bluuebunny/crossref_metadata_2025 huggingface dataset:
High fat diet induces hypertension but not vascular and cardiac remodeling in female Dahl sensitive rats.
Authors: Fernandes Roxanne , Bonilla Patricia A Perez , Garver Hannah , Galligan James J , Fink Gregory D , Xu Hui
Published: November 2023

Mechanisms responsible for obesity associated hypertension may be different in males and females. Altered vascular reactivity to sympathetic neurotransmitters and inflammation‐induced vascular remodeling/fibrosis contribute to obesity associated hypertension in males, but recently elevated leptin and aldosterone levels have been proposed to cause obesity associated hypertension in females. We and others have found that high fat (HF) feeding significantly increases mean arterial blood pressure (MAP) in male Dahl salt sensitive (SS) rats. Here we sought to test whether HF diet increases MAP in female Dahl SS rats, and if the mechanisms responsible are similar in males and females. Female and male Dahl SS rats were fed either normal fat diet (NF, 10% kcal from fat with 0.24% NaCl, 0.4% K+) or HF diet (60% kcal from fat with 0.3% NaCl, 1% K+) for 24–26 weeks starting at weaning (3 weeks of age). At the end of the study MAP was significantly and similarly higher in HF versus NF males (n=9–10, 158±1 mmHg vs 130±1 mmHg) and HF versus NF females (n=4–5, 157±3 mmHg vs 125±7 mmHg) (P<0.05). Neither HF males nor females had larger final body weights than their respective NF groups. Hyperlipidemia and increased visceral fat were found in HF males, but not females, compared with NF rats. No differences in plasma levels of fasting glucose, insulin, leptin, aldosterone, or BUN were found between HF versus NF males or females. Male HF rats showed a greater acute depressor response to ganglion blockade than did male NF rats, but this was not seen in female rats. HF feeding did not cause alterations in mesenteric arterial (MA) contractile responses to sympathetic neurotransmitters (norepinephrine and ATP) in males or females. HF feeding significantly increased MA wall‐thickness and cardiac mass in males, but the changes were much less dramatic in females. Morphological studies did not reveal greater fibrosis in adventitia of mesenteric, intrarenal and coronary arteries from HF fed males or females. Our studies show that HF diet promotes hypertension in Dahl SS males and females without causing overt metabolic dysfunction in either sex. Females, however, are less prone to develop hypertension associated vascular and cardiac remodeling, and only males exhibit a neurogenic component to their hypertension.

Support or Funding Information

NIH2P01HL070687‐11A1
Microvascular endothelial function in lean versus obese women with polycystic ovary syndrome: role of the endothelin B receptor
Authors: Usselman Charlotte W , Taylor Hugh S , Stachenfeld Nina S
Published: November 2023

Polycystic ovary syndrome (PCOS) affects 10% of women and is associated with myriad cardiovascular risk factors, including obesity and endothelial dysfunction. PCOS is also associated with elevated levels of plasma endothelin‐1 (ET‐1), one of several circulating indicators of endothelial injury and dysfunction. Women with PCOS have reduced vasodilatory responses to ET‐1 as mediated through the endothelin receptor subtype B (ETBR). We hypothesized that impairments in ETBR‐dependent and independent vasodilation are more severe in obese than in lean women with PCOS. We tested 3 lean (age 30 ± 6 y; BMI 23 ± 1 kg/m2) and 3 obese (33 ± 5 y; 36 ± 4 kg/m2) women diagnosed with PCOS according to the Rotterdam criteria. We assessed cutaneous vascular conductance (CVC) with laser Doppler flowmetry during intradermal microdialysis perfusions of ET‐1 (1, 3, 4, 5, and 7 pmol) with either lactated Ringer's solution, ETBR inhibition (BQ‐788), or nitric oxide inhibition (L‐NMMA). Dose‐response curves (%CVCmax) as a function of the log concentration of ET‐1 demonstrated reduced vasodilatory responses in lean PCOS (logEC50 0.61 ± 0.07) relative to obese PCOS (logEC50 0.52 ± 0.47). Inhibition of ETBR decreased the ET‐1‐induced vasodilation in both lean (i.e., logEC50 0.66 ± 0.11) and obese (logEC50 0.62 ± 0.21) groups. Contrary to our hypothesis, this inhibition was two‐fold greater in obese PCOS women, indicating a greater role for ETBR‐induced vasodilation under basal conditions relative to lean PCOS. Conversely, nitric oxide inhibition had little effect in either lean (logEC50 0.61 ± 0.32) or obese PCOS women (logEC50 0.54 ± 0.18) compared to ET‐1 alone. Our data suggest that impairments to endothelial function are more severe in lean than obese women with PCOS, and that ETBR plays an important role in this potentially adverse cardiovascular outcome.

Support or Funding Information

Supported by The John B. Pierce Laboratory and the American Heart Association (15POST24990003).
Sex differences in Mesenteric Arterial Function at Pre‐Diabetic and Diabetic Stage of UC Davis Type 2 Diabetic Mellitus Rats
Authors: Shaligram Sonali , Akther Farjana , 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 exhibiting etiology very similar to clinical T2DM seen in humans compared to other rodent models. It is characterized by polygenic adult‐onset obesity and diabetes observed in both sexes along with development of insulin resistance, impaired glucose tolerance, and subsequent β‐cell decompensation. The objectives of this study were to investigate whether 1) the mesenteric arterial function is impaired and 2) sex differences exist in the development of abnormal vascular responses in UCD‐T2DM rats at pre‐ diabetic and diabetic stage.

Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5 M) was measured in mesenteric arteries (MA) pre‐contracted with phenylephrine (PE, 2 μM). Concentration response curves (CRC) to sodium nitroprusside (SNP) (Nitric oxide (NO) donor, 10−9 to 10−5 M) and contractile agents such as PE (10−7 to 3×10−5 M) or endothelin‐1 (ET‐1, 10−10 to 10−7 M) were also determined.

ACh‐induced relaxation was significantly impaired in MA of pre‐diabetic and diabetic rats, regardless of sex. However, in pre‐diabetic stage, the extent of impairment was greater in males compared to that of female rats. When animals become diabetic, MA from female rats demonstrated a greater suppression of EDV than that of males. Similar to ACh responses, the relaxation responses to NO donor (SNP) were also impaired in MA of pre‐diabetic and diabetic rats, in both sexes. Furthermore, female diabetic rats exhibited more impairment compared to male diabetic. In addition, arteries from female diabetic but not male diabetic showed a larger shift of SNP CRC to the right compared to their respective pre‐diabetic animals. Finally, the sensitivity of MA to contractile agents such as PE and ET‐1 was significantly enhanced in female diabetic rats compared to other groups.

Our data, for the first time, show that the mesenteric arterial function of UCD‐T2DM rats is impaired in both sexes as early as pre‐diabetic stage. Moreover, we demonstrated a greater predisposition of MA to vascular injury in female diabetic rats, might be due to an altered sensitivity of arteries to contractile agents and NO.

Support or Funding Information

Supported by NIH/NIHLBI
Increases in Cytochrome P450 Expression and 20‐HETE Production Reduce Mean Arterial Pressure in an Animal Model of Preeclampsia
Authors: Cotton Joshua , Wu Wenjie , Murphy Sydney
Published: November 2023

Recent data suggests cytochrome P450 (CYP450) vasoactive eicosanoids in the pathophysiology of preeclampsia. Thus, we investigated whether alterations in CYP450 expression and 20‐HETE production contribute to maternal hypertension during pregnancy. In order to address this hypothesis, we utilized a newly characterized model of hypertension during pregnancy also documented to have a genetic deficiency in 20‐HETE production, the Dahl S rat. In addition, we utilized a CYP4A1 overexpression transgenic rat. Mean arterial pressure, measured via telemetry, and proteinuria were measured in 12 week old Spraque Dawley (SD), Dahl SSJr, and CYP4A1 Tg rats at baseline, prior to pregnancy, and gestational day (GD) 5, 12, and 19. Consistent with previous reports, a reduction in MAP at term during normal pregnancy was not seen in the Dahl S rat (1.5±6 mmHg, p<0.05; SD, 14±1 mmHg; CYP4A1 Tg, 18±2 mmHg). Proteinuria was elevated in both Dahl SSJr (107±45 mg/day, p<0.05) and CYP4A1 Tg (53±5 mg/day, p<0.05) rats relative to SD (10±1 mg/day) pregnant rats on GD19. Pup number was reduced in the Dahl S (9±1, p<0.05) and increased in the CYP4A1 Tg (11±1, ns) rat compared to the SD (12±1), with no differences noted in placenta to pup weight. CYP450 enzymatic activity, as measured by production of 20‐HETE, within the placenta and kidney were significantly increased in the placenta (0.002±0.0006 vs 0.4±0.2 pmol/min/mg protein) and kidney of CYP4A1 Tg rats (4.4±0.5 vs 1.9±0.7 pmol/min/mg protein). Therefore, alterations in CYP450 enzyme activity and 20‐HETE may contribute to the increases in MAP during pregnancy and may be a potential therapeutic target in the treatment of preeclampsia.

Support or Funding Information

14SDG20160020; HL121042
Pre‐Conception Leptin Resistance Characterizes the BPH/5 Mouse Model of Preeclampsia
Authors: Sutton Elizabeth F , Lob Heinrich E , Liu Chin‐Chi Liford , Redman Leanne M , Davisson Robin L , Sones Jenny Liford
Published: November 2023

Preeclampsia (PE) is a devastating disorder of pregnancy that presents with maternal hypertension and proteinuria after 20 weeks of gestation. Despite being a leading cause of maternal and fetal morbidity/mortality, the underlying mechanisms are unknown. PE is often described as an overall increased inflammatory state without a clear cause, and pre‐conception obesity has been proposed as a risk factor. Women with an elevated body mass index (>35 kg/m2) at conception have a 30% increased risk of developing PE compared to lean counterparts. We utilized the obese BPH/5 mouse model to investigate pre‐conception risk factors that may contribute to PE. BHP/5 mice are hyperphagic, have increased white adipose tissue (WAT), and spontaneously develop the maternal syndrome of hypertension and proteinuria, as well as the poor fetoplacental outcomes associated with PE. Reduction of WAT through pair‐feeding matched with C57BL/6 mice resulted in improved fetoplacental outcomes in late gestation. These improvements led us to hypothesize that obese BPH/5 female mice have derangements in leptin signaling contributing to their adverse metabolic phenotype and their risk of developing PE. To test this, female, virgin BPH/5 and C57BL/6 mice were fed an ad libitum standard chow diet and studied at 6–12 weeks of age. BPH/5 mice had significantly more circulating leptin as compared to C57BL/6 mice (12,600±3098 vs. 1165±271 pg/mL; n=4–6, p<0.05). Following three days of leptin administration (i.p., 30μg twice a day), BPH/5 mice exhibited severely blunted reductions in body weight (<5% v 10% in C57BL/6; n=4–6, p<0.05). Furthermore, food intake in BPH/5 remained significantly elevated as compared to C57BL/6 (n=4–6, p<0.05). Taken together this data suggests BPH/5 mice are leptin resistant. Chronic hyperleptinemia has been shown to characterize other models of PE; however, the preexisting maternal metabolic profile prior to pregnancy has not been fully understood as it relates to the pathogenesis of PE. Further studies will involve challenging the physiologic metabolic state of BPH/5 mice through pair‐feeding to reduce WAT before pregnancy and determining its effect on inflammation, placentation, and hypertension during gestation.
Expression of MEF2 transcription factors in human placenta and involvement in trophoblast invasion and differentiation
Authors: Li Lucy , Gong Xiaoming , Rubin Lewis P
Published: November 2023

Introduction

The important and common adverse pregnancy outcome, preeclampsia, is characterized by placental trophoblast dysfunction and insufficient (shallow) trophoblast uterine invasion/remodeling during early pregnancy. Myocyte enhancer factor 2 (MEF2) family transcription factors play an important role in cell differentiation and invasion in a variety of cell types and tissues. The objective of this study is to explore the role of MEF2s in trophoblast differentiation and uterine invasion in normal placental development and preeclampsia.

Methods

Human cytotrophoblasts (CTBs) were isolated from term placentas by mechanical and enzymatic digestion and purified using Percoll gradient centrifugation and differential adhesion. Highly purified (>95%) CTBs in acute cultures (1–4 d) were used to analyze expression of MEF2 isoforms and MEF2‐dependent cell signaling pathways. The effects of MEF2 on human trophoblast invasion and differentiation and cellular pathways were examined principally in two relevant human trophoblast cell lines: HTR8/SVneo (first trimester human extravillous trophoblast phenotype) and BeWo (choriocarcinoma CTB‐like) cells. Invasion/migration was evaluated by Transwell (including chemotactic) invasion and “scratch” migration 2D assays; differentiation was assessed by trophoblast syncytial formation and expression of trophoblast differentiation genes.

Results

MEF2 isoforms are differentially expressed in first and third trimester placental trophoblast. In particular, MEF2D and 2B are highly expressed during first trimester placental development. In other experiments, transient overexpression of MEF2D in HTR8/SVneo cells promotes trophoblast invasion and migration. This invasive property is blocked by exogenous expression of a dominant negative MEF2. In contrast, MEF2A is the major isoform expressed in term trophoblast. MEF2A expression is associated with CTB differentiation into syncytiotrophoblast; transient overexpression of MEF2A enhances cytodifferentiation/syncytia formation. Based on p38 MAPK null experimental animals, p38 is the principal upstream regulator of MEF2. During in vitro differentiation of primary human CTB cultures, we found the course of p38 activity (phosphorylation) parallels MEF2A expression. Placental tissues obtained from pre‐eclamptic and normal (non‐pre‐eclamptic) pregnancies exhibit differences in MEF2 isoform expression.

Conclusion

These findings suggest individual MEF2 family members differentially regulate trophoblast invasion and differentiation. Dysregulation of MEF2 expression or signaling in early pregnancy may be associated with placenta‐related pregnancy disorders, including preeclampsia.

Support or Funding Information

Seed grant from Laura W. Bush Institute for Women's Health, Texas Tech University Health Sciences Center
NMDA receptor‐mediated synaptic transmission in the spinal dorsal horn and spinal nerve ligation‐induced nociceptive behavior are enhanced in serine racemase knockout mice
Authors: Fukushima Teruyuki , Kato Eiko , Konno Ryuuichi , Hori Yuuichi
Published: November 2023

N‐methyl‐D‐aspartate (NMDA) receptors are reported to play an important role in synaptic plasticity that may underlie not only learning and memory but also persistent pain. Peripheral nerve injury or tissue inflammation often induces an abnormal pain state, so‐called neuropathic pain. The activation of NMDA receptors mediates the development and maintenance of neuropathic pain. D‐serine co‐localizes with NMDA receptors to a high degree and acts as an endogenous coagonist of the NMDA receptor “glycine site”. D‐serine is synthesized from L‐serine by serine racemase (SR), an enzyme that is abundant in the central nervous system (CNS). To elucidate the contribution of SR to synaptic transmission in the CNS, we generated SR knockout (KO) mice.

Animal experimental procedures were reviewed and approved by the institutional animal care and use committees at Dokkyo Medical University. The care and use of the animals were in accordance with the guidelines of the International Association for the Study of Pain. In the present study, we investigated NMDA receptor‐mediated synaptic transmission in the superficial dorsal horn (SDH) of spinal cord slices by means of tight‐seal whole‐cell recordings in SR‐KO mice and wild type (WT) mice. We also analyzed nociceptive behavior, which is mediated by NMDA receptors in the SDH, induced by spinal nerve ligation (SNL). The amplitude of the NMDA receptor‐mediated component of excitatory postsynaptic currents (EPSCs), normalized to that of the non‐NMDA receptor‐mediated component of EPSCs, was smaller in SR‐KO mice than in WT mice. In contrast, the time decay course and the synaptic charge transfer of the NMDA component were slower and larger in SR‐KO mice than in WT mice, respectively. Mechanical allodynia induced by the SNL was augmented in SR‐KO mice compared to WT mice. It is speculated that SR‐KO mice have a different NMDA receptor subunit composition compared to WT mice, which might explain the differences in synaptic transmission and nociceptive behaviors between the SR‐KO mice and WT mice. Real‐time RT‐PCR analysis indicated that the expression level of NR2B subunit of NMDA receptors was slightly but distinctly increased in SR‐KO mice.

Finally, the present observations could suggest the possibility that the SR activity modulates the neuropathic nociception by changing the subunit composition of NMDA receptors. SR‐KO mice may provide a unique tool to investigate the synaptic action of D‐serine in the CNS.
Inactivation of the PLA2 Activity of Prdx6 Ameliorates Sepsis‐Induced Acute Lung Injury
Authors: Vazquez‐Medina Jose Pablo , Tao Jian‐Qin , Patel Priyal , Dodia Chandra , Sorokina Elena , Feinstein Sheldon I , Chatterjee Shampa , Fisher Aron B
Published: November 2023

Prdx6 is a multi‐functional protein that expresses peroxidase, phospholipase A2 (PLA2) and LPC acyltransferase activities. While the three activities of Prdx6 contribute to either the scavenging of lipid hydroperoxides or to the repair of peroxidized membranes, the PLA2 activity of Prdx6 (aiPLA2) is also required for NOX2 activation in pulmonary microvascular cells (PMVECs), alveolar macrophages and polymorphonuclear neutrophils. During sepsis‐induced acute lung injury (ALI), NOX2 is activated generating superoxide radicals that contribute to vascular inflammation. To test whether inactivation of aiPLA2 ameliorates sepsis‐induced ALI, we used a knock‐in mouse model that lacks aiPLA2 activity due to a mutation of a key member of the PLA2 catalytic triad (Prdx6‐D140A). Prdx6‐D140A mice treated IP with LPS (10 mg/Kg) exhibited increased survival rates compared to WT mice (75% vs 25% 4 days after LPS). Total nucleated cells in BALF, lung VCAM‐1 expression, and lung permeability increased significantly (p<0.05) after 24 h treatment with LPS in WT but not in Prdx6‐D140A mice. In PMVECs, LPS treatment (1 μg/mL for 8h) increased Prdx6 phosphorylation and aiPLA2 activity by two‐fold (p<0.05). Furthermore, phosphorylated Prdx6 was localized at the plasma membrane after LPS treatment. LPS also increased extracellular hydrogen peroxide generation (Amplex Red oxidation) by 70% in WT but not in NOX2 null or Prdx6‐D140A PMVECs (p<0.05). Similarly, LPS increased intracellular hydrogen peroxide generation by 90% in WT PMVECs stably‐expressing the pHyPer‐Cyto sensor. Increased intracellular hydrogen peroxide generation was prevented by pre‐treatment with MJ33, a pharmacological inhibitor of aiPLA2. These results demonstrate that aiPLA2 is needed for LPS‐induced, NOX2‐driven oxidant generation in PMVECs. Results also show that genetic inactivation of aiPLA2 reduces LPS‐induced vascular inflammation, increased vascular permeability, and mortality in an animal model. Furthermore, these studies suggest that aiPLA2 could be a target for prevention or treatment of sepsis‐induced ALI.

Support or Funding Information

Research funded by NHLBI L105509 and HL102016.
Acute High Fat Diet Exposure Results in Discrete Shifts in the Adipose Tissue Inflammation Profile in Mice
Authors: Hetrick Byron , Huffard Adrian , McCurdy Carrie
Published: November 2023

Obese adipose tissue is characterized by chronic, low grade inflammation that is currently recognized to play a direct role in adipocyte insulin resistance. Lean adipose tissue is characterized by a variety of leukocytes that maintain tissue homeostasis, for example, anti‐inflammatory M2 macrophages which are supported by IL‐4 secretion by Eosinophils. A pro‐inflammatory set of leukocytes, for example, M1 macrophages, neutrophils, and CD8+ T‐cells are elevated in obese adipose tissue. While the inflammation profile of lean and obese adipose tissue has been characterized to a large extent, little is known about the temporal changes and relative importance of inflammatory factors during the transition from lean to obese state. We have characterized changes to the inflammatory phenotype of mouse epididymal and subcutaneous adipose depots upon exposure to an acute (3 day) high fat diet in order to understand early changes to adipose tissue inflammation that may contribute to insulin resistance prior to the full onset of obesity. C57BL/6J mice were fed either a high fat diet (45% calories from fat) or control diet. After three days, the mice were euthanized and the epidiymal and subcutaneous adipose tissue was removed, and digested into a cell suspension. The stromal vascular fraction was isolated by differential centrifugation, and the leukocyte content of the tissue was measured by multi‐color flow cytometry. We observed a decrease in anti‐inflammatory leukocytes, such as eosinophils and regulatory T‐cells in the epididymal fat depot. While we did not see an increase in the amount of M1 polarized macrophages, the number of TNFα expressing macrophages was increased, which may indicate early stages of a polarization shift in the macrophage subset from M2 to M1. We also observed an increase in B‐cells and Natural Killer T‐cells indicating they play a role in the initiation of a high fat diet induced pro‐inflammatory phenotype. Surprisingly, we saw very different effects in the subcutaneous adipose depot. Eosinophils and IL‐10 expressing macrophages were increased upon high fat diet exposure indicating that this fat depot responds to prevent or delay the pro‐inflammatory phenotype associated with insulin resistance. In summary, only a subset of inflammatory factors are affected by acute exposure to high fat diet, indicating a complex network of signaling orchestrates the transition of adipose tissue from a lean state to a pro‐inflammatory, obese state. From our observations, we predict that high fat diet exposure initially causes a reduction in anti‐inflammatory factors, causing proliferation, infiltration, or polarization of pro‐inflammatory factors that block the insulin signal transduction cascade in adipocytes, resulting in insulin resistance.

Support or Funding Information

NIH R01 DK095926
Phenotypic abnormalities in microvascular endothelial cells isolated from the Sugen/Hypoxia rat
Authors: Suresh Karthik , Servinsky Laura , Huetsch John , Shimoda Larissa
Published: November 2023

The mechanisms behind microvascular endothelial dysfunction in PAH are unclear, although mitochondrial dysfunction, increases in reactive oxygen species (ROS) and increased intracellular calcium concentration ([Ca2+]i) have all been implicated in endothelial cell samples from humans with PAH. To better understand the mechanisms behind endothelial dysfunction in PAH, we recently isolated lung microvascular endothelial cells (LMVEC) from normoxic rats (N‐LMVEC) and rats undergoing Sugen/Hypoxia (SuHx‐LMVEC), an experimental model of PAH.

To induce PH, rats were injected with SU5416 (Sugen), a VEGFR inhibitor, and then placed in hypoxia for 3 weeks, followed by return to normoxia for 2 weeks. Following hemodynamic measurements, LMVEC were isolated from rats at 5 weeks using a magnetic bead approach, with anti‐CD31 and Griffonia simplicifolia (GS‐II) lectin conjugated beads. Calcium measurements were made in Fura 2‐AM loaded cells placed in a heated flow chamber perfused with Krebs buffer. Immunofluorescence was performed using primary and secondary antibodies and DAPI nuclear stain on rat LMVEC. To measure ROS, LMVEC were infected with a baculovirus vector containing a plasmid encoding roGFP, a ratiometric ROS dye. Cells were imaged 48 h after infection. Mitochondrial respiration was measured using the Seahorse metabolic analyzer system.

N‐LMVEC rats appeared phenotypically similar to mouse and human LMVEC, while SuHx‐LMVEC appeared more spindle‐shaped. N‐ and SuHx‐LMVEC stained positive for GS‐II and CD31. Unlike N‐LMVEC, SuHx‐LMVEC additionally stained positive for smooth muscle actin and myosin heavy chain. Mitochondrial respiration was decreased in SuHx‐LMVEC compared to N‐LMVEC, whereas ROS, basal [Ca2+]I, migration and proliferation were all increased. However, basal [Ca2+]i, migration and proliferation in SuHx‐LMVEC were attenuated with treatment with TEMPOL, a ROS scavenger.

We conclude that LMVEC isolated from SuHx rats exhibit metabolic and phenotypic differences from control cells that result from elevations in ROS, possibly due to mitochondrial dysfunction.

Support or Funding Information

F32HL124930
Chronic Inflammation Deactivated the MAPK pathway and Decreased the TRPV1 Protein Expression in the 50B11 Dorsal Root Ganglion Cell Line
Authors: WANG HANJUN , Rozanski George J. , Zucker Irving H.
Published: November 2023

It has been shown that skeletal muscle metabolic afferents are desensitized in the chronic heart failure (CHF) state, which is associated with a decreased transient receptor potential vanilloid 1 (TRPV1) receptor expression in the lumbar dorsal root ganglia (DRG). However, the molecular mechanisms underlying the downregulated TRPV1 protein expression in lumbar DRG neurons in CHF remain unclear. Because CHF is associated with chronic inflammation, we decided to investigate the potential effects of acute and chronic inflammation on TRPV1 protein expression in the 50B11 DRG Cell line. We used tumor necrosis factor (TNF) alpha (a pro‐inflammatory cytokine) to create an inflammatory environment. Since evidence suggests that the mitogen‐activated protein kinases (MAPK) pathway is involved in the regulation of TRPV1 channel function, we first investigated the time and dose‐dependent effects of TNF alpha on the phosphorylation of ERK (one of the MAPK components). Our time‐course experiments showed that TNF alpha (100 ng/ml) caused peak phosphorylation of ERK at 15 min and lasted up to 3 hours. TNF alpha also dose‐dependently (0.01, 0.1, 1, 10 and 100ng/ml) increased the phosphorylation of ERK with a peak effect at 10 ng/ml (1.6±0.26 at 10 ng/ml and 1.7±0.26 at 100ng/ml vs. Vehicle (1.0±0), n=6, p<0.01) at 15 min. However, either 20‐hour or 5‐day of continuous treatment with TNF alpha decreased the phosphorylation of ERK by 30% compared to vehicle treatment. Furthermore, a 20‐hour treatment with TNF alpha (100 ng/ml) decreased protein expression of TRPV1 by 30% whereas a 5‐day continuous TNF alpha treatment (10 ng/ml) further decreased TRPV1 protein expression by ~90% (0.10±0.04 vs. Vehicle (1.0±0.0), n=8, p<0.01). These data suggest that chronic inflammation causes a significant reduction in TRPV1 protein expression in DRG neurons which is associated with decreased phosphorylation of ERK. Ongoing experiments will determine if the decreased phosphorylation of ERK by chronic treatment with TNF alpha contributes to the loss of TRPV1 receptors in DRG neurons.

Support or Funding Information

This work was supported by by grants from the National Heart, Lung, and Blood Institute (1R01HL121012‐01A1 and R01 HL116608‐01A1).
Acute Electrical Vagal Nerve Stimulation Exerts Powerful Anti‐inflammatory Effects Through its Nicotinic Action in Rats with Pulmonary Arterial Hypertension
Authors: Yoshida Keimei , Saku Keita , Tanaka Mariko , Abe Kohtaro , Kishi Takuya , Tsutsui Hiroyuki , Sunagawa Kenji
Published: November 2023

Backgrounds

The inflammatory mechanism plays a crucial role in the pathogenesis of pulmonary arterial hypertension (PAH). Since autonomic dysregulation, that has been reported in PAH, aggravates the inflammatory responses, we hypothesized that electrical vagal nerve stimulation (VNS) may help restore autonomic balance, thereby prevent the worsening of PAH. To clarify the beneficial mechanism of VNS, we investigated how acute VNS impacts on the inflammatory mechanism involved in PAH.

Methods

We used Fischer344 rats weighing 180–200g. As a model of PAH, Sugen5416 was injected (VEGF receptor inhibitor, 20mg/kg, subcutaneously), followed by hypoxia (O2 10%) for 3 weeks and normoxia for 2 weeks. We attached the electrode to the right cervical vagal nerve, applied VNS (20Hz, 0.1–0.15mA, n=5) or sham stimulation (n=4) for 90 minutes, measured hemodynamics and sampled tissues of lung at the end of experiment. We performed western blotting for eNOS and real time PCR for pro‐inflammatory cytokines, anti‐inflammatory cytokine, endothelin‐1 (ET‐1), and α7 nicotinic acetylcholine receptor (α7nAchR).

Results

VNS significantly reduced heart rate by about 10 % from baseline, while did not change mean pulmonary arterial pressure (before VNS: 39.3±8.8, after VNS: 35.9±8.7 mmHg, n.s.) or pulmonary vascular resistance (Fig. 1). Compared to sham, VNS did not alter the eNOS level or mRNA level of ET‐1, whereas attenuated the expressions of pro‐inflammatory cytokines, especially in Tnfa (p<0.05), and that of Il10, an anti‐inflammatory cytokine (p<0.01). In addition, VNS upregulated α7nAchR, the upstream regulatory factor of pro‐inflammatory cytokines (p<0.05) (Fig. 2).

Conclusions

VNS exerted the powerful anti‐inflammatory effect through the nicotinic action in PAH model rats. Because the excessive inflammatory responses accelerate pulmonary vascular remodeling in PAH, VNS may serve as a potential therapeutic strategy for PAH. However, how those acute anti‐inflammatory effect of VNS translates into the improvement of PAH remains to be seen.

Support or Funding Information

Nothing to declare.
Disruption of Mitochondrial Redox and Metabolic States in Norepinephrine‐Exposed T‐lymphocytes
Authors: Case Adam J. , Collins Christopher W. , Roessner Colton T. , Tian Jun , Zimmerman Matthew C.
Published: November 2023

Increased sympathoexcitation and norepinephrine (NE) outflow are hallmarks of chronic diseases such as heart failure, hypertension, diabetes, and even psychological stress. NE has been demonstrated to modulate the immune system, but mechanisms controlling this cross‐talk between the autonomic nervous and immune systems are complex and poorly defined. Recently, using a mouse model of elevated NE we elucidated that NE utilizes superoxide (O2•−)‐dependent redox signaling to modulate T‐lymphocyte function. However, the source of this O2•− and how it mechanistically contributes to T‐lymphocyte function remain elusive. Due to growing evidence that metabolism is a key regulator of T‐lymphocyte activation and differentiation, we hypothesized that NE mediates its effects on T‐lymphocytes via disruption of mitochondrial bioenergetics and redox state. To address this hypothesis, we utilized purified mouse splenic CD4+ and CD8+ T‐lymphocytes stimulated with NE and assessed subcellular O2•− levels, mitochondrial metabolism, cellular proliferation/differentiation, and cytokine profiles. First, we demonstrated the source of O2•− was indeed the mitochondria as evidenced by a 50% increase (p<0.01 vs vehicle) in MitoSOX Red oxidation as well as the attenuation of this signal by the addition of the mitochondrial‐targeted O2•− scavenging antioxidant MitoTempol. NE‐stimulated T‐lymphocytes also demonstrated a 25% (p<0.05 vs vehicle) decrease in mitochondrial respiratory capacity, which suggests disruption of mitochondrial metabolism and the potential source of increased mitochondrial O2•−. Next, NE inhibited proliferation of T‐lymphocytes by approximately 50% (p<0.01 vs vehicle), which was accompanied by a concomitant increase in the percentage of cells remaining in their naïve state (49.5% with NE vs 26.4% with vehicle, p<0.01). Interestingly, NE lead to significant increases (ranging from 1.8–4.1 fold, p<0.05) in O2•− levels in all T‐lymphocytes independent of activation state (i.e. naïve, memory, and effector). Last, mitochondrial O2•− was shown to be mechanistic to the NE‐mediated T‐lymphocyte phenotype as supplementation of MitoTempol could significantly reverse the approximate 50% decreases in IL‐2 and interferon gamma (p<0.01) and 2‐fold increases in IL‐6 and IL‐17 (p<0.01) levels observed with NE treatment. However, the decreases in proliferation could not be reversed by increased mitochondrial O2•− scavenging. Overall, our data suggest the potential for a new paradigm involving mitochondrial redox and metabolism as mechanistic elements in the regulation of T‐lymphocyte function by NE.

Support or Funding Information

This work is supported by the National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) R00HL123471 (to AJC) and R01HL103942 (to MCZ).
Acute kidney injury induces oxidative stress and distant organ dysfunction due to glutathione depletion
Authors: Shang Yue , Siow Yaw L. , Isaak Cara K. , Karmin O
Published: November 2023

Ischemia‐reperfusion is a common cause for acute kidney injury and often complicated by distant organ dysfunction. Oxidative stress plays an important role in ischemia‐reperfusion induced local and distant organ injury. However, the mechanisms of AKI induced distant organ injury are not well understood. Glutathione is a major endogenous antioxidant and its depletion directly correlates to ischemia‐reperfusion injury. The liver has a high capacity for producing glutathione and is a key organ that regulates local and systemic redox balance. Liver function is often compromised in patients with acute kidney injury. In the present study, we investigated the mechanism by which kidney ischemia‐reperfusion induced oxidative stress and downregulated glutathione biosynthesis in the liver. The left kidney of Sprague‐Dawley rats was subjected to 45min ischemia followed by 6h reperfusion. Renal ischemia‐reperfusion impaired kidney and liver function as indicated by increased plasma creatinine, aspartate aminotransferase and alanine aminotransferase levels. This was accompanied by a significant reduction in glutathione levels in the liver and plasma. There was a marked elevation of hepatic lipid peroxidation and plasma homocysteine levels. Renal ischemia‐reperfusion caused a significant decrease in mRNA and protein levels of glutamate‐cysteine ligase (catalytic and modifier subunits) which regulated the rate‐limiting reaction in glutathione biosynthesis. A decrease in gene expression of glutamate‐cysteine ligase subunits was mediated through inhibition of a transcription factor Nrf‐2. Renal ischemia‐reperfusion also inhibited hepatic expression of cystathionine‐gamma‐lyase (CSE), an enzyme responsible for producing cysteine (an essential precursor for glutathione synthesis) through the transsulfuration pathway. In conclusion, our study has identified that decreased expression of glutamate‐cysteine ligase, a key enzyme for glutathione biosynthesis as well as reduced CSE‐mediated cysteine production through the transsulfuration pathway may be responsible for hepatic glutathione depletion upon renal IR. This, in turn, dampens the antioxidant defense mechanism and contributes to renal ischemia‐reperfusion induced oxidative stress.

Support or Funding Information

This study was supported, in part, by NSERC and St. Boniface Hospital Research Centre.
Intra‐Operative Urinary Hypoxia During Cardiac Surgery on Cardiopulmonary Bypass Predicts Later Development of Acute Kidney Injury
Authors: Evans Roger G. , Zhu Michael Z. , Smith Julian A. , Harrop Gerard K. , Thrift Amanda G. , Cochrane Andrew D.
Published: November 2023

Renal medullary hypoxia may be a common pathway in the development of acute kidney injury (AKI). There are no validated methods to detect medullary hypoxia in patients. However, experimental findings indicate that changes in urinary oxygen tension (UPO2) reflect changes in medullary PO2. Therefore, we evaluated the relationship between intra‐operative UPO2 and the development of AKI after cardiac surgery requiring cardiopulmonary bypass (CPB). From January 2015 to July 2016, thirty‐five adult patients undergoing on‐pump cardiac surgery were prospectively enrolled. UPO2 was continuously recorded intra‐operatively via a fiber‐optic probe deployed through the lumen of the urinary catheter, with the end of the probe at the catheter tip, where it was in contact with bladder urine. UPO2 fell during surgery, particularly during CPB. The lowest (nadir) UPO2 was most frequently observed during the rewarming phase of CPB, or shortly after weaning from CPB (n=25, 71%). Fourteen patients (40%) developed AKI as defined by an increase in serum creatinine from baseline of either > 26.5 μmol/L (0.3 mg/dL) within 48 hours or > 50% within 5 days. Nadir intra‐operative UPO2 was lower in patients who later developed AKI (8.5 ± 1.6 mmHg, mean ± SE) than in those who did not (16.5 ± 4.2 mmHg, P = 0.02). UPO2 below 10 mmHg at any time during surgery was associated with a 4.5‐fold [95% confidence limits 1.6 – 19.1] greater risk of AKI (P = 0.03). Furthermore, urinary PO2 below 15 mmHg for longer than the median time for all patients (5.6 min per hour of surgery) was associated with a 7.3‐fold [1.8 – 35.1] greater risk of AKI (P = 0.01) and an area under the receiver operator curve of 0.73 [0.56 – 0.90] (P = 0.03). We conclude that low UPO2 during cardiac surgery requiring CPB is strongly associated with later development of AKI. Continuous intra‐operative monitoring of UPO2 is simple and relatively non‐invasive. It may provide a real‐time biomarker of risk of AKI. Early detection of risk of AKI may in‐turn provide a window of opportunity to intervene and thus avoid development of AKI in patients undergoing cardiac surgery.
Atractylodes lancea Up‐Regulates ERK1/2 and p‐ERK1/2 Expression on Hypoxia/Reoxygenation‐Injured H9c2 Cells
Authors: Nusuetrong Punnee , Gerdprasert Orapin
Published: November 2023

Atractylodes lancea (Thung) DC. has been used for treatment of fever, flu, sore throat, and common cold. Since, A. lancea contains antioxidant phenolics and flavonoids, A. lancea should be able to protect cardiomyoblasts against hypoxia/reoxygenation (H/R) injury. In the present study, the cardioprotective effect of A. lancea extract (AL) on H/R‐damaged H9c2 cells was elucidated. Incubation of AL (0.01–1 mg/mL) for 24 h showed no toxic effect on H9c2 cells, measured by MTT assay. Cardioprotective effect of AL was determined on the cells exposed to hypoxic buffer for 6 h followed by reoxygenation in complete medium for 24 h. Treated cells were measured by MTT assay as well as under fluorescent microscopy using Hoechst33342. AL at a concentration of 0.1 mg/mL showed an inhibition against H/R‐damaged H9c2 cells, using 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid as a positive control. Nuclear morphology of H/R‐exposed cells showed shrinking in size, meanwhile normal cells and AL (0.1 mg/mL)‐treated H/R‐exposed cells were found healthy. AL at the same concentration was able to restore the protein expression of ERK1/2 and pERK1/2 when compared to the H/R control. In conclusion, AL inhibited H/R‐injured cardiomyoblasts, which was partly through up‐regulation of ERK1/2 and pERK1/2.

Support or Funding Information

This work was supported by research grant from the National Research Council of Thailand (NRCT 2009‐113) and grant from HRH Princess Maha Sirindhorn Medical Center, Faculty of Medicine, Srinakharinwirot University, Thailand (N0.359/2556). We specially thank Dr. Sanya Hokputsa for giving us the extracts. The authors gratefully acknowledge help and assistance Mr. Pirapas Yingdamnoona for laboratory manipulation of cell culture.
Osteocytes, Muscle and Exercise: Role in Healthy Aging
Authors: Bonewald Lynda
Published: November 2023

The osteocyte is a mechanosensory, multifunctional cell regulating mineral and skeletal metabolism and functions as an endocrine cell secreting factors that target distant organs such as kidney. These cells form a highly connected intricate network within the bone matrix that can communicate with cells on the bone surface. Osteocytes can live for decades in the bone matrix, however, with age, the osteocyte is subjected to increased reactive oxygen species making the cell more susceptible to apoptosis. Whereas some cells develop a hypermineralized perilacunar matrix and some die resulting in empty lacunae that fill in with mineral, called micropetrosis, the majority develop a senescence‐associated secretory phenotype, SASP. In proliferating cells, SASP halts proliferation of dysfunctional cells, however, osteocytes are not proliferating cells, so it is not clear why they develop this phenotype. One reason may be to avoid apoptosis which would lead to micropetrosis and reduced connectivity of the osteocyte lacunocanalicular network. Development of the senescense phenotype may serve to maintain the function of the network.

We have found that muscle factors, and one in particular, called beta‐aminoisobutyric acid are more potent than estrogen or n‐acetyl cysteine in preventing or reducing osteocyte cell death due to reactive oxygen species. These muscle factors are elevated and secreted with muscle contraction. Exercise in the form of voluntary wheel running maintains the capacity of muscle to secrete these osteocyte protective factors. The mechanism of protection is through preservation of mitochondrial structure and function in osteocytes. Conversely, we have found that the young, healthy osteocyte secretes molecules such as Wnt3a and PGE2 that are elevated in response to shear stress to promote muscle myogenesis and function. In summary, exercise can significantly ameliorate or delay the effects of aging through the maintenance of muscle secretory function to maintain an intact osteocyte lacunocanalicular network.

Support or Funding Information

NIH NIA 1PO1AG039355
Berlin Questionnaire is a Useful Tool to Identify Risk of Obstructive Sleep Apnea in Metabolic Syndrome Patients
Authors: Cepeda Felipe Xerez , Virmondes Leslie , Rodrigues Sara , Dutra‐Marques Akothirene C.B. , Carvalho Jefferson C. , Toschi‐Dias Edgar , Alves Maria Janieire N.N. , Rondon Maria Urbana P.B. , Lopes‐Vicente Wanda R.P. , Hussid Maria Fernanda , Lorenzi‐Filho Geraldo , Trombetta Ivani C.
Published: November 2023

Obstructive sleep apnea (OSA) is a risk factor frequently present in patients with metabolic syndrome (MetS). Despite of MetS, moderate and severe OSA has been associated with cardiovascular diseases, increasing 80% the risk of cardiovascular event. The gold standard diagnostic method for OSA is the overnight polysomnography (PSG), that is in general not accessible. The aim of the present study was to evaluate whether Berlin Questionnaire (BQ) is a good tool for assessing the risk of OSA in patients with MetS. We submitted 97 patients, untreated and newly diagnosed with MetS (ATP‐III) to a PSG. OSA was characterized by apnea‐hypopnea index (AHI). BQ was administered before PSG and we evaluated the sensitivity, specificity, positive and negative predictive values and accuracy. Of the 97 patients with SMet, 81 patients had OSA, being 47 (48.5%) with moderate and severe OSA. For all SMet with OSA (AHI>5 events/hour), the BQ showed good sensitivity (0.65) and fair specificity (0.38) with a positive predictive value of 0.84, negative predictive value of 0.18 and accuracy of 84%. Similarly, for moderate‐severe OSA (AHI>15 events/hour) we found good sensitivity (0.73) and fair specificity (0.40). Interestingly, for severe OSA (AHI>30 events/hour), there was a very good sensitivity (0.91) and moderate specificity (0.42). In conclusion, BQ is a good predictor for the screening of OSA in MetS patients in general, but it is a very good tool for predicting severe OSA.

Support or Funding Information

Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) #2011/175336, #2015/175336 and #2016/168317
Cardiorespiratory Responses to Severe Arterial Hypoxemia with Increasing Remifentanil Plasma Concentrations in the Rabbit
Authors: Quail Anthony W , Cottee David BF , Johnstone Janice M , O'Hara Kate , White Saxon W
Published: November 2023

Remifentanil is an ultra‐short acting synthetic opiate employed clinically as an infusion for total intravenous anesthesia, sedation and analgesia. We have previously reported that vagal control of heart rate (HR) with remifentanil during severe arterial hypoxia (PaO2 < 35 mm Hg) is maintained, while its effects on control of minute ventilation (VE), tidal volume (VT) and respiratory frequency (f) are selective (Quail et al., 2016). Although opiates can act at the carotid body (McQueen and Ribeiro 1980; Mayer, et al., 1989) and the NTS (Zhang et al. 2011), our data suggest the actions of remifentanil on cardiorespiratory control during hypoxia are mainly in the CNS, rather than at the carotid body. We now report additional data concerning the plasma concentrations of remifentanil corresponding to each dose of the drug producing these effects. An ear artery and vein of NZ White rabbits (n=6) were cannulated (lidocaine 1%). Measurements of VE, VT, f, HR, mean arterial pressure (MAP) and oxygen saturation (ear SpO2) were made during normoxia, followed by 5 minutes of hypoxia (FiO2 7–8%). After a one hour recovery, rabbits breathed air during an intravenous remifentanil infusion and the hypoxia was repeated. Arterial blood samples were taken for blood gas analysis and measurement of plasma concentrations of remifentanil (HPLC and mass spectrometry). Two doses of remifentanil were studied on each of two days, one week apart. Data were analysed using repeated measures ANOVA. In rabbits breathing air remifentanil mean plasma concentrations after 12 minutes of infusion (0.1, 0.2, 0.3, or 0.4 mcg/kg/min) were respectively 1.3, 3.1, 4.5 and 11.7 ng/ml. Remifentanil caused dose‐related falls in f, VE and HR (P<0.01), while VT and MAP were unchanged. Remifentanil concentrations during hypoxia were similar to normoxia and the reflex bradycardia observed in conscious rabbits was maintained at the lower concentrations (P<0.05). The VT response remained intact but the increase in VE was attenuated with f increasing from a lowered baseline. We conclude that at similar target plasma concentrations to those employed clinically, vagal control of heart rate with remifentanil during severe hypoxia is maintained, while the components of ventilation are selectively modified.

Support or Funding Information

John Hunter Charitable Research Trust and Hunter Medical Research Institute
Voltage‐dependent K+ channels (KV) suppress hypoxia‐induced elevation of [Ca2+]i in rat carotid body glomus cells
Authors: Wang Jiaju , Hogan James O. , Kim Donghee
Published: November 2023

Voltage‐gated K+ channels (KV) and a large‐conductance Ca2+‐activated K+ channel (BK) are highly expressed in the glomus cells, but their role in hypoxia‐induced excitation of glomus cells is not clear. We used iberiotoxin (IBTX) and tetraethylammonium/4‐aminopyridine (TEA/4‐AP) to block BK and Kv, respectively, and measured changes in [Ca2+]i in normoxia and hypoxia. In normoxia (21% O2), the K+ channel inhibitors did not affect the basal [Ca2+]i, indicating that BK and Kv are mostly closed at rest. TEA/4‐AP enhanced (range: 125–300%) the increase in [Ca2+]i in response to mild‐severe levels of hypoxia. IBTX weakly enhanced (45%) the increase in [Ca2+]i produced by severe hypoxia but not by mild‐moderate hypoxia. Guangxitoxin (GXTX, Kv2 inhibitor) decreased the whole‐cell current in glomus cells by 49%, indicating that Kv2 is a major Kv in rat glomus cells. GXTX increased the hypoxia‐induced [Ca2+]i rise by 130%. These results show that BK and Kv are closed in isolated glomus cells, and their activation by hypoxia dampens the rise in [Ca2+]i. The suppression of the hypoxia‐induced rise in [Ca2+]i by BK alone is weak, whereas that by BK and Kv together is strong.