bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced endothelial permeability by free fatty acid due to lysosomal cathepsin B‐mediated activation of Nlrp3 inflammasome
Authors: Li Xiang , Wang Lei , Nguyen Thaison , Zhang Yang 
Published: November 2023

Recent studies have demonstrated that free fatty acid (FFA) palmitate induces insulin resistance leading to Type 2 diabetes via the activation of Nlrp3 inflammasome with a focus on inflammatory cells. Our findings demonstrated that enhanced vascular permeability in the myocardium in mice on the high fat diet (HFD) compared to mice on the normal diet, whereas such HFD‐induced impairment of vascular integrity was significantly attenuated in Nlrp3−/− mice. In microvascular endothelial cells (MVECs), FFA markedly induces Nlrp3 inflammasome complex formation leading to caspase‐1 activation and IL1β production. However, FFA has no effects on the transcription level of Nlrp3 inflammasome genes including Nlrp3, Asc, and caspase1. By fluorescence microscopy and flow cytometry, we observed that such FFA‐induced Nlrp3 inflammasome activated was accompanied by a reduction in interendothelial tight junction proteins ZO1/2. Such FFA‐induced decrease of ZO1/2 was also correlated with an increase in the permeability of endothelial monolayers treated with FFAs. Moreover, FFA‐induced alterations in ZO1/2 or permeability were significantly reversed by an inflammasome activity inhibitor, YVAD, or a high mobility group box 1 (HMGB1) activity inhibitor. Lastly, blockade of a lysosomal protease, cathepsin B, with its inhibitor Ca‐074Me significantly abolished FFA‐induced activation of Nlrp3 inflammasomes, down‐regulation of ZO1/2, and enhanced permeability in MVECs or their monolayers. Taken together, these data strongly suggest that activation of endothelial Nlrp3 inflammasomes due to increased FFAs produces HMGB1, which disrupts inter‐endothelial junctions and increase paracellular permeability of endothelium contributing to early onset of endothelial injury during metabolic disorders such as obesity.Support or Funding InformationNIH grants HL122769 and HL122937
Retinoic Acid Metabolites and Isomers have Differential Effects on Lipid Metabolism
Authors: Tripathy Sasmita , Han Chang Yeop , Isoherranen Nina 
Published: November 2023

All‐trans retinoic acid (atRA) and 13‐cisRA, and their metabolites 4‐oxo‐atRA and 4‐oxo‐13‐cisRA are found in circulation in humans as endogenous compound and following clinical doses of atRA and 13‐cisRA. Of these four compounds, only atRA is believed to be biologically important due to its high affinity towards RARs and activation of PPARδ. 13‐cisRA and 4‐oxo‐atRA also activate RARs but less is known about the 4‐oxo‐13‐cisRA. The activation of PPARs by 13‐cisRA and 4‐oxo‐metabolites has not been studied. The aim of this study was to determine whether atRA, 4‐oxo‐atRA, 13‐cisRA and 4‐oxo‐13‐cisRA have biological effects via RAR and other non‐RAR mediated pathways in human hepatocytes, HepG2 cells and adipocytes. At 1 μM concentration atRA, 4‐oxo‐atRA, 13‐cisRA and 4‐oxo‐13‐cisRA each significantly increased the mRNA levels of RARβ demonstrating that all four compounds activate RARs. atRA treatment increased expression of PPARδ targeted genes PGC1α, PGC1β and NRF1 and fatty acid oxidation, but had no effect on fatty acid and triglyceride synthesis genes SREBP1, MTTP, DGAT2 and PPARγ. In contrast, 13‐cisRA, 4‐oxo‐atRA, and 4‐oxo‐13‐cisRA had no effect on PPARδ targeted genes PGC1α, PGC1β and NRF1 and fatty acid oxidation, but significantly increased the expression of SREBP1, MTTP, DGAT2 and PPARγ. This suggests that 13‐cisRA, 4‐oxo‐atRA, and 4‐oxo‐13‐cisRA increase triglyceride synthesis and secretion. In addition, 4‐oxo‐atRA antagonized the induction of PGC1α, PGC1β and NRF1 and fatty acid oxidation by atRA demonstrating an independent activity of the 4‐oxo‐atRA when compared to atRA. Collectively, these results indicate that atRA promotes lipid catabolism via PPARδ, and 13‐cisRA and 4‐oxo‐RAs increase triglyceride synthesis, possibly via SREBP1 and PPARγ. Our findings regarding 13‐cisRA and its main circulatory metabolite 4‐oxo‐13‐cisRA explain the clinical data of increased incidence of hypertriglyceridemia and hypercholesterolemia following 13‐cisRA dosing. In addition, our data supports the mouse findings that atRA increases fatty acid oxidation in vivo. This study demonstrates that the 13‐cisRA isomer and 4‐oxo‐metabolites of RA found in circulation regulate lipid metabolism independent of atRA and RAR activation, and may play an important role in vivo in modulating lipid homeostasis in human tissues.Support or Funding InformationNIH R01 GM111772, R01 GM081569, R01 GM081569‐S1 and P30 DK035816
Inhibition of in vivo oxidation of native low‐density lipoprotein by N‐acetylcysteine is associated with decreased atherosclerosis
Authors: Liu Patrick Z , Cui Yuqi , Narasimhulu Chandrakala A , Li Xin , Xiao Yuan , He Guanglong , Liu Zhenguo , Parthasarathy Sampath 
Published: November 2023

BackgroundLow‐density lipoprotein (LDL) is not atherogenic itself. Oxidized LDL (ox‐LDL) is a potent oxidative agent, produces reactive oxidative species (ROS), and critical to the development of atherosclerosis. N‐acetylcysteine (NAC) has anti‐atherosclerotic effect. The mechanisms for its anti‐atherosclerotic actions remain largely unknown.ObjectiveThe present study aimed to determine if NAC could attenuate in vivo oxidation of LDL and inhibit atherosclerosis.Methods and resultsA single dose of human native LDL (50 μg) was injected intravenously into male C57BL/6 mice with and without NAC treatment. Human ox‐LDL levels in the blood were measured at different time after LDL injection using ELISA kit. Measurable serum level of human ox‐LDL was detected 30 min after the injection, reached the peak in 3 hours, started to decline afterwards, and became undetectable in 12 hours. NAC treatment significantly reduced serum ox‐LDL level with no detectable ox‐LDL in blood 6 hours after injection. No difference in ox‐LDL clearance was observed in NAC‐treated animals. Intracellular and extracellular ROS production was significantly increased in the animals treated with ox‐LDL and in hyperlipidemic LDL receptor knockout (LDLR−/−) mice that was completely prevented with NAC treatment. NAC also significantly reduced atherosclerotic plaque formation in hyperlipidemic LDLR−/− mice.ConclusionNative LDL was converted to ox‐LDL in vivo. NAC attenuated in vivo oxidation of LDL and ROS formation from ox‐LDL that might contribute to decreased atherosclerotic plaque formation in hyperlipidemia.Support or Funding InformationNIH
Investigation of the cardiovascular toxicity of oxidized cholesterols using the zebrafish as an animal model
Authors: Kurogi Katsuhisa 
Published: November 2023

Zebrafish has been used as an animal model for the investigation of cardiovascular disorders. One of the major pathogenic factors for cardiovascular diseases, such as atherosclerosis, is oxidized cholesterols. The cytotoxicity of oxidized cholesterols for the vascular endothelial cells has been proposed to be an etiological factor for atherosclerosis. However, it is unclear which type(s) of oxidized cholesterol may exhibit more serious pathogenic effects. In the current study, the zebrafish was employed to verify the cardiovascular toxicities of oxidized cholesterols.Zebrafish larvae at 48 hours postfertilization (hpf) were exposed to cholesterol and 10 different oxidized cholesterols by the soaking method or by microinjection into yolk sac. At every 24 hours during exposure up to 7 dpf, heart beats, blood flow, and mortality were examined. To investigate further the effect on the cardiovascular system, o‐dianisidine staining was performed.5,6α‐Epoxycholesterol and 5,6β‐epoxycholesterol, tested using both the soaking and the microinjection methods, showed the strongest cardiovascular toxicities, including lower heart beats, decreased blood flow, and higher mortality rate, among the oxidized cholesterols tested. Intriguingly, 22‐hydroxycholesterol exhibited a severe cardiovascular toxicity when tested using the soaking method, but not the microinjection method. Collectively, these results indicated that epoxycholesterols caused stronger cytotoxicity toward vascular endothelial cells than other oxidized cholesterols such as 7β‐hydroxycholesterol and 7‐ketocholesterol. Our results suggested that the zebrafish larva may serve as a convenient and useful model for investigating the pathogenicity and/or prevention of atherosclerosis caused by oxidized cholesterols.
Cyclosporine Ameliorates Reductions in Blood Pressure and Heart Rate Variability Caused by Endotoxemia in Rats: Modulation by Central PI3K/sGC/MAPKs Signaling
Authors: El‐Mas Mahmoud M , Sallam Marwa Y , Abdel‐Galil Abdel‐Galil A , El‐Gowilly Sahar M 
Published: November 2023

Cyclosporine A (CSA) improves survivability in endotoxemia and offsets the associated loss in vascular reactivity and hypotension. We tested the hypothesis that CSA abrogates the reduced blood pressure (BP) and cardiac autonomic dysfunction in endotoxic rats via the modulation of phosphoinositide‐3‐kinase (PI3K)/soluble guanylate cyclase (sGC)/mitogen activated protein kinases (MAPKs) cascade in central neurons. The effects of individual pharmacologic inhibition of these molecular substrates on the interaction of CSA with hemodynamic and cardiac autonomic responses, as reflected by heart rate variability (HRV) measurement, evoked by lipopolysaccharide (LPS) were evaluated. CSA (10 mg/kg i.v.) reversed the decreases in BP and increases in HR elicited by LPS (10 mg/kg i.v.) in conscious rats. Likewise, the decreases caused by LPS in time‐domain (standard deviation of beat‐to‐beat intervals, SDRR, and the root mean square of successive beat‐to‐beat differences in R‐R intervals, rMSSD) and frequency domain (total power and low and high‐frequency spectral powers) measures of HRV were abolished by CSA. These advantageous effects of CSA disappeared in endotoxic rats treated intracisternally (i.c.) with ODQ (sGC inhibitor) in contrast to no effect for wortmannin (PI3K inhibitor). We also report divergent roles for central MAPKs in the CSA‐LPS interaction. The reversal by CSA of the hypotensive effect of LPS disappeared in rats treated concurrently with i.c SB203580 (MAPKp38 inhibitor) or PD98059 (MAPKERK inhibitor), in contrast to little effect for SP600125 (MAPKJNK inhibitor). Alternatively, the amelioration by CSA of the LPS‐evoked reductions in HRV was abolished in presence of SP600125 or PD98059, but not SB203580. Collectively, the facilitation of central circuits of sGC/MAPKs contributes to CSA counteraction of the cardiovascular actions of endotoxemia. However, the identity of the MAPK isoform involved in the CSA‐LPS interaction largely depends on the specificity of the cardiovascular response.Support or Funding InformationSupported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895).
Central GABAA Receptors Provoke Inflammatory and Cardiovascular Consequences of Endotoxemia in Conscious Rats
Authors: El‐Mas Mahmoud M , Sallam Marwa Y , Abdel‐Galil Abdel‐Galil A , El‐Gowilly Sahar M 
Published: November 2023

Gamma‐aminobutyric acid (GABA), the principal brain inhibitory neurotransmitter, plays a key role in inflammatory and neurodegenerative disease. In this study, we tested the hypothesis that central GABAergic neurotransmission mediates the detrimental inflammatory, hemodynamic, and cardiac autonomic actions of endotoxemia. The effects of drugs that block GABA receptors or interfere with GABA uptake or degradation on blood pressure (BP), heart rate (HR), and HR variability (HRV) responses elicited by i.v. lipopolysaccharide (LPS) were assessed in conscious rats. The BP lowering effect of LPS (10 mg/kg) was blunted after intracisternal (i.c.) administration of bicuculline (GABAA receptor antagonist, 3 μg/rat) or saclofen (selective GABAB receptor antagonist, 50 μg/rat). By contrast, the concomitant LPS‐evoked tachycardia and decreases in time‐ and frequency domain indices of HRV, measures of cardiac autonomic control, were abolished upon treatment with bicuculline but not saclofen. The increases in serum tumor necrosis factor‐α and interleukin‐6 caused by LPS disappeared in rats treated concurrently with bicuculline or saclofen, whereas the LPS‐evoked increases in serum nitric oxide metabolites was reversed by bicuculline only. We also report that none of the effects of endotoxemia was altered in rats treated with i.c. tiagabine (GABA reuptake inhibitor, 100 μg/rat) or vigabatrin (GABA transaminase inhibitor, 200 μg//rat). These data suggest a major role for central GABAA receptors in the inflammatory and cardiovascular effects of endotoxemia.Support or Funding InformationSupported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895).
Doxorubicin Acutely Inhibits Lymph Flow in Rat Mesenteric Lymph Vessels
Authors: Stolarz Amanda J , Fletcher Terry W , Marecki John C , Sarimollaoglu Mustafa , Galanzha Ekaterina , Zharov Vladimir , Klimberg Suzanne , Rusch Nancy J 
Published: November 2023

Arm lymphedema is a major complication after surgery and/or radiation for breast cancer. This life‐long and disfiguring complication can cause serious medical problems including lymphangitis, cellulitis, and other recurrent infections. Doxorubicin (DOX) is often a central component in chemotherapy to prevent metastases or recurrence of breast cancer, but this cytotoxic drug increases the incidence and severity of lymphedema as an off‐target effect. Interestingly, the mechanisms by which DOX contributes to arm lymphedema have not been elucidated. We hypothesized that DOX directly suppresses lymphatic drainage by disrupting lymph vessel (LV) contractile function. Rats were anesthetized and one loop of mesentery was exposed on a heated microscope stage. High‐resolution optical imaging of rat mesenteric LVs in vivo revealed that continuous superfusion of DOX (0.05 to 10 μmol/L) progressively reduced positive volumetric lymph flow to ~30% of baseline (t=20 min, n=6) with an IC50 of 0.66 μmol/L. This effect of DOX was partially reversed by drug washout. Data were corrected for solvent and time controls (n=5–6). In a subsequent study, baseline measurements were recorded for 10 min, and then rats were injected via tail vein with a clinically relevant dose of DOX (10 mg/kg) or equivalent volume of saline. Mesenteric lymph flow was measured continuously for 2 hr post‐injection. At 0.75–1.25 hr post‐ injection, DOX significantly reduced positive volumetric flow to ~45% of baseline, whereas saline injection increased positive volumetric flow by ~35% from baseline (n=4–5). HPLC was used to determine corresponding DOX plasma levels during the measurement period. The average plasma concentration of DOX was 0.15±0.06 μmol/L (86 ± 34 ng/mL) and 0.10±0.02 μmol/L (59±13 ng/mL) at 1 and 2 hr after injection, respectively (n=5). These values were similar to DOX concentrations used in the superfusate studies and equivalent to plasma DOX concentrations achieved during chemotherapy in patients. Thus, clinically relevant concentrations of DOX appear to directly and acutely inhibit LV contractile function in vivo, and the resulting compromise of lymph flow represents a possible contributing mechanism of drug‐induced lymphedema.Support or Funding InformationFunded by Rho Chi‐AFPE Graduate Fellowship, UAMS Pilot Grant, and NIH/NCI R21CA131164 Grant.
The Effect of the CXCR1/2 Antagonist SCH257123 in a Mouse Model of Severe Asthma
Authors: Young Alan , Nath Puneeta , Sadler Emma , Thang Kevin , Russell Vince 
Published: November 2023

ObjectiveSevere asthma patient phenotypes are characterised as hard to control by low dose inhaled corticosteroids (ICS) and patients often require high doses of oral steroids for optimum symptom control. There is a high unmet medical need for novel therapies for the treatment of this disease. Neutrophilic airway inflammation has also been associated with corticosteroid insensitivity in the severe asthmatic population. In the current study, we have investigated the effect of a CXCR1/2 antagonist, SCH257123 (SCH), in a mouse severe asthma model known to be steroid insensitive.MethodsBALB/c mice were sensitised subcutaneously (s.c.) with house dust mite (HDM) emulsified in Complete Freunds adjuvant (CFA) and 14 days later animals were administered HDM via the intranasal (i.n.) route. Animals were dosed with SCH (10 or 30 mg/kg, p.o., 1hr pre‐ and 5 hrs post‐HDM challenge), Dexamethasone (DEX) 1 mg/kg, p.o., 1hr pre‐HDM challenge), or the appropriate vehicle. The mice were killed 24hrs post‐HDM challenge, and a bronchoalveolar lavage (BAL) was performed to determine total and differential cell numbers. Additionally, levels of IFNγ, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐10, IL‐12p70, KC/GRO and TNFα were measured in the BAL fluid.ResultsHDM‐challenge caused a significant increase in pulmonary inflammation, comprised of a mixed cell population of neutrophils and eosinophils. Pre‐treatment with 10 and 30mg/kg SCH only inhibited neutrophil cell numbers by a maximum of 30% in the BAL which was not significantly different when compared to the HDM‐vehicle treated group. As is consistent in this model, DEX did not significantly inhibit either the eosinophilic or neutrophilic inflammation. HDM‐challenge increased IFNγ, IL‐10 and TNFα levels in the BALF (p<0.0001) which were decreased by DEX (p<0.01) but not SCH. The levels of IL‐1β, IL‐4, IL‐6, and IL‐12p70 were also increased by HDM‐challenge, but were unaffected by both SCH and DEX. Of interest, BAL fluid levels of IL‐2, IL‐5 and KC/GRO were increased following HDM challenge, unaffected by DEX, but further increased by SCH (all p<0.001).In summary, the CFA/HDM severe asthma model has a cellular infiltration insensitive to both steroids and a CXCR1/2 antagonist, at doses known to work in mouse models of neutrophilic inflammation (Chapman et al, JPET 2007 322(2):486–93). Of interest was the increase in the levels of several mediators associated with neutrophil recruitment in the HDM‐challenged mice following SCH administration, which may have limited its efficacy. In the clinic, SCH has been shown to reduce neutrophil numbers in the sputum of severe asthmatics by approximately 36%, which is of similar magnitude to that seen in this model (Nair et al Clin Exp Allergy, 2012 42(7):1097–103).Support or Funding InformationFunded by Charles River
Preclinical Evaluation of Flt3 Ligand to Improve T Cell Adaptive Immune Response During Sepsis
Authors: Patil Naeem K , Bohannon Julia K , Luan Liming , Guo Yin , Sherwood Edward R 
Published: November 2023

BackgroundSepsis is a leading cause of death in the United States. Severely burned patients are highly susceptible to opportunistic infections and sepsis, leading to multi‐organ failure and death. Studies by Hotchkiss and colleagues show that T cell dysfunction significantly contributes to impaired immune response during sepsis in non‐burn subjects. However, the changes in T cell adaptive immunity after burn wound sepsis have not been investigated in detail. We used a clinically relevant mouse model of burn wound infection to assess adaptive immune system function. Furthermore, we evaluated the cytokine, Fms‐like tyrosine kinase‐3 ligand (Flt3L), to improve T cell adaptive immunity post burn wound sepsis. Flt3L is known to stimulate the expansion of hemopoietic progenitors, particularly dendritic cells.Methods8–10 weeks old male BALB/c mice were subjected to 35% total body surface area full thickness burn and the wound was infected with Pseudomonas aeruginosa on day 4 post‐burn. Flow cytometry was used to characterize the cell counts and phenotype of CD4+ and CD8+ T and dendritic cells in spleen and wound draining lymph nodes. Flt3L (10 μg, i.p.) was administered daily for 4 days following burn injury.ResultsWound infection led to severe sepsis in the mice as evidenced by detection of bacteria in blood and lungs, and multi‐organ injury (increased markers of liver and kidney injury) on day 2 after infection and mortality rate of approximately 80% by seven days. We observed a significant decline (>80%) in blood absolute lymphocyte count and CD4+ and CD8+ T cell counts in the spleen and burn‐draining lymph nodes of wound infected mice as compared to non‐infected burn mice on day 2 post infection. Flt3L significantly attenuated the infection induced decline in CD4+ and CD8+ T cell counts in the spleen. Wound infection also caused a decline in the CD11c+ dendritic cell numbers in lymph nodes. Flt3L stimulated the expansion of both the splenic and lymph node dendritic cells and protected against the dendritic cell loss in the lymph nodes. Furthermore, wound infection caused an increase in the expression of Programmed Death‐Ligand 1 (PD‐L1) expression on dendritic cells, F4/80+ macrophages and Ly6C+ inflammatory monocytes in the spleen. PD‐L1 is known to interact with its binding partner PD‐1 on T cell surface leading to T cell inhibition. Flt3L attenuated the increase in PD‐L1 expression and thus indirectly improves T cell function.ConclusionBurn wound sepsis causes significant adaptive immune system dysfunction and Flt3L represents a novel therapeutic cytokine to protect T cell function during sepsis and merits further investigation.Support or Funding InformationSupported by NIH R01 GM66885
DNA‐Dependent Protein Kinase Inhibition Prevents Manifestation of Acute Lung Injury in Mice, a Novel Target Associated with Th1 Type Disease
Authors: Pyakurel Kusma , Ghonim Mohamed , Ibba Salome , Tarhuni Abdelmetalab , Wang Jeffery , Boulares Hamid 
Published: November 2023

BackgroundThe acute respiratory distress syndrome (ARDS), a clinical complication of severe acute lung injury (ALI) in humans, is a leading cause of morbidity and mortality in critically ill patients. ALI is characterized by disruption of the lung alveolar‐capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudates which is explained by injury to both the vascular endothelium and alveolar epithelium. We previously reported that DNA‐dependent Protein Kinase (DNA‐PK) inhibition prevents in‐vitro differentiation of T‐helper 1(Th1) cells and Th1 specific cytokine production under Th1 skewing condition.Materials and methodsLPS‐induced ALI, Th1 type, murine disease model was used. Wild‐type or DNA‐PK heterzygous animals were anesthetized using i.p administration of xylazine/ketamine after which 10mg/kg LPS was intratracheally administered. Some group of mice received 5mg/kg of DNA‐PK inhibitor, NU 7441, 30 min after LPS administration. Cytospin and subsequent diff‐quick staining, myeloperoxidase assay, cytokine analysis and recording of dry and wet lung weights were performed.ResultsAdministration of DNA‐PK inhibitor, NU 7441, reduced production of inflammatory cytokines in the broncheoalveolar lavage fluid (BALF). Such effects correlated with a marked reduction in inflammatory cells such macrophages, neutrophils and lymphocytes in the lungs. Reduction in myeloperoxidase expression in lung homogenates in NU 7441 treated groups further confirmed the inhibition of neutrophils recruitment in the lungs. Furthermore, administration of NU 7441 reduced pulmonary edema in the mice, as measured by lung water retention volume (wet to dry weight ratio). DNA‐PK heterozygosity also prevented manifestation of ALI symptoms similar to those observed in NU 7441 treated animals.ConclusionDNA‐PK plays a critical role in ALI and, therefore, may constitute a novel therapeutic target for the treatment of the disease.Support or Funding InformationSupport: NIH; AHA; LCRC funds