bluuebunny/crossref_metadata_2025 huggingface dataset:
Shear Stress Induces Apoptosis via Cytochrome C Release from Dynamic Mitochondria in Endothelial Cells
Authors: Hu Ying‐Li , Hur Sung Sik , Lei Lei , Wang Yingxiao , Chien Shu
Published: November 2023

Mechano‐sensors on endothelial cells (ECs) detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. The variations in shear stress patterns can result in different consequences of mechanotransduction and propensity for atherosclerosis. In the regions of disturbed, oscillatory flow with low shear stress, excess cell turnover generates spatial differences in apoptosis in the endothelium. The aims of the present study were to determine whether oscillatory shear stress (OS) and laminar shear stress (LS) could induce EC apoptosis and modulate the cytochrome c (Cyt c) release from mitochondria. ECs were subjected to oscillatory and laminar flows, with shear stresses of 0.4±4 and 12 dynes/cm2, respectively. A significantly increased apoptosis was seen following OS over 24 h, compared with LS.

Apoptosis (%)
4 h
24 h

Oscillatory Flow / Static
1.85±0.06
2.89±0.05*

Laminar Flow / Static
1.73±0.03
1.24±0.04

Values are mean±SEM.

P<0.05 for Oscillatory vs. Laminar Flow.

Endothelial mitochondria are essential to the functional integrity of ECs as they integrate a wide range of cellular processes. The mitochondrial morphology changes dynamically due to their frequent fusion and fission in response to various cellular conditions. Mitochondrial fission is an early event during apoptosis and it occurs very close in time to Cyt c release, and the fragmented mitochondria can readily be seen to retain Cyt c. Cyt c staining of the mitochondria was detected in ECs under both types of flow. In comparison to static control, Cyt c was significantly reduced in the mitochondria of ECs exposed to both flow patterns for 4 h and 24 h. The reduction of Cyt c in endothelial mitochondria was greater under oscillatory flow for 24 h.

Mitochondrial Cyt c
4 h
24 h

Oscillatory Flow / Static
0.16±0.05
0.59±0.05*

Laminar Flow / Static
0.19±0.03
0.90±0.03

Values are mean±SEM.

P<0.05 for Oscillatory vs. Laminar Flow.

We used time‐lapse imaging to monitor the spatial‐temporal dynamics of mitochondria in live ECs transfected with RFP‐mito. During the shearing of live ECs, mitochondria networks are dramatically reorganized from long filamentous, interconnected tubules into small punctate spheres under both flow patterns. Under oscillatory flow, more small fragmentations of endothelial mitochondria were observed than under laminar flow. The results show that mechanical forces affect the mitochondria dynamics in ECs to cause Cyt c release, which was greater under oscillatory than laminar flow. The findings suggest that shear stress induces apoptosis via mitochondrial pathway. Understanding of the endothelial mitochondria responses to different flow patterns will help to elucidate the mechanism of the region‐specific localization of atherosclerosis in the arterial system.

Support or Funding Information

This work was supported by NHLBI Research Grants HL‐104402 and HL‐106579 (S.C.)
Microparticle Delivery of Cyclic AMP to Pulmonary Microvascular Endothelial Cells
Authors: Scruggs April K , Leavesley Silas J , Annamdevula Naga S , Rich Thomas C , Bauer Natalie N
Published: November 2023

Microparticles (MPs) are important for the exchange of information between cells. MPs are extracellular vesicles that range in size from 0.1 to 1 μm in diameter. They are constitutively released from many cell types and are thought to play a role in vascular homeostasis. Further, the number and content of MPs are altered in pathologies involving inflammation and vascular cell activation. MPs carry intracellular constituents from parent cells that affect target cell function. However, the mechanisms of delivery of the MP payload to target cells are unknown. Understanding the payload delivery system between MPs and target cells is the focus of this project.

We recently discovered that cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger, is found in MPs released from pulmonary microvascular endothelial cells (PMVECs). Additionally, MPs isolated from PMVECs treated with β adrenergic agonists and phosphodiesterase (PDE) inhibitors have increased cAMP levels. This suggests that different levels of cAMP are packaged in MPs from the parent cell. MPs containing maximal cAMP were therefore used to study the delivery of MP payload to target cells.

We developed mathematical models of MP payload delivery to subcellular compartments of target PMVECs to better understand how the cAMP signaling pathway may be altered by MP‐mediated cAMP delivery. Simulations indicate that if the cAMP contained in one MP were released into a near‐membrane compartment of a target cell, cAMP levels within that compartment would be sufficient to activate protein kinase A (PKA) and Exchange protein activated by cAMP (Epac) for several minutes. Release of cAMP from a single MP released into the larger volume of the bulk cytoplasm would have little effect on target cell signaling. However, several MPs are often internalized by a target cell and their combined cAMP content would sufficiently elevate bulk cytosolic cAMP levels to activate PKA.

To experimentally test the predictions of this model, MPs were collected from PMVECs treated with rolipram (a PDE type 4 inhibitor; 10 μM, 5 min) and isoproterenol (a β adrenergic agonist; 1 μM, 10 min). These MPs were added to cells transfected with a cytosolic cAMP FRET sensor. Time lapse hyperspectral image stacks were acquired using a Nikon A1R confocal microscope. Custom hyperspectral analysis scripts in MatLab (MathWorks) were used to unmix fluorescent spectra and quantify cAMP concentration changes throughout the target cell. Preliminary results indicate that MPs significantly increased cAMP levels in target cells compared to control. Together, these data indicate that MPs can deliver cAMP as a functional payload. Future studies will determine whether the MP‐delivered cAMP is sufficient to trigger sustained responses in target cells and contribute to MP‐mediated signaling events.

Support or Funding Information

This work was supported by NIH T32HL076125, P01HL066299, S10RR027535, and AHA 11SDG7390037.
Calcium Influx Through Cav1 and TRP Channels Induced by the Calcium Release Via RyR Activates BKCa in Mouse Mesenteric Artery
Authors: Garcia Daniela Cibeli Guimarães , Lopes Miguel José , Lemos Virgínia Soares , Cortes Steyner França
Published: November 2023

The sarcoplasmic reticulum (SR)/plasma membrane (PM) junction plays an important role in the control of the excitation‐contraction coupling of the vascular smooth muscle cells (VSMCs). The present work investigated the PM/SR interaction involved in the modulation of the contraction induced by the activation of ryanodine receptors (RyRs) in mouse mesenteric artery. 2nd branch of endothelium‐denuded small mesenteric arteries from male Swiss mice (10–12 weeks old) were studied using wire myography, epifluorescence microscopy, and electrophysiology methods. All experimental protocols were approved by the local ethics committee (protocol n° 86/2015). Caffeine (10 mM) induced a transient contraction in mesenteric arteries and increased of the intracellular calcium signal. In Ca2+‐free solution, the contraction and the calcium signal induced by caffeine were reduced. Nifedipine (10 μM), a selective Cav1 blocker, increased the area under the curve (AUC) resulting from the reduction of the contraction decay time while reducing the intracellular calcium signal. 2‐APB (10 μM), a TRPC blocker, increased the peak and the AUC of the contraction and reduced the calcium signal. Paxilline (1 μM), a BKCa channel blocker, also increased the AUC of the contraction. The contractile effect of caffeine was not changed by apamin (50 nM) and TRAM‐34 (1 μM), respective blockers of SKCa and IKCa. Caffeine evoked a hyperpolarization, which was blocked by nifedipine, 2‐APB, and paxilline. The present work demonstrated that the release of calcium stores sensitive to the activation of RyRs induces an influx of calcium through Cav1 and TRP channels responsible for a BKca‐dependent hyperpolarization, triggering a negative feedback of the contraction induced by caffeine in mouse mesenteric artery.

Support or Funding Information

Supported by the Brazilian Council for Scientific and Technological development (CNPq).
Sex Differences in Obesity‐Associated Vascular/Perivascular Remodeling and Adipokine Production in Obese Patients Undergoing Roux‐en‐Y Bariatric Surgery
Authors: Mui Ryan K , Fernandes Roxanne N , Galligan James J , Fink Gregory D , Pereira‐Hicks Cristiane N , Watson Ralph E , Lane Mindy K , Xu Hui
Published: November 2023

Introduction

In obese humans, inflammation and adipokine production affect mortality and cardiovascular tissue remodeling independent of BMI. Studies of plasma adipokine levels suggest that anti‐inflammatory adipokines including omentin‐1, secreted frizzled‐related protein 5 (SFRP5), and adiponectin improve cardiovascular function while pro‐inflammatory adipokines, like resistin, impair cardiovascular function. Furthermore, adipokine production and function differ in males and females. Therefore, we tested the hypothesis that in human obesity, the degree of vascular/perivascular pathology is sex‐dependent and associated with a distinct adipokine profile.

Methods

Mesenteric vessels and perivascular adipose tissue were obtained from 26 obese patients (4 males, 22 females) undergoing Roux‐en‐Y bariatric surgery. A blinded semi‐quantitative approach was used to assess 4 criteria: Fibrosis, adventitial wall thickening, angiogenesis, and immune cell infiltration. Samples with 2+ criteria were considered abnormal (pathology). Expression levels of adiponectin, omentin‐1, resistin, and SFRP5 in perivascular fat (PVAT) were evaluated by qRT‐PCR. All collected data were analyzed by histopathology and sex.

Results

60% of female tissue samples displayed significant pathology, which consistently involved fibrosis and adventitial wall thickening. Significant pathology was not observed in male tissues. When categorized by histopathology alone, females with pathology had 3‐fold lower omentin‐1 mRNA and 2‐fold higher resistin mRNA vs. females and males without pathology. When compared to males, females without pathology had 10‐fold higher omentin‐1 mRNA whereas obese females with pathology had only 1.6‐fold higher omentin‐1 mRNA and 1.6‐fold higher resistin mRNA. In females only, females with pathology had 5‐fold lower omentin‐1 mRNA, 2‐fold higher resistin mRNA and 2‐fold higher SFRP5 mRNA vs. females without pathology. None of these findings were associated with age, BMI, type II diabetes, dyslipidemia, or hypertension.

Conclusions

In the mesentery, females are more susceptible to obesity‐associated vascular/perivascular tissue remodeling than males. When compared to obese females without pathology, obese females with pathology have lower omentin‐1 mRNA and higher mRNA levels of resistin and SFRP5. Therefore, omentin‐1, resistin and SFRP5 may be useful adipokines in identifying obese females at risk of worsening cardiovascular tissue quality.

Support or Funding Information

Supported by NIH 2P01HL070687
Reduced Uterine Perfusion Pressure‐Induced Preeclampsia Acquires Salt‐Sensitivity Characteristics in Postpartum
Authors: Matsuura Taku , Shinohara Keisuke , Kishi Takuya , Tsutsui Hiroyuki , Hirooka Yoshitaka
Published: November 2023

Background

Preeclampsia, a hypertensive disorder during pregnancy, occurs in 3% to 8% of pregnancies, and the risk of future cardiovascular diseases is 2 to 4 times higher in women with preeclampsia than in women without. Women with a history of severe preeclampsia also have a tendency toward salt‐sensitive hypertension. However, it is not yet clear whether preeclampsia leads to salt‐sensitive hypertension later in life as an acquired characteristic or as a manifestation of the original predisposition. We hypothesized that preeclampsia results in postpartum salt‐sensitive hypertension as an acquired characteristic.

Methods and results

We established a rat with reduced uterine perfusion pressure model (RUPP) as a model of acquired preeclampsia, by placing a silver clip (0.203‐mm ID) around the aorta above the iliac bifurcation and branches of bilateral ovarian arteries (0.100‐mm ID). Pregnant Sprague‐Dawley rats were divided into two groups that underwent the RUPP operation or sham operation (SHAM) at day 14 after gestation. Mean arterial pressure (MAP) at day 19 after gestation was significantly higher in RUPP than in SHAM rats (116 ± 4 mmHg vs. 100 ± 5 mmHg, n's=7 and 3, respectively; p<0.05). At 2 weeks postpartum, MAP similarly returned to normal levels between RUPP and SHAM rats (92.9±3.0 mmHg vs. 87.4±5.4 mmHg, n=6 and 4). At 3 weeks postpartum, a high‐salt (8% NaCl) diet for 1 week was initiated. Surprisingly, the high‐salt diet further increased MAP in RUPP (110.8±3.2 mmHg, n=6), compared to SHAM (99.8±5.0 mmHg, n=4).

Conclusion

Preeclampsia results in postpartum salt‐sensitive hypertension as an acquired characteristic.
Reciprocal control of apoptotic cell death and synaptic plasticity by DAPK1 depletion in focal cerebral ischemic brain
Authors: Won Jinyoung , Park Sookyoung , Hong Yonggeun
Published: November 2023

Death‐associated protein kinase 1 (DAPK1) has been identified as a key player in different types of cell death that includes apoptosis, autophagy and necrosis. Recent studies indicated a close relationship between DAPK1 and ischemic brain injury. The present study investigated the effect of DAPK1 deficiency on the synaptic plasticity of cortical pyramidal neurons in DAPK1 knockout mice after focal cerebral ischemia. The subjected C57BL/6 mice exhibited neurological deficits and altered behavioral outcomes after ischemic brain injury. Progressive dendritic change and synaptic degeneration were observed in cortical pyramidal neuron. Apoptosis signaling pathway activated by increase of dephosphorylated DAPK1, which is active form under pathological condition including ischemic stroke. However, DAPK1 knockout mice exhibit significantly reduced infarct volumes and enhanced functional recovery. In addition, abnormal dendrite and dendritic spine morphology were decreased in DAPK1 knockout mice. The expression of BAX and caspase‐3 as pro‐apoptotic marker was significantly decreased and BCL‐2 as anti‐apoptotic marker was significantly increased in DAPK1 knockout mice. These results indicate that activation of DAPK1 during ischemic insult was exclusively associated with dendritic pathology and neurodegeneration via DAPK1 dependent apoptotic cell death. Thus, regulation of the expression and activity of DAPK1 could be a novel interventional strategy for ischemic brain injury.

Support or Funding Information

NRF 2013R1A2A2A01067169 to Y.H., NRF‐2014R1A1A3051724 to S.P., KRIBB Research Initiative Program (KGM4611613), Republic of Korea
Behavioral and Inflammatory Response in Animals Exposed to a Low‐Pressure Blast Wave and Supplemented with β‐Alanine
Authors: Hoffman Jay R , Zuckerman Amitai , Ram Omri , Sadot Oren , Stout Jeffrey R , Ostfeld Ishay , Cohen Hagit
Published: November 2023

This study investigated the benefit of β‐alanine (BA) supplementation on reducing behavioral and cognitive responses relating to mild traumatic brain injury (mTBI) and post‐traumatic stress disorder (PTSD) in rats exposed to a low‐pressure blast‐wave. In addition, inflammatory, neurotrophin and tau protein expression in the hippocampus was also examined. Animals were fed a normal diet with or without (PL) BA supplementation (100mg·kg−1) for 30‐days, prior to being exposed to a low‐pressure blast‐wave. Diets were maintained until the end of the study. A third group of animals served as a control (CTL). These animals were fed a normal diet, but were not exposed to the blast. Validated cognitive‐behavioral paradigms were used to assess both mTBI and PTSD‐like behavior on days 7–14 following the blast. Brain‐derived neurotrophic factor (BDNF), neuropeptide Y, glial fibrillary acidic protein (GFAP), and tau protein expressions were analyzed a day later. In addition, brain carnosine and histidine content was assessed as well. The prevalence of animals exhibiting mTBI‐like behavior was significantly lower (p=0.044) in BA than PL (26.5% and 46%, respectively), but no difference (p=0.930) was noted in PTSD‐like behavior between the groups (10.2% and 12.0%, respectively). Carnosine content in the cerebral cortex was higher (p=0.048) for BA compared to PL, while a trend towards a difference was seen in the hippocampus (p=0.058) and amygdala (p=0.061). BDNF expression in the CA1 subregion of PL was lower than BA (p = 0.009) and CTL (p<0.001), while GFAP expression in CA1 (p=0.003) and CA3 (p=0.040) subregions were higher in PL than other groups. Results indicated that BA supplementation for 30‐days increased resiliency to mTBI in animals exposed to a low‐pressure blast‐wave.

Support or Funding Information

Natural Alternatives International (Carlsbad, CA, USA)
The Glycolysis Regulatory Enzyme PFK‐FB3 Enhances Endothelial Barrier Restoration in Acute Lung Injury
Authors: Wang Li , Chaturvedi Pallavi , Krantz Sarah , Klomp Jennifer , Pohl‐Avila Nicole M , Ray Anne‐Marie , Toth Peter T , Carmeliet Peter , Karginov Andrei , Malik Asrar B , Rehman Jalees
Published: November 2023

Acute lung injury (ALI) is a devastating inflammatory complication of infectious lung disease, characterized by a breakdown of the pulmonary vascular barrier and the subsequent formation of protein‐rich alveolar edema. In recent years, considerable advances have been made in the understanding of how intracellular signaling regulates the disruption and re‐assembly of adherens junctions (AJs) which are mediators of the endothelial barrier function. Little is known about the role of endothelial metabolism as a modulator of endothelial barrier function and its restoration in the setting of lung vascular injury. We hypothesized that the glycolysis regulatory enzyme PFK‐FB3 would support re‐annealing of the endothelial barrier because glycolysis allows for the local generation of ATP at the AJs. We observed a 2–3‐fold upregulation of PFK‐FB3, an enzyme which is a critical regulator of glycolytic activity, in response to inflammatory injury in human lung endothelial cells (p<0.05). ATP levels also increased at the AJs during recovery after LPS‐induced injury. Depletion of the hypoxia‐regulator transcription factor Hif1α attenuated the upregulation of PFK‐FB3, suggesting that hypoxic signaling activates PFK‐FB3 mediated upregulation of glycolysis. Suppression of PFK‐FB3 activity by either pharmacologic inhibition or genetic depletion delayed restoration of the endothelial barrier. In vivo delivery of liposomal PFK‐FB3, on the other hand, enhanced restoration of lung vascular barrier function in a model of experimental acute lung injury by 24 h (p<0.05). These findings suggest that upregulation of glycolysis via enhanced PFK‐FB3 activity serves as an endogenous metabolic adaptation to inflammatory vascular injury and facilitates endothelial barrier restoration.

Support or Funding Information

supported by NIH grant P01 HL60678‐16A1.
Divergent effects of DHA treatment and AMPK activation on protein synthesis rate and signaling in palmitate‐treated skeletal muscle
Authors: Perry Ben Douglas , Rahnert Jill A , Xie Yang , Zhang Peng , Espinosa Daniel , Price S. Russ
Published: November 2023

Circulating levels of saturated fatty acids, such as palmitate (PA), are elevated in type II diabetes mellitus. In skeletal muscle, PA impairs insulin sensitivity, induces ER stress and impairs protein synthesis. Both insulin resistance and ER stress are reversed with treatment of n‐3 polyunsaturated fatty acids like docosahexaenoic acid (DHA). However, it remains unclear whether DHA reverses the PA‐induced impairment in protein synthesis. This study investigated the effects of PA and DHA on protein synthesis and related signaling proteins, including eukaryotic initiation factor 2α (eIF2α) and p70S6K in skeletal muscle cells. Due to its potential role in reducing ER stress and altering protein synthesis through mTOR signaling, we also investigated the effects of 5′ AMP‐activated protein kinase (AMPK) on these processes. C2C12 myotubes were treated with vehicle (2% BSA), DHA (100 μM), PA (500 μM) or co‐treated with PA+DHA. Protein synthesis was inhibited after 6h (−34.7%) and 24h (−40.8%) of treatment with PA and co‐treatment with DHA restored protein synthesis to the control level; DHA alone increased protein synthesis. PA treatment for 6–24 h also increased eIF2α phosphorylation, a response that reduces protein translation; co‐treatment with DHA prevented the responses. Surprisingly, the phosphorylation of p70S6K was increased after 6 h of PA treatment but restored to normal levels after 24 h. Moreover, AMPK phosphorylation was also increased after treatment with PA for 6 h but reduced after 24 h; in both cases, DHA co‐treatment normalized AMPK phosphorylation. Co‐treating myotubes with PA plus metformin, an activator of AMPK, caused a drastic decrease in protein synthesis and p70S6K phosphorylation after 24 h. In summary, PA induced a global reduction in protein synthesis rate that was prevented by co‐treatment with DHA. Changes in the phosphorylation of eIF2α, but not p‐p70S6K coincided with those in protein synthesis after both 6 and 24 h. Despite the widely reported beneficial effects of AMPK activation in PA‐treated cells, impaired protein synthesis and increased p‐eIF2α preceded the PA‐induced reduction in AMPK.

Support or Funding Information

Supported by NIH RO1 DK95610, NIH T32 DK007656 and VA Merit I01BX001456
Contribution of Membrane Repair Defects to Skeletal Muscle Inflammation
Authors: McElhanon Kevin E. , Young Nicholas , Hampton Jeffrey , Beck Eric X , Sahenk Zarife , Aggarewal Rohit , Oddis Chester V. , Jarjour Wael N. , Weisleder Noah E.
Published: November 2023

Inflammation is a significant contributor to multiple skeletal muscle diseases, however the mechanisms that facilitate these inflammatory responses have not been fully characterized. Idiopathic immune myopathies are a group of disorders involving chronic damage to skeletal muscle due to extensive inflammation triggered by autoimmune responses and provides a model to further understand general mechanisms of inflammation. Chronic inflammation leads to degeneration of muscle structure and function resulting in severe proximal muscle weakness. T‐cell‐ and antibody‐mediated processes are involved in the progression of disease, however the mechanism leading to pathogenesis remains unknown. Two animal models have recently provided insight into the pathogenic mechanisms of myositis. Synaptotagmin VII null (SytVII−/−) mice display impaired sarcolemmal repair capacity and develop mild myositis, suggesting that antigen presentation of internal skeletal muscle proteins may play a role in initiating an autoimmune response. A more robust model of myositis combines the Syt VII−/− model with a FoxP3 mutation (FoxP3−/Y/Syt VII−/−). This mouse strain combines impaired membrane repair with regulatory T‐cell deficiency. Adoptive transfer of lymph node cells from FoxP3−/Y/Syt VII−/− mice to RAG‐1 mice lacking T‐ or B‐cells results in significant muscle inflammation. The current project examined the contribution of defective sarcolemmal repair to the pathogenesis of myositis. Sarcolemmal membrane repair is a conserved process where disruptions in the lipid bilayer are actively resealed to maintain cellular integrity. Previous studies established the cellular framework of the membrane repair process utilizes exocytotic and endocytotic vesicle trafficking to restore membrane integrity. Previous work by our lab and others identified TRIM72, a member of the Tripartite Motif family of E3 ubiquitin ligases, as a critical component of the membrane repair process. Our studies here identified novel autoantibodies against TRIM proteins in patient sera. Using an adoptive transfer model to induce myositis in mice, we show that membrane repair is compromised in skeletal muscle distal to the initial site of inflammation. We also demonstrate that exogenous delivery of antibodies against TRIM proteins can compromise membrane repair in healthy skeletal muscle. Our data indicate that defects in membrane repair associated with the progression of myositis lead to TRIM protein exposure to the extracellular space and that the autoantibodies produced against TRIM proteins further compromise membrane repair and exacerbate inflammation.

Support or Funding Information

National Institute of Arthritis and Musculoskeletal and Skin Diseases, MDA, The Ohio State University Center for Clinical and Translational Science
Muscle gp130 Receptor Regulation of Oxidative Metabolism During Cancer Cachexia
Authors: Fix Dennis , Hardee Justin , Gao Song , VanderVeen Brandon , Carson James A.
Published: November 2023

Introduction

Muscle oxidative metabolism is disrupted by the cachectic environment brought on by cancer. Processes related to mitochondrial biogenesis, dynamics, and mitophagy are all altered. The IL‐6 family of cytokines have an established regulatory role in cachectic signaling in several preclinical mouse models through the activation of intracellular signaling pathways initiated by receptor complexes involving glycoprotein 130 (gp130). Therefore, we examined muscle gp130 role in the disruption of muscle oxidative metabolism induced by a cachectic environment.

Methods

Male, C57BL/6 (B6, N=5), ApcMin/+ (MIN; N=5), skeletal muscle specific gp130 knockout (KO; N=5), ApcMin/+ KO (MIN KO; N=5) mice were sacrificed at 20 weeks of age. Additionally, Lewis Lung Carcinoma (LLC) injected B6 (N=5) and KO (N=5) mice were examined 30 days after injection. C2C12 myotubes were treated with a small molecule STAT3 inhibitor (C188‐9) and incubated in LLC conditioned media (24 hours). Oxidative metabolism, mitochondrial content, and dynamics were examined.

Results

LLC and MIN increased muscle gp130 protein expression and STAT3, which was suppressed by gp130 KO. COX activity was decreased in LLC and MIN, and not rescued by the KO. MIN and LLC increased FIS‐1 protein expression, and this induction was ablated by KO in both mice. MIN decreased MFN‐1 protein expression which was not altered by KO. Beclin‐1 and 4HNE protein expression increased in MIN and LLC, and the KO blocked this induction. LLC media activation of myotube STAT3 was blocked using the C188‐9 inhibitor. Inhibiting the LLC induction of STAT3 in cultured myotubes did not affect the induction if FIS‐1, Beclin‐1 and 4HNE protein expression.

Conclusions

These results implicate direct signaling through the muscle gp130 receptor for the cachexia regulation of muscle mitochondrial dynamics and autophagy, and this regulation may be independent of STAT3.

Support or Funding Information

This work was supported by National Institutes of Health grant # NCI‐R01CA121249 (JAC)
Reversal of ciprofloxacin‐induced testosterone reduction by probiotic microbes in mouse testes
Authors: Xie Congcong , Gao Fulu
Published: November 2023

Ciprofloxacin (CPFX) is a second‐generation fluoroquinolone antibiotic used to treat infections of various Gram‐negative organisms. Although CPFX is a highly effective antibiotic, it has been reported to significantly impair both testicular function and structure in rats. Probiotics are beneficial microorganisms to the host, and dietary probiotics may provide an alternative natural approach to the prevention of late‐onset male hypogonadism. However, whether probiotic microbes can reverse CPFX‐induced testosterone reduction has not been reported. In this study, male mice were administered saline for 6 days (Sal6), CPFX for 6 days (A6 group), CPFX for 6 days and then bifidobacteria for the next 6 days (A6+P6 group), CPFX for 6 days and then saline for the next 6 days (A6+Sal6 group). We detected levels of testosterone in the serum, as well as levels of steroidogenic enzymes [cholesterol side‐chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR)] in testes, deleted in breast cancer 1 (DBC1), Silent Mating Type Information Regulation 2 Homolog 1 (Sirt1), nuclear factor kappa beta (NF‐κB), and inflammatory and anti‐inflammatory cytokines in the testes. The results showed that the A6+P6 group had significantly higher serum testosterone levels and steroidogenic enzymes, higher Sirt1 and anti‐inflammatory cytokine expression, and lower NF‐κB, DBC1, inflammatory cytokine expression compared with the A6 and A6+Sal6 groups. The results suggest that probiotic microbes can reverse the reduction of CPFX‐induced testosterone reduction, and these effects may be carried out through the increase of Sirt1 expression which inhibit the NF‐kB inflammatory signaling pathway.
Hydrogen Sulfide Attenuates Homocysteine‐Induced Mitochondrial Toxicity in MC3T3‐E1 Pre‐osteoblastic Cells
Authors: Zhai Yuankun , Behera Jyotirmaya , Tyagi Suresh , Tyagi Neetu
Published: November 2023

INTRODUCTION

Osteoporosis is a bone disorder which leads to an increased risk of fracture, and the main treatments always aimed at increasing bone formation but inhibiting bone resorption. Homocysteine (Hcy) have been reported to induce bone loss, but the detailed mechanism still not very clear. In this study, we want to clarify the negative effects of Hcy on bone formation and the rescue effects of hydrogen sulfide (H2S).

METHODS

MC3T3‐E1 cells were cultured in vitro, and were divided into 5 groups, contained control, 300μM homocysteine, 30μM H2S, 300μM Hcy plus 30μM H2S, 100μM H2O2, and then cell viability were measured by MTT method and Hoechst 33342 staining, while ALP activity and positive colony formation were detected after osteogenic induced for 9 days, and calcified nodules were stained by alizarin red S after osteogenic induced 21 days. Total mRNA and protein were isolated, some related factors were measured by western‐blot and RT‐PCR, including OPG, RANKL, HO‐1, Nrf‐2, COX‐2, NOX‐2 and NOX‐4 etc. ROS production were detected by flow cytometry and confocal, the mitochondria function were present by several assays, such as JC‐1 intake, expression of Mfn2 and Drp1, ATP production and also oxygen consumption. Meanwhile, MMPs (MMP3, MMP9, and MMP13) expression also were measured by RT‐PCR, western‐blot and zymography.

RESULTS

We found Hcy could decrease the MC3T3‐E1 osteoblastic cells viability, ALP activity, and calcified nodules, decrease OPG but increase RANKL expression, both increase expression of antioxidant and oxidant factors, including HO‐1, Nrf2, COX‐2, NOX‐2 and NOX‐4 respectively. Moreover, we also found Hcy promote the intracellular ROS production, decreased in mitochondria membrane potential, mitochondria copy number, ATP production, oxygen consumption, also less Mfn2 and more Drp1 expression which represents mitochondria fusion and fission, therefore induced dysfunction of mitochondria. NaHS, an H2S donor, increased cell viability, reduced ROS production and mitochondria dysfunction, reverse the factor changes induced by Hcy, and then protect MC3T3‐E1 cell from apoptosis. Meantime, we also found NaHS inhibit the enhancing of MMP3 and MMP13 which induced by Hcy, and MMPs were known as degradation factors for extracellular bone matrix.

CONCLUSION

In summary, Hcy stimulate intracellular ROS production and mitochondria dysfunction, then decrease the cell viability and bone formation activity, the mechanism may depend on increasing MMPs, ROS production, and also dysfunction of mitochondria, but all these effects could be inhibited by the H2S supplement. So our findings suggested that H2S may have a potentially therapeutic value for osteoporosis patients which also with hyperhomocysteine levels.

Support or Funding Information

Acknowledgement: Financial support from National Institute of health grant AR‐067667 and HL‐107640 are greatly acknowledged.
Mitochondrial fragmentation is a novel feature of adherent‐invasive E. coli evoked pathophysiology in enteric epithelia
Authors: Mancini Nicole , Wang Arthur , Shearer Jane , McKay Derek
Published: November 2023

Introduction

Mitochondria are not distinct organelles; they exist as a dynamic network that constantly remodels via the processes of fission and fusion to meet the cells energy demands and allow recycling of damaged mitochondria. Mitochondrial dysfunction has been described in inflammatory bowel disease, where adherent‐invasive E. coli (AIEC) have been described as a pathobiont that can drive epithelial apoptosis. Little is known of microbial regulation of enterocytic mitochondrial dynamics and nothing of the potential of AIEC to perturb the critical metabolic function that mitochondria fulfill for the cell.

Hypothesis

AIEC induce mitochondrial fragmentation in intestinal epithelial cells in an invasion‐dependent manner that requires ROS, and drives the loss of epithelial permeability.

Aim

Explore the relationship between mitochondrial dynamics and epithelial function in vitro and determine if, then how, infection with AIEC affects mitochondrial dynamics and any consequence for epithelial barrier function.

Methods

Human colon‐derived epithelial lines were cultured with AIEC or a non‐invasive E. coli and readouts assessed: (a) visualization of mitochondria morphology and membrane potential with confocal microscopy; (b) immunoblotting of whole cell protein extracts for the mitochondrial fusion protein Optic Atrophy Factor 1 (OPA1); (c) ROS neutralized by the antioxidants; (d) treatment with spent medium from bacterial cultures; and, (e) blockade of fission using specific pharmacological inhibitors and measurement of epithelial permeability in response to AIEC.

Results

AIEC infection resulted in reduced mitochondrial membrane potential and ATP, and dramatic mitochondrial fragmentation accompanied by OPA1 cleavage in gut epithelia in a time‐ and dose‐dependent manner. The mitochondrial fragmentation was not reproduced by exposure to AIEC conditioned medium and was not abrogated by treatment with a general (vitamin C) or a mitochondrial‐specific (mitoTEMPO) anti‐oxidant co‐treatment. Invasion of epithelial cells by AIEC and transepithelial resistance were unaffected by co‐treatment with pharmacological inhibitor of mitochondria fission.

Conclusion

As a putative cause or contributor to Crohn's disease, AIEC were found to drive massive mitochondrial fragmentation in model gut epithelia independent of ROS generation or AIEC soluble factors. Neither the invasion of the E. coli nor the increase in paracellular permeability they evoke is sensitive to treatment with inhibitors of mitochondrial fission.

Support or Funding Information

Acknowledgements: Supported by: CIHR operating grant to DMM, U. Calgary Eyes High Doctoral Studentship to NLM.
Renal Denervation Prevents Nicotine‐Induced Hypertension and Reduces Renal IL‐17a
Authors: Harwani Sailesh C , Ratcliff Jason , Cicha Michael Z , Chapleau Mark W , Abboud Francois M
Published: November 2023

Interaction between the nervous and immune systems has become a major focus in the pathophysiology of essential hypertension. Cholinergic activation of nicotinic acetylcholine receptors are known to exert an anti‐inflammatory effect. However, we recently demonstrated that in vivo cholinergic activation with nicotine induces renal inflammation and premature development of hypertension in young Spontaneously Hypertensive Rat (SHR). We hypothesized that renal nerves contribute to nicotine‐induced renal inflammation and premature hypertension in young SHR. Following bilateral renal nerve denervation (RND) (or Sham surgery) in young (3–5 week old SHR), we continuously infused nicotine bitartrate (15mg/kg/day) or saline subcutaneously for 2 weeks. Systolic blood pressure was measured by tail cuff. Norepinephrine (NE) and IL‐17a were measured in kidneys by ELISA and Luminex assay, respectively. RND prevented the development of hypertension in response to nicotine infusion in young SHR (n=6), compared to nicotine‐infused Sham operated young SHR (n=3) (140 ± 4 vs 180 ± 5 mmHg, respectively, p<0.001). RND also significantly decreased IL‐17a in kidneys nicotine‐infused SHR, compared with kidneys from sham operated SHR (12 ± 0.7 pg/mL vs 17 ± 0.9 pg/mL, p<0.01). NE analysis of renal tissues confirmed renal denervation with over an 85% reduction in renal NE levels, compared to sham operation (31±11 ng/mg vs 225±43 ng/mg, P<0.05). We conclude that renal nerves mediate nicotine‐induced renal inflammation and hypertension.

Support or Funding Information

University of Iowa, Carver College of Medicine

NIH/NHLBI‐HL119588‐01A1 and HL014388‐48
The Therapeutic Potential of Modified Deep‐Ocean Water on 2,4‐dinitrochlorobenzene Induced Atopic Dermatitis in Balb/c Mice
Authors: Ho Ching‐Jung , Chien Chiang‐Ting
Published: November 2023

Atopic dermatitis is a chronic skin disease characterized with continuous inflammation and pruritic skin lesions. Repeating epicutaneous application of 2,4‐dinitrochlorobenzene (DNCB) treated on the dorsal skin of Balb/c mice can induce atopic dermatitis‐like symptoms and skin lesions. Deep ocean water (DOW), which is rich in micronutrients and minerals and with antioxidant and anti‐inflammatory qualities, may be developed as marine drugs to provide skin protection against atopic dermatitis. Because of the unique properties of DOW, in the present study, we explored its therapeutic potential on DNCB‐induced atopic dermatitis in Balb/c mice. The DOW with hardness at 2400 ppm was orally administrated for 2 weeks to the mice. Our results showed that there was no significant difference in scratching behavior between DNCB group and DOW group. However, the skin wound caused by DNCB was healing sooner in DOW group compared to tap‐water‐treated group. In histopathological analysis, oral DOW administration significantly alleviated DNCB‐induced epidermal thickness and lymphocyte extravasation. Caspase‐3, one of the apoptosis related proteins, was significantly increased in DNCB‐induced dermatitis skin tissue. The enhanced caspase‐3 expression was efficiently decreased after oral DOW treatment. Spleen, an organ plays important roles in atopic dermatitis. The spleen weight of DNCB group was significantly higher. However, the spleen weight did not decrease in DNCB group with DOW treatment. We also found that the margin between red pulp and white pulp of spleen was blurred in DNCB group but was alleviated in DOW group. In conclusion, these results suggest that oral DOW treatment may be a potentially therapeutic strategy in ameliorating atopic dermatitis.
The impaired vasodilatation in rat renal interlobar arteries after hypoxia/re‐oxygenation is endothelium independent
Authors: Braun Diana , Ludwig Marion , Persson Pontus Börje , Dietze Stefanie , Patzak Andreas
Published: November 2023

Renal vasculature plays an important role in renal ischemia/reperfusion injury. We showed recently that hypoxia/re‐oxygenation combined with low dose norepinephrine treatment increases the angiotensin II responses in interlobar arteries. Since angiotensin II activates constrictor and dilator systems we hypothesized an impaired vasodilatation in this context.

To test the hypothesis rat interlobar arteries were studied by using wire myography. The dilatory capacity was investigated by application of acetylcholine (10‐9 to 10‐5 mol/l). Hypoxia (95% N2, 5% CO2 for 60 min) alone followed by re‐oxygenation (95% O2, 5% CO2, 10 min) did not affect vasodilatation. However, norepinephrine in a non‐constrictor dose (10‐9 mol/l) applied during hypoxia resulted in a decreased response to acetylcholine. The contribution of the nitric oxide system, COX‐products, and endothelial derived hyperpolarizing factor to reduced dilatation was tested by pretreatment with L‐NAME (10‐4 mol/l), indomethacin (10‐5 mol/l), TRAM‐34 (10‐6 mol/), and UCL1684 (10‐7 mol/l), respectively. L‐NAME decreased the acetylcholine response after hypoxia/re‐oxygenation stronger compared to control conditions, while indomethacin, TRAM‐34, and UCL1684 did not affect it. Endothelium‐independent effects were analyzed by using the NO‐donor SNP and simultaneous blocking of endothelium‐dependent NO production. SNP significantly reduces vasodilation after hypoxia+norepinephrine/re‐oxygenation (H+NE/R). Passive stiffness of arteries was not altered in our experimental setup.

H+NE/R provokes an impairment of the dilatory response to acetylcholine in renal arteries. Our data suggest that the endothelial nitric oxide system, COX products, or TRAM‐34 and UCL1684 inhibitable potassium channels do not contribute to reduced dilatation. The results indicate changed dilatory signaling pathways in vascular smooth muscle cells.

Support or Funding Information

This study was financially supported by a grant of the German Research Council to A.P and P.B.P (FG 1368, DFG PA 479/10‐1 and PA 479/10‐2).
The effects of telmisartan on ischemia/reperfusion injury of rat kidney
Authors: Lee Pei Ying
Published: November 2023

Telmisartan is a kind of selective angiotensin II receptor type I inhibitor that can reduce both the systolic pressure and diastolic blood pressure. There are many studies show that telmisartan is effective in reversing tissue damage induced by ischemia/reperfusion in different tissues. However, treatments of telmisartan were given for several days before ischemia injury. To evaluate the effects of telmisartan as an antioxidant and for its tissue protective properties in ischemia/reperfusion injuries in rat kidneys. 10 mg/kg dose of telmisartan was injected (ip.) into rats with ischemia‐reperfusion (I/R) AKI and compared with PBS‐treated AKI rats. Renal function, histopathological findings, and the levels of ROS‐related factors and inflammatory cytokines were measured and analyzed. Our finding showed that telmisartan injected into kidney immediately after I/R injury didn't reduced the impairment of renal functioning and tissue injury compared to PBS‐treated rats.
Assessment of Protective Effect of Phikud Navakot Extract against Hypoxia/Reoxygenation Injury on H9c2 Cardiomyoblasts
Authors: Gerdprasert Orapin , Nusuetrong Punnee
Published: November 2023

Phikud Navakot is composed of nine main medicinal plants in Yahom Navakot, which has been used as traditional medicine for treatment of cardiovascular symptoms such as dizziness and fainting. The present study was to investigate the protective effect of the ethanolic extract of Phikud Navakot (PN) against hypoxia/reoxygenation (H/R)‐injured H9c2 cardiomyoblasts. The cytotoxicity of PN (0.001–0.5 mg/mL) on H9c2 cells was measured by MTT assay, showing no cytotoxicity to H9c2 cells. Cardioprotective ability of PN was investigated during exposure to 6‐h hypoxic buffer in ischemic box followed by 24‐h reoxygenation in complete medium in CO2 incubator. The results showed that PN at a concentration of 0.1 mg/mL was able to increase the percentage of cell survival in H/R‐induced cell death when compared with the H/R control, using 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid as a positive control. PN at the same concentration was also decreased the intensity of reactive oxygen species (ROS) production measured by 2′,7′‐dichlorofluorescin diacetate, when compared to the H/R control. In conclusion, the present results revealed that PN (0.1 mg/mL) was able to decrease H/R‐induced cell injury in H9c2 cardiomyoblasts, which should be partly through inhibition of the intracellular generation of ROS.

Support or Funding Information

This work was supported by research grant from the National Research Council of Thailand (NRCT 2009‐113). We specially thank Dr. Sanya Hokputsa for giving us the extracts. The authors gratefully thank Mr. Weerasak Ussawawongaraya for preliminary of hypoxic‐simulated H9c2 cells.
Identification of Vesicular Monoamine Transporter 2 (vmat2)‐Containing O2 Chemoreceptors in the Gills of Zebrafish
Authors: Pan Wen , Scott Angela L , Nurse Colin A , Jonz Michael G
Published: November 2023

All vertebrates “sense” changes in oxygen (O2) via specialized cells, called chemoreceptors, associated with the peripheral nervous system. Aquatic vertebrates are especially prone to fluctuations in environmental O2 levels. Thus it is essential for them to readily detect low O2 and make appropriate ventilatory adjustments. Teleost species, such as zebrafish, have O2 chemoreceptive neuroepithelial cells (NECs) embedded in the gill epithelium. These cells exhibit membrane depolarization and vesicular recycling upon exposure to hypoxia, and express the monoamine neurotransmitter, serotonin (5‐HT). Our research objectives are to identify gill O2 chemoreceptors in live tissue preparations and determine the role of intracellular Ca2+ in O2 sensing. We are currently employing a transgenic zebrafish line, expressing green fluorescent protein (GFP) under the control of vesicular monoamine transporter 2 (vmat2) regulatory elements, in order to identify NECs and characterize their hypoxic responses in vitro and in situ. Immunohistochemistry and confocal microscopy were used to determine the distribution and identity of GFP‐positive cells in gills. Our results show that the population of GFP‐positive cells in our transgenic line significantly overlaps with serotonergic NECs in the gill filaments. Preliminary studies using fura 2‐based imaging indicate that isolated GFP‐positive cells stimulated by hypoxia undergo an increase in intracellular Ca2+ concentration. Identification of NECs both in vitro and in situ using a vital marker will have a great impact on future work addressing the physiological properties of these cells.

Support or Funding Information

This research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).
Effects of Different Oxygen Concentrations on Human Differentiated Preadipocytes Lipogenic and Lipolytic functions
Authors: MAHAT BIMIT , Mauger Jean‐François , Imbeault Pascal
Published: November 2023

Context

Fat stored in white adipose tissue is the human body's largest energy reservoir. Adipocytes, the predominant cell population in white adipose tissue, play a central role in the clearance of circulating triglyceride (TG) and the delivery of fatty acids for use by peripheral tissues. Impaired regulation of adipose tissue functions is thought to stimulate lipid deposition in non‐adipose tissues, inducing lipotoxic response and the development of metabolic disorders. During weight gain, adipocytes expand in size, decreasing adipose tissue capillary density and creating areas characterized by excessively low oxygen concentrations (hypoxia). It is not clear how hypoxia modulates adipose tissue TG clearance and fatty acid delivery.

Objective

The purpose of this study was to investigate the effects of different hypoxia concentrations on lipogenic (store TG) and lipolytic functions (deliver fatty acids) of human differentiated preadipocytes.

Methods

Differentiated human preadipocytes were exposed to different oxygen concentrations (3%, 10%, and 21%) either during differentiation (14d, continuous exposure) or acutely for 24h after differentiation (acute exposure). Gene expression of lipogenic enzymes and regulating factors will be determined by real‐time PCR. Lipoprotein lipase (LPL) activity was measured using the EnzChek Lipase Substrate and lipolytic rate was determined by measuring glycerol release over 3 hours.

Results

In differentiated human preadipocytes, a dose‐dependent reduction in LPL activity was observed in both acute and continuous hypoxia (6‐fold reduction under 3% O2 p<0.001, 2‐fold reduction under 10% O2, p<0.05, compared to 21% O2). Basal lipolysis was significantly higher during acute hypoxia (3%) session (p<0.05). No differences in isoproterenol‐ and epinephrine‐stimulated lipolytic responses were observed in subcutaneous abdominal preadipocytes between normoxia and acute hypoxia (3%).

Conclusions

These preliminary results suggest that acute and continuous hypoxia strongly inhibits LPL activity and increases basal lipolysis. These observation supports the hypothesis that hypoxia may adversely affect adipose tissue storage functions, which could in turn contribute to ectopic fat storage, lipotoxicity and a greater prevalence of metabolic disorders in individual with excess adiposity.

Support or Funding Information

This study was supported by the Natural Sciences and Engineering Research Council of Canada as well as the University of Ottawa.