1/2 MAP‐Kinases, and increased the activity of the pro‐survival signaling cascade Akt. Immunohistochemistry staining showed that EGFRtk inhibition and TUDCA treatment lowered macrophages and neutrophil infiltration after I/R injury in diabetic hearts compared to untreated mice.ConclusionOverall, we showed for the first time that inhibition of EGFRtk and ER stress protect diabetic mice against ischemia‐reperfusion injury by activating the reperfusion injury survival pathway and inhibiting apoptotic signaling and inflammation. These findings suggest the importance of targeting ER stress and EGFRtk as a potential therapy for heart complication in type 2 diabetes.Support or Funding InformationThis study was supported by EVMS Research Enhance Grant
Sympathetic Modulation Decreases in Rats with Renovascular Hypertension Treated with Chronic Administration of Catalase Inhibitor
Authors: Lauar Mariana Ruiz , De Luca Laurival Antonio , De Paula Patricia Maria , Colombari Débora S A , Colombari Eduardo , Andrade Carina A F , Menani José Vanderlei 
Published: November 2023

Previous studies have shown that the increase of endogenous hydrogen peroxide (H2O2) with the administration of the catalase inhibitor 3‐amino‐1,2,4‐triazol (ATZ) intravenously acutely reduced mean arterial pressure (MAP) and the pressor response induced by angiotensin II intracerebroventricularly in renal hypertensive rats. Recent data have also shown that chronic subcutaneous (sc) administration of ATZ reduced MAP in 2‐kidneys, 1‐clip (2K1C) hypertensive rats. In the present study, we analyzed the autonomic modulation of cardiovascular function in 2K1C hypertensive rats treated with chronic sc injection of ATZ. Male Holtzman rats (initial weight 150–180 g, n=7–11/group) received a silver clip around the left renal artery to generate 2K1C hypertension. After 6 weeks of the surgery, rats were treated with ATZ or saline for 7 days before the recording of arterial pressure and the analysis of systolic blood pressure (SBP) and pulse interval (PI) variability using power spectral analysis. Baseline MAP was increased in 2K1C rats compared to sham (207 ± 9, vs. 100 ± 2 mmHg, respectively). ATZ (600 mg/kg/day) in 2K1C rats reduced MAP (162 ± 10 vs. saline 209 ± 5 mmHg), the low frequency (LF) of SBP (3.3 ± 0.5, vs. saline 7.6±1.5 mmHg2) and LF of PI (14.6±3, vs. saline: 31±3 normalized units). ATZ also increased high frequency (HF) of PI (85±3, vs. saline: 68±3 normalized units) and improved the sympathovagal balance calculated for LF/HF ratio of PI variability (0.2±0.05, vs. saline: 0.52±0.09). The results suggest that the treatment with ATZ reduces MAP in 2K1C hypertensive rats by reducing the modulation of sympathetic activity.Support or Funding InformationFAPESP, CNPq and PROPE/FUNDUNESP.
Sympathetic Vasomotor Activation To Different Targets In A Model Of Hypertension Induced By Nitric Oxide Synthase Blockade
Authors: Zambrano Lysien Ivania , Pontes Roberto Braz , Garcia Michelle Louvaes , Nishi Erika Emy , Campos Ruy R. , Bergamaschi Cassia T. 
Published: November 2023

High blood pressure is a global public health problem. Several factors are involved in the generation and maintenance of hypertension, such as increased sympathetic nerve activity (SNA), although controversies have been reported. Our aims were to evaluate sympathetic activation to two targets, the kidneys (rSNA) and other visceral bed, the splanchnic (sSNA), in animals with hypertension induced by nitric oxide synthase blockade (L‐NAME). Wistar rats were treated with L‐NAME (20mg/kg/day, once a day, for 8 days). L‐NAME‐treated group presented increased blood pressure (154±13 mmHg, N=16), compared with control group (102±2 mmHg, N= 10). Both rSNA (156±37.4 spikes/s, N=16) and sSNA (145±42.7 spike/s, N=16) were higher in L‐NAME‐treated group, compared with control group (111±20.6, N=10 and 63±20.6 spikes/s, N=10, respectively). Taken altogether, our data show that the hypertension induced by nitric oxide synthase blockade is characterized by sympathoexcitation to more than one bed and that can be involved in the generation and/or maintenance of high blood pressure.Support or Funding InformationCAPES, CNPq, Universidad Nacional de Honduras (UNAH).
Kv1 Channels are Critical Regulators of Coronary Blood Flow in Swine
Authors: Goodwill Adam G , Noblet Jillian N , Sassoon Daniel J , Fu Lijuan , Schepers Luke , Sturek Joshua , Tune Johnathan D , Dick Gregory M 
Published: November 2023

Despite numerous investigations, the mechanisms regulating the relationship between coronary vascular flow and myocardial oxygen consumption remain poorly understood. Recent attention has been given to voltage gated potassium channels (Kv), as data suggest that they mediate redox‐sensitive coronary vasodilation. Through this mechanism, myocardial production of H2O2 increases coronary blood flow, providing a direct mechanistic linkage. The purpose of this study was to establish the role of Kv1 channels in the porcine coronary circulation through blockade of Kv1 channels with the selective channel blocker, correolide. Kv1 transcripts were present in porcine coronary arteries, with KCNA5 (Kv1.5) being the most abundant (p<0.001). Immunohistochemical analyses demonstrated expression of Kv1.5 protein in the vascular smooth muscle layer of both porcine and human coronary arteries. Whole cell electrophysiology demonstrated significant correolide‐sensitive current in coronary smooth muscle. In vivo studies involved direct intracoronary infusion of correolide into the pressure‐clamped left anterior descending artery of healthy swine while simultaneously measuring coronary blood flow. Correolide had no effect on heart rate or systemic pressure development at any tested dose (coronary plasma concentration 0.3–3 μM). Correolide reduced coronary blood flow in a dose‐dependent manner relative to vehicle control (ΔFlow, Vehicle +0.19 ± 0.08 vs. Correolide −0.12 ± 0.03 mL/min/g; p< 0.01). To determine the role of Kv1 channels in coronary metabolic vasodilation, swine received systemic infusion of the sympathomimetic compound dobutamine (0.3–10 μg/kg/min) in the presence or absence of correolide (3 μM). Correolide significantly reduced the coronary flow response to increasing myocardial demand across the range of dobutamine concentrations (~20%; p<0.001). Coronary artery occlusions (15 s) elicited reactive hyperemia and correolide reduced the flow volume repayment by approximately 50% (p=0.02). Taken together, these data support a role for Kv1 channels in modulating baseline coronary vascular tone, in regulating responses to ischemia, and in balancing myocardial oxygen delivery with metabolism.Support or Funding InformationThis study was supported by U01HL118738 (D. Beard and G. Kassab, PI) and R01HL11760 (J. Tune and K. Mather, PI). Dr. Goodwill was supported by American Heart Association 13POST1681001813 (A. Goodwill, PI). Ms. Noblet and Mr. Sassoon were supported by TL1 TR000162 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Attenuated Response of hCD39 Overexpressing Transgenic Mice to High‐fat Diet‐induced Obesity and Glucose Intolerance
Authors: Brandes Anna U , Zhang Yue , Robson Simon C , Kishore Bellamkonda K 
Published: November 2023

CD39 (ENTPD1) is an ecto‐nucleotidase that scavenges extracellular nucleotides, and thus terminates P2 receptor signaling. Previously we reported that genetic deletion ATP/UTP‐activated P2Y2 receptor confers significant protection against high‐fat diet (HFD)‐induced obesity and insulin resistance (IR). In order to understand the role of extracellular nucleotides in diet‐induced obesity and IR, we subjected wild type C57/Bl6 (WT) and syngeneic transgenic (TG) mice overexpressing human CD39 (hCD39) to either regular diet (10% of calories as fat; n = 5/genotype) or HFD (60% calories as fat; n = 7/genotype) feeding for 18 weeks with free access to drinking water and food. Body weights (BW), food and water consumption and urine output were measured every two weeks. Glucose tolerance test (GTT) was performed during 15th week. At the end of the experimental period all mice were humanely euthanized. Both genotypes showed significant increases in BW over time, and reached comparable mean BW toward the end of the study. However, until week 14, the slope of gain in BW was higher in WT vs. TG mice. No significant differences were found in daily food intake between the two groups. And there were no signs of steatorrhea, indicating no malabsorption of fat. Significant increases in the amount of brown and white adipose tissues were recorded in both groups, although the increases were not comparable. Liver and kidney weights, when adjusted to BW, are modestly, but significantly higher in HFD‐fed TG mice vs. WT mice. Glucose tolerance was significantly better in HFD‐fed TG vs. WT mice. Our results demonstrate that scavenging of extracellular nucleotides has the potential to delay the onset of diet‐induced obesity and glucose intolerance, and open potential avenues for the development of new therapies for obesity and insulin resistance.Support or Funding InformationDept. of Veterans Affairs (BKK) & PRIA Foundation (SCR)
Contribution of prolyl hydroxyl binding proteins (PHD) to MYD88/NFkB pathways in Nitric Oxide deficiency‐induced hypertensive renal injury
Authors: Choi Myung , Oyekan Adebayo 
Published: November 2023

Oxidative stress and associated inflammation, features of hypertension and kidney disease, involve many mediators that include Nrf2, a transcription factor, the primary cellular defense against oxidative stress, MYD88, an adaptor protein implicated in NFkB‐mediated inflammation. PHD, oxygen sensor in hypoxia is intricately linked to NO. This study evaluated the role played by PHD and its association with Nrf2, MYD88 and NFKB in hypertension‐induced renal injury.Adult rats were placed on LNNA, inhibitor of NO synthase and/or DMOG, an inhibitor of PHD. Blood pressure (BP) was determined on Days 1, 4, 7, and 14 and 24‐hour urine collected for protein excretion. Kidneys were collected to determine expression of PHD1 and PHD2, Nrf2, p65, and MYD88 in nuclear fractions. KIM‐1 expression, index of tubular damage, was assessed in whole kidneys. Plasma TNFa and IL‐1b levels, markers of inflammation, were measured by ELISA. Renal generation of oxidant species was evaluated as xanthine (3,5,10 mg/kg)‐induced generation of H2O2. LNNA, inhibitor of NO synthase increased BP (35%, P<0.05), kidney weight (15%, P<0.05), protein excretion (3.5‐fold, P<0.05) and KIM‐1 expression (3.8‐fold, P<0.05). DMOG blunted the increases. LNNA also reduced PHD2 (20%) but not PHD1 expression or Nrf2 expression. There was increased nuclear expression of p65 (34%, P<0.05) and MYD88 (32%, P<0.05) and increased TNFa and IL‐1b (P<0.05) in LNNA‐treated rats. DMOG blunted these effects. Renal production of H2O2 by xanthine was greater (P<0.05) in LNNA‐treated rats. These data suggest a role for PHD2 in hypertensive renal injury and inflammation induced by NO deficiency. The accompanying changes in MYD88/p65 expression and cytokine production confirm the ongoing inflammatory process. However, despite the increase in production of reactive oxygen species, Nrf2 does not appear to play a part in NO deficiency‐induced cardiovascular effect.Support or Funding InformationNational Institutes of Health grant 5G12 MD007605
Proximal Tubular Cell Angiotensinogen Upregulation by High Glucose is Mediated by Reactive Oxygen Species and not Dependent on AT1 Receptor Activation
Authors: Cypress Michael , Satou Ryousuke , Hopfer Ulrich , Navar Luis Gabriel 
Published: November 2023

Enhanced proximal tubular angiotensinogen (AGT) levels have been shown in diabetes mellitus, which can be a key factor in intrarenal RAS activation. AGT is upregulated in proximal tubular cells (PTC) by high glucose (HG), but the detailed mechanisms are unclear. Sodium‐glucose cotransporter 2 (SGLT2) is expressed in PTC and is responsible for glucose reabsorption and consequent ROS generation. Therefore, changes in SGLT2 levels may alter the regulation of AGT in PTC under diabetic conditions. The goal of this study was to determine the effects of HG‐induced ROS and angiotensin II type 1 receptor (AT1R) activation, which also generates ROS, on AGT augmentation by HG in PTC. Immortalized mouse PTC derived from the early proximal tubular segment were treated with 5 mM (normal), 15 mM, or 25 mM D‐glucose for up to 24 hours. D‐Mannitol was used as an osmotic control. PTC were treated with sodium pyruvate (1 mM) to determine if enhanced glycolysis stimulates AGT. Moreover, 2.5 mM tempol, an antioxidant, was used to evaluate if HG‐induced AGT augmentation is ROS‐dependent. To determine the role of AT1R activation in AGT upregulation by HG, 1 mM olmesartan, an AT1R blocker, was used. Furthermore, PTC were treated with 10−10–10−7 M angiotensin II (Ang II) and/or HG. In addition, the effect of HG and Ang II on SGLT2 expression was investigated. AGT and SGLT2 protein levels were quantified by Western blot analysis, and AGT mRNA was measured by qRT‐PCR. AGT protein levels were increased by 15 mM (4.4±0.2‐fold compared to control) and 25 mM (4.6±0.2‐fold) glucose. AGT mRNA augmentation was also observed (31.1±3.5‐fold) by HG. On the other hand, AGT levels were not increased by mannitol, indicating that elevated osmolality does not change AGT levels. Pyruvate stimulated AGT expression (10.7±1.0‐fold) under normal glucose conditions, supporting a role for glycolysis in AGT augmentation. Pretreatment with tempol attenuated AGT augmentation in HG‐treated cells (77% suppression compared to HG without tempol), suggesting that ROS mediates HG‐induced AGT upregulation. AT1R blockade and Ang II did not alter AGT responses to HG. Moreover, SGLT2 protein levels were unchanged by HG or Ang II. These findings indicate that AGT upregulation by HG occurs independently of AT1R activation, but is mediated by glycolysis and ROS in PTC. In addition, SGLT2 upregulation is not required for HG‐induced AGT augmentation.Support or Funding InformationThis study was supported by the National Institute of General Medical Sciences IDeA Program (COBRE, P30GM103337) and the American Heart Association Predoctoral Fellowship (15PRE25090156).