Expression Of Inflammatory Molecules Research Articles

Endothelial IGFBP6 suppresses vascular inflammation and atherosclerosis.

Beyond dyslipidemia, inflammation contributes to the development of atherosclerosis. However, intrinsic factors that counteract vascular inflammation and atherosclerosis remain scarce. Here we identify insulin-like growth factor binding protein 6 (IGFBP6) as a homeostasis-associated molecule that restrains endothelial inflammation and atherosclerosis. IGFBP6 levels are significantly reduced in human atherosclerotic arteries and patient serum. Reduction of IGFBP6 in human endothelial cells by siRNA increases inflammatory molecule expression and monocyte adhesion. Conversely, pro-inflammatory effects mediated by disturbed flow (DF) and tumor necrosis factor (TNF) are reversed by IGFBP6 overexpression. Mechanistic investigations further reveal that IGFBP6 executes anti-inflammatory effects directly through the major vault protein (MVP)-c-Jun N-terminal kinase (JNK)/nuclear factor kappa B (NF-κB) signaling axis. Finally, IGFBP6-deficient mice show aggravated diet- and DF-induced atherosclerosis, whereas endothelial-cell-specific IGFBP6-overexpressing mice protect against atherosclerosis. Based on these findings, we propose that reduction of endothelial IGFBP6 is a predisposing factor in vascular inflammation and atherosclerosis, which can be therapeutically targeted.

Extracellular vesicles of ADSCs inhibit ischemic stroke-induced pyroptosis through Gbp3 regulation: A role for the NLRP3/GSDMD signaling pathway.

Mounting data indicates that extracellular vesicles (EVs) have the potential to improve the injury after a stroke. Pyroptosis is a recently identified kind of programmed cell death that initiates an inflammatory reaction. We aimed to ascertain the therapeutic implications and possible molecular processes of EVs obtained from adipose-derived stem cells (ADSCs) in inhibiting pyroptosis in ischemic stroke. The investigation employed transient middle cerebral artery occlusion (tMCAO) rat model and a BV2 of oxygen-glucose deprivation/reoxygenation (OGD/R) to ascertain ADSCs-EVs implications on inflammation and pyroptosis as assessed by neurological deficit scores, TTC staining, IHC, HE, CCK8, WB, ELISA, and immunofluorescence. RNA-Seq was performed on BV2 cells in the control, OGD/R, and OGD/R + ADSCs-EVs groups. Using sequencing data analysis, in the OGD/R group, we screened the upregulated genes regulated by EVs, overlapped with 74 pyroptosis-related genes, and identified Guanylate-binding protein 2 (Gbp2) and Guanylate-binding protein 3 (Gbp3) as key genes. Following the validation of the sequencing results in vivo and in vitro, Gbp3 was selected for further study. To test its regulatory effects on inflammation and pyroptosis, Gbp3 was knocked down and overexpressed in vitro. The administration of ADSCs-EVs resulted in a significant reduction in neurological involvement scores and reduced infarct volume in rats with tMCAO. They were also protective against BV-2 cells after OGD/R. In vivo and in vitro, ADSCs-EVs inhibited inflammatory response and pyroptosis after stroke. The outcomes of the RNA-Seq data analysis manifested that the protective implications of EVs after stroke are mediated by the modulation of inflammation-related mechanisms. Moreover, treatment with EVs led to a significant reduction in Gbp3 expression in post-ischemic brain tissue and cells. When Gbp3 was knocked down, the expression of inflammatory molecules and proteins linked to pyroptosis had a significant decline. When Gbp3 was overexpressed, the opposite results were obtained. ADSCs-EVs modulate the NLRP3/GSDMD signaling pathway via Gbp3 to attenuate the inflammatory response and reduce pyroptosis that occurs after stroke.

Synergistic effects of combined lead and iprodione exposure on P53 signaling-mediated hepatotoxicity, enterotoxicity and transgenerational toxicity in zebrafish.

Environmental heavy metal contamination, combined with inappropriate use of fungicides, has led to the co-existence of lead (Pb) and iprodione (IPR), presenting signification risks to ecosystems and human health. The toxic effects resulting from concurrent exposure to Pb and IPR, however, remain poorly understood. In the study, we conducted a comprehensive 60-day subchronic study to investigate the toxic effects on the liver and gut in parental male zebrafish through employing multi-omics analyses. We also explored the potential transgenerational toxicity to unexposed offspring embryos. The results demonstrated that exposure to both Pb and IPR exacerbated intestinal pathological damage, decreased the expression of intestinal tight junction molecules, and activated the expression of intestinal inflammatory molecules in the gut. Metabolic and microbial analyses, utilizing 16S rRNA sequencing and non-targeted metabolic profiling, revealed alterations in the intestinal flora structure and disruptions in metabolite synthesis. Notably, we observed a significant negative correlation between the abundance of the Lactobacillus genus and uracil synthesis. Furthermore, liver RNA-seq analysis identified a marked enrichment of the P53 signaling pathway, confirmed by the activation of P53-mediated apoptotic markers, which was consistent with the observed increase in inflammatory infiltration and pathological damage within the liver. Importantly, P53-mediated apoptosis and inflammatory responses were activated in offspring embryos, suggesting that long-term parental exposure to Pb and IPR may induce transgenerational toxicity, potentially impacting offspring health. Despite the identification of these molecular changes, the phenotypic effects remain to be elucidated. Future studies are necessary to evaluate the potential phenotypic changes in offspring to fully understand the long-term effects of Pb and IPR exposure. Overall, these findings enhance the understanding of the molecular mechanisms underlying the toxic effects of Pb and IPR and emphasize the importance of a comprehensive risk assessment of environmental pollutants.

RAD001-mediated mTOR targeting in human monocyte-derived dendritic cells shifts them toward an immunogenic phenotype.

Dendritic cells (DCs) are essential for promoting T lymphocyte responses since they are specialist antigen-presenting cells. In order to maintain tolerance or initiate immune responses, DCs must be activated in a balanced and regulated manner via diverse signaling pathways. By using a variety of pharmacological components, we can interfere with their different signaling pathways such as the mammalian target of rapamycin (mTOR) to appropriately modulate DC activity. In the current study, we administered RAD001 to DCs to examine the impact of mTOR inhibition on both the maturation stage and the expression of inflammatory and anti-inflammatory molecules in DCs. Pure monocytes were cultivated and stimulated with GM-CSF and IL-4 to generate immature DCs, which were then treated with RAD001. The phenotype of the DCs was determined by labeling surface markers and analyzing them using flow cytometry. Afterward, real-time PCR was carried out to evaluate the expression of inflammatory and anti-inflammatory genes. The administration of RAD001 to DCs led to a significant upregulation in the gene expression of inflammatory molecules such as IL-12, IL-1β, tumor necrosis factor (TNF)-α, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB). Conversely, RAD001 treatment resulted in a decrease in the gene expression of anti-inflammatory factors IL-10 and indoleamine 2,3-dioxygenase (IDO). However, the expression of differentiation and antigen presentation-related markers CD11c and human leukocyte antigens (HLA)-DR in RAD001-treated DCs was lower and higher compared to the control group that did not receive the treatment, respectively. Taken together, our findings indicated that RAD001 treatment of DCs can be a promising therapeutic approach for the generation of immunogenic DCs in order to barricade tumor growth. However, there is a need for further investigation to evaluate the impacts of mTOR inhibition by RAD001 in DCs on cellular immune responses in vitro and in vivo.

Abstract 4138745: Blockade of Mineralocorticoid Receptor by Esaxerenone Improves Insulin Sensitivity in Obese Mice

Background: Aldosterone regulates blood pressure and electrolyte balance. However, overactivation of the mineralocorticoid receptor (MR) and elevated aldosterone levels are implicated in the pathogenesis of cardiometabolic disorders, though the underlying mechanisms are not fully understood. In this study, we investigated whether a novel, non-steroidal selective MR blocker, esaxerenone, ameliorates the development of metabolic disorders in obese mice models and examined the underlying mechanism. Methods: Esaxerenone (3 mg/kg/day) was orally administered to high-fat diet (HFD)-fed C57BL/6J or normal chow-fed db/db mice. Metabolic parameters, tissue morphology, and effects of esaxerenone on insulin sensitivity were assessed. Gene expression and insulin signaling, determined by Akt phosphorylation, were examined using quantitative RT-PCR and western blotting, respectively. In in-vitro experiments, insulin targets cell lines such as 3T3-L1 adipocytes, HepG2 cells, and C2C12 myotubes were used. Insulin-responsive cells were treated with 10 nM or 100 nM esaxerenone or eplerenone for 30 minutes before 4-hour 1000 nM aldosterone stimulation. Insulin signaling was assessed 15 minutes after stimulation with 17 nM insulin. Results: Esaxerenone improved insulin sensitivity (P<0.05) without altering metabolic parameters in HFD-induced obese and db/db mice. Esaxerenone suppressed fat accumulation, adipocyte size, and liver lipid droplets compared with the vehicle-treated group (P<0.01). In adipose tissue, esaxerenone reduced macrophage infiltration (P<0.01) and the expression of inflammatory molecules (P<0.05). In in-vitro experiments, aldosterone significantly decreased the expression levels of PPARγ and adiponectin in 3T3-L1 adipocytes. Moreover, aldosterone attenuated insulin-induced Akt phosphorylation in 3T3-L1 adipocytes, HepG2 cells, and C2C12 myotubes in a dose-dependent manner (P<0.01). Pretreatment with esaxerenone reversed the effect of aldosterone, whereas eplerenone, an MR antagonist, did not show a similar effect at the same dose. Conclusion: Inhibition of MR by esaxerenone improved insulin sensitivity in HFD-induced obese and genetically diabetic mice. The reduction of inflammation and fat accumulation and enhancement of insulin signaling were suggested to be underlying mechanisms. Esaxerenone might be a beneficial therapeutic approach for managing metabolic disorders.

STING-mediated DNA damage response in diabetes-induced vascular complications

Abstract Background Hyperglycemia drives endothelial dysfunction, a key factor in the development of diabetes vascular complications. The stimulator of interferon genes (STING) signal, plays a crucial role in the immune system and is implicated in the pathogenesis of inflammatory diseases. Therefore we investigated the role of STING in endothelial dysfunction in streptozotocin (STZ)-induced diabetic mice. Methods and Results Diabetes was induced by single-dose administration of STZ in 8-week-old C57BL/6 and Sting-/- mice. STZ injection promoted the expression of STING and DNA damage markers in the aorta of C57BL/6 mice. Genetic deletion of STING ameliorated endothelial dysfunction as determined by vascular reactivity assay (P < 0.001) and increased aortic eNOS phosphorylation (P < 0.05) in STZ-injected mice. Stimulation of STING agonist (cGAMP), or mtDNA increased inflammatory molecules expression (e.g., VCAM1 and IFNB) and decreased eNOS phosphorylation in HUVECs. In ex-vivo experiments, cGAMP significantly impaired endothelial function which was ameliorated in the presence of an STING inhibitor or a neutralizing IFN-β antibody. Furthermore, the administration of STING inhibitor ameliorated endothelial dysfunction in STZ-induced diabetic mice (P < 0.01). Conclusion The DNA damage response regulated by STING aggravated diabetes-induced endothelial dysfunction. STING signaling may be a potential therapeutic target for diabetic vascular complications.

Expression of key molecules of the classical inflammation activation pathway in blood leukocytes of autistic children

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders, whose causes are currently not fully understood. Research suggests that inflammation and changes in immune functions may play an important role in the development of autism. Increased levels of proinflammatory cytokines in the brains of autistic children lead to negative regulation of synaptic plasticity, as well as impaired proliferation and differentiation of neurons through activation of the nuclear factor kappa B (NF-κB) signaling pathway. The purpose of the work is to analyze the levels of mRNA: TLR2, TLR4, MyD88, IκBα, NF-κB p50, NF-κB p65 in peripheral blood leukocytes of children in comparison with the severity of autism spectrum disorders. The study included 126 children aged from 3 to 13 years (the ratio of boys to girls was 4:1): 45 children with typical neurodevelopment, and 81 children with a clinically confirmed diagnosis of autism. According to the Childhood Autism Rating Scale, 51 children had mild to moderate ASD (CARS score: 29-36), and 30 children had severe autism (CARS score: 36-60). The expression of inflammatory signal transduction pathway molecules was determined in peripheral blood leukocytes using real-time polymerase chain reaction with SYBRGreen. To compare the samples, one-way ANOVA and Tukey’s test were used. It was found that in leukocytes of children with severe ASD, the expression of the adapter protein MyD88 and the p65 subunit of the nuclear transcription factor NF-κB was significantly reduced, and the expression of the NF-κB inhibitor, IκBα, was significantly increased, compared to the control group. In leukocytes of children with mild ASD, a decrease in NF-κB p65 expression was found at a trend level. When comparing groups of children with different severity of autism symptoms (mild/severe), no significant differences were found in the levels of mRNA of key signaling molecules of the classical inflammation activation pathway in blood leukocytes. Thus, in the blood leukocytes of children with severe ASD, suppression of the expression of key molecules of the classical inflammation activation pathway (NF-κB) is observed, which leads to a decrease in the expression of pro-inflammatory cytokines: IL-1β, IL-18 and IL-2, against the background of increased expression of key cytokine of Th1 cells – IFNγ.

Inflammatory Gene Expression in Livers Undergoing Ex Situ Normothermic Perfusion Is Attenuated by Leukocyte Removal From the Perfusate.

Ex situ normothermic perfusion (ESNP) is a method to evaluate and potentially recondition organs before transplantation. However, increased expression of inflammatory molecules, including by tissue-resident immune cells, may occur during the perfusion process, potentially negating the beneficial effects of perfusion. We used RNA sequencing to assess gene expression in 31 livers undergoing ESNP, including 23 donated after circulatory death (DCD) and 8 donated after brain death. In 7 DCD livers, a leucocyte filter was added to the circuit during perfusion. Biopsies were available for transcriptomic assessment in all cases at the start of perfusion and at varying time points postperfusion. During ESNP in DCD livers, we observed an increase in proinflammatory, profibrinolytic, and prorepair pathway genes. SERPINE1, encoding plasminogen activator inhibitor-1, was among the genes most significantly upregulated during perfusion in DCD livers, potentially promoting fibrin clot persistence in vasculature. We also found increased expression of monocyte and neutrophil recruiting chemokine and proinflammatory cytokine transcripts during ESNP, but several prorepair molecules, including thymic stromal lymphopoietin, were also upregulated. In both DCD and donation after brain death livers, interferon-gamma response genes were enriched, whereas oxidative phosphorylation genes decreased in organs with high perfusate alanine transaminase, a biomarker associated with adverse clinical outcomes. The inclusion of a leukocyte filter in the perfusion circuit mitigated the induction of inflammation/immune pathway genes during perfusion and was associated with enrichment in oxidative phosphorylation genes. Leukocyte removal during ESNP abrogates transcriptional changes that are associated with unfavorable clinical outcomes, potentially benefiting human livers undergoing ESNP.

The Role of TNF- α Inhibitors in The Management of Gout Arthritis and Its Comparison with Other Modalities

Background: Gout arthritis is caused by the deposition of monosodium urate crystals in the joints, which can cause significant pain, swelling, and recurring inflammation, particularly in the big toe. Inflammatory mediators are critical to the beginning and duration of the inflammatory response in gout arthritis. Gout is mediated by TNF-α, ILs, and the NLRP3 inflammasome, which promotes macrophages, monocytes, and neutrophils, leading to inflammatory reactions. TNF-α acts as a potent inflammatory mediator by stimulating the expression of other inflammatory cytokines, adhesion molecules, and matrix metalloproteinases (MMPs). TNF-α interacts with particular receptors and activates intracellular signaling pathways, leading to leukocyte recruitment, NLRP3 inflammasome activation, and the release of other inflammatory mediators. Objective: This article is a review article in which the researchers examine the current state of understanding regarding the role of TNF-α as a major inflammatory mediator in gout arthritis, deepen the understanding of TNF-α in gout inflammation, and explore its potential as a therapeutic target. Method: It involvedsystematically searching relevant databases, selecting studies based on inclusion criteria, and synthesizing findings to identify trends, gaps, and the current state of knowledge on the topic. Result: The results of this review article indicated that TNF-α plays a crucial role in joint inflammation by triggering the release of pro-inflammatory cytokines and stimulating neutrophil infiltration into the joints. Conclusion: Thus, Inhibiting TNF-α can reduce inflammation and improve symptoms in people with gout arthritis.

Evidence of Browning and Inflammation Features in Visceral Adipose Tissue of Women with Endometriosis

Patients with endometriosis tend to have a low body mass index, suggesting an inverse relationship between body fat and risk of disease. This is supported by evidence that miRNAs differentially expressed in endometriosis induce browning of pre-adipocytes in vitro. Thus, we hypothesize that endometriosis may underlie adipose tissue (AT) dysfunction and browning. Identify inflammation and browning processes in AT collected from endometriosis patients. Visceral and subcutaneous AT samples were obtained during endometriosis (n = 32) or uterine myoma (n = 14; controls) surgery. Blood catecholamines were determined by high-performance liquid chromatography while IL-6 and TGF-β levels were quantified by ELISA. Adipocyte cross-sectional areas were analyzed in H&E-stained sections by computer-assisted morphometry. Macrophages (F4/80; Galectin-3) and browning activation (UCP-1; PGC-1α) in tissues were identified by dual label immunofluorescence. Expression of inflammatory (IL-6; MCP-1; Galectin-3; CD206; TIMP1; TGF-β) and browning-related (UCP-1; PGC-1α; DIO2; CITED1; CIDEA; TMEM26; TBX1; PRDM16; PPAR-γ) molecules in AT were assessed by RT-PCR and Western blotting. Compared to controls, patients presented smaller adipocytes, especially in VAT, and lower norepinephrine levels. Serum IL-6, but not TGF-β, was increased in patients. UCP-1, PGC-1α, IL-6, and MCP-1 were upregulated in VAT from endometriosis women, which also evidenced a reduction of CD206, relative to controls. However, no differences were found in mRNA expression of IL-6, TIMP1, and TGF-β nor Galectin-3 protein levels. In SAT, protein expression remained unchanged between patients and controls. Our findings support an endometriosis' role as a pro-catabolic state along with local signals of VAT browning and inflammation.

Mouse hepatitis virus JHMV I protein is required for maximal virulence.

Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.

Flaxseed Oil Alleviates Trimethyltin-Induced Cell Injury and Inhibits the Pro-Inflammatory Activation of Astrocytes in the Hippocampus of Female Rats.

Exposure to the neurotoxin trimethyltin (TMT) selectively induces hippocampal neuronal injury and astrocyte activation accompanied with resultant neuroinflammation, which causes severe behavioral, cognitive, and memory impairment. A large body of evidence suggests that flaxseed oil (FSO), as one of the richest sources of essential omega-3 fatty acids, i.e., α-linolenic acids (ALA), displays neuroprotective properties. Here, we report the preventive effects of dietary FSO treatment in a rat model of TMT intoxication. The administration of FSO (1 mL/kg, orally) before and over the course of TMT intoxication (a single dose, 8 mg/kg, i.p.) reduced hippocampal cell death, prevented the activation of astrocytes, and inhibited their polarization toward a pro-inflammatory/neurotoxic phenotype. The underlying protective mechanism was delineated through the selective upregulation of BDNF and PI3K/Akt and the suppression of ERK activation in the hippocampus. Pretreatment with FSO reduced cell death and efficiently suppressed the expression of inflammatory molecules. These beneficial effects were accompanied by an increased intrahippocampal content of n-3 fatty acids. In vitro, ALA pretreatment prevented the TMT-induced polarization of cultured astrocytes towards the pro-inflammatory spectrum. Together, these findings support the beneficial neuroprotective properties of FSO/ALA against TMT-induced neurodegeneration and accompanied inflammation and hint at a promising preventive use of FSO in hippocampal degeneration and dysfunction.

Synergistic risk in the gut and liver: Insights into the toxic mechanisms and molecular interactions of combined exposure to triazophos and fenvalerate in zebrafish

The simultaneous or sequential application of pesticides such as triazophos (TRI) and fenvalerate (FEN) in agriculture results in their residues co-existing in the environments. However, the impact of co-exposure to TRI and FEN on the gut-liver axis, along with the underlying mechanisms, remains unclear. Our results showed that exposure to FEN (96 h-LC50 value of 0.096 mg a.i. L−1) was more toxic to adult zebrafish compared to TRI (96 h-LC50 value of 6.75 mg a.i. L−1). Furthermore, the study aimed to reveal the toxic potencies of individual and combined exposure to TRI and FEN on the liver-gut axis in zebrafish (Danio rerio). Our results also indicated that pesticide exposure decreased tight junction molecule expression and increased intestinal inflammatory molecule expression in D. rerio, with co-exposure demonstrating enhanced toxicity. Co-exposure altered gut flora structure and species abundance. RNA-Seq sequencing revealed changes in liver gene expressions, particularly enrichment of P53 signaling. Molecular docking demonstrated FEN's stronger binding to P53 and Caspase3, correlating with its higher toxicity. Liver pathology confirmed exacerbated liver damage by individual and co-exposures, with co-exposure inducing more severe liver injury. qPCR results showed increased pro-apoptotic gene expression and decreased anti-apoptotic gene expression, with co-exposure exhibiting an interactive effect. Overall, this study identifies specific targets and pathways influenced by these pesticides, revealing toxicity mechanisms involving the gut-liver axis, which is crucial for environmental risk assessment of pesticide mixtures.

A mild increase in nutrient signaling to mTORC1 in mice leads to parenchymal damage, myeloid inflammation and shortened lifespan.

The mechanistic target of rapamycin complex 1 controls cellular anabolism in response to growth factor signaling and to nutrient sufficiency signaled through the Rag GTPases. Inhibition of mTOR reproducibly extends longevity across eukaryotes. Here we report that mice that endogenously express active mutant variants of RagC exhibit multiple features of parenchymal damage that include senescence, expression of inflammatory molecules, increased myeloid inflammation with extensive features of inflammaging and a ~30% reduction in lifespan. Through bone marrow transplantation experiments, we show that myeloid cells are abnormally activated by signals emanating from dysfunctional RagC-mutant parenchyma, causing neutrophil extravasation that inflicts additional inflammatory damage. Therapeutic suppression of myeloid inflammation in aged RagC-mutant mice attenuates parenchymal damage and extends survival. Together, our findings link mildly increased nutrient signaling to limited lifespan in mammals, and support a two-component process of parenchymal damage and myeloid inflammation that together precipitate a time-dependent organ deterioration that limits longevity.

Nuclear factor κB overactivation in the intervertebral disc leads to macrophage recruitment and severe disc degeneration.

Persistent inflammation has been associated with severe disc degeneration (DD). This study investigated the effect of prolonged nuclear factor κB (NF-κB) activation in DD. Using an inducible mouse model, we genetically targeted cells expressing aggrecan, a primary component of the disc extra cellular matrix, for activation of the canonical NF-κB pathway. Prolonged NF-κB activation led to severe structural degeneration accompanied by increases in gene expression of inflammatory molecules (Il1b, Cox2, Il6, and Nos2), chemokines (Mcp1 and Mif), and catabolic enzymes (Mmp3, Mmp9, and Adamts4). Increased recruitment of proinflammatory (F4/80+,CD38+) and inflammatory resolving (F4/80+,CD206+) macrophages was observed within caudal discs. We found that the secretome of inflamed caudal disc cells increased macrophage migration and inflammatory activation. Lumbar discs did not exhibit phenotypic changes, suggestive of regional spinal differences in response to inflammatory genetic overactivation. Results suggest prolonged NF-κB activation can induce severe DD through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and activation of macrophage cell populations.

Effect of Lupeol on the Expression of Neuro Inflammatory Signalling Molecules in Brain Tissues of High Fat Diet and Sucrose Fed Type-2 Diabetic Rats

Lupeol has anti- inflammatory, antimicrobial, anti-protozoal, anti-proliferative, anti-invasion, anti-angiogenic and cholesterol-lowering properties. However, the mechanisms underlying the effect of lupeol on neuroinflammatory signalling molecules have not yet been identified. The study was designed to study the effect of lupeol on the expression of inflammatory signalling molecules in brain tissue of high-fat diet and sucrose-fed type-2 diabetic rats. Adult male albino rats of Wistar 150–180 days old with 180–200 g body weight (b.wt) were divided into four groups of six rats each. Group I: Control (vehicle-treated): Group II: High fat diet-induced type-2 diabetic rats; Group III: Type-2 diabetic rats treated with lupeol (25 mg/kg b.wt/day) orally for 30 days and Group IV: Type-2 diabetic rats treated with metformin (50 mg/kg, b.wt/day orally for 30 days. After 30 days of treatment, the animals were anaesthetized, and brain tissue was dissected and used for the assessment of mRNA expression analysis. Type-2 diabetic animals showed a significant increase (p<0.05) in TNF-α and IL-6 mRNA levels in brain tissue in high-fat diet-induced type-2 diabetic animals. However, lupeol treatment, effectively reduced (p<0.05) the neuroinflammatory signaling molecules (TNF- α and IL-6 mRNA) showing that lupeol has significant role over the control of neuroinflammatory signaling. Our present findings clearly show that lupeol has a significant role in reducing neuroinflammation via the downregulation of TNF-α and IL-6 in brain tissues and hence, lupeol can be a potential natural drug for the treatment of diabetic neuropathy.

Targeting the High-Density Lipoprotein Proteome for the Treatment of Post-Acute Sequelae of SARS-CoV-2.

Here, we target the high-density lipoprotein (HDL) proteome in a case series of 16 patients with post-COVID-19 symptoms treated with HMG-Co-A reductase inhibitors (statin) plus angiotensin II type 1 receptor blockers (ARBs) for 6 weeks. Patients suffering from persistent symptoms (post-acute sequelae) after serologically confirmed SARS-CoV-2 infection (post-COVID-19 syndrome, PCS, n = 8) or following SARS-CoV-2 vaccination (PVS, n = 8) were included. Asymptomatic subjects with corresponding serological findings served as healthy controls (n = 8/8). HDL was isolated using dextran sulfate precipitation and the HDL proteome of all study participants was analyzed quantitatively by mass spectrometry. Clinical symptoms were assessed using questionnaires before and after therapy. The inflammatory potential of the patients' HDL proteome was addressed in human endothelial cells. The HDL proteome of patients with PCS and PVS showed no significant differences; however, compared to controls, the HDL from PVS/PCS patients displayed significant alterations involving hemoglobin, cytoskeletal proteins (MYL6, TLN1, PARVB, TPM4, FLNA), and amyloid precursor protein. Gene Ontology Biological Process (GOBP) enrichment analysis identified hemostasis, peptidase, and lipoprotein regulation pathways to be involved. Treatment of PVS/PCS patients with statins plus ARBs improved the patients' clinical symptoms. After therapy, three proteins were significantly increased (FAM3C, AT6AP2, ADAM10; FDR < 0.05) in the HDL proteome from patients with PVS/PCS. Exposure of human endothelial cells with the HDL proteome from treated PVS/PCS patients revealed reduced inflammatory cytokine and adhesion molecule expression. Thus, HDL proteome analysis from PVS/PCS patients enables a deeper insight into the underlying disease mechanisms, pointing to significant involvement in metabolic and signaling disturbances. Treatment with statins plus ARBs improved clinical symptoms and reduced the inflammatory potential of the HDL proteome. These observations may guide future therapeutic strategies for PVS/PCS patients.

Inflammation-induced TRPV4 channels exacerbate blood–brain barrier dysfunction in multiple sclerosis

BackgroundBlood–brain barrier (BBB) dysfunction and immune cell migration into the central nervous system (CNS) are pathogenic drivers of multiple sclerosis (MS). Ways to reinstate BBB function and subsequently limit neuroinflammation present promising strategies to restrict disease progression. However, to date, the molecular players directing BBB impairment in MS remain poorly understood. One suggested candidate to impact BBB function is the transient receptor potential vanilloid-type 4 ion channel (TRPV4), but its specific role in MS pathogenesis remains unclear. Here, we investigated the role of TRPV4 in BBB dysfunction in MS.Main textIn human post-mortem MS brain tissue, we observed a region-specific increase in endothelial TRPV4 expression around mixed active/inactive lesions, which coincided with perivascular microglia enrichment in the same area. Using in vitro models, we identified that microglia-derived tumor necrosis factor-α (TNFα) induced brain endothelial TRPV4 expression. Also, we found that TRPV4 levels influenced brain endothelial barrier formation via expression of the brain endothelial tight junction molecule claudin-5. In contrast, during an inflammatory insult, TRPV4 promoted a pathological endothelial molecular signature, as evidenced by enhanced expression of inflammatory mediators and cell adhesion molecules. Moreover, TRPV4 activity mediated T cell extravasation across the brain endothelium.ConclusionCollectively, our findings suggest a novel role for endothelial TRPV4 in MS, in which enhanced expression contributes to MS pathogenesis by driving BBB dysfunction and immune cell migration.

Abstract 6791: Investigation of sex hormone effects in an organ chip model of endothelial barrier dysfunction in the triple negative breast cancer microenvironment

Abstract Inflammation and pathological vasculature are common features of the tumor microenvironment (TME). Sex hormones potently modulate vascular physiology and immune cell functions, and there are established correlations with the outcomes of triple negative (TNBC) despite the defining feature of receptor negativity. Circulating estrogens and androgens have been hypothesized as drivers of TNBC through the actions exerted by receptor-positive cells in the TME. Here, we present an organ chip model of the TNBC microenvironment for quantifying the impact of configurable TME variables such as the ECM composition and the relative densities and phenotypes of tumor cells, cancer associated fibroblasts, and myeloid cells on endothelial barrier function. After establishing baselines of vascular permeability in the presence and absence of TNBC cells, we tested the effect of estradiol, estrone, and dihydrotestosterone on barrier permeability. Multilayer polydimethylsiloxane (PDMS) organ chips were fabricated using standard soft lithography. The layers of the device were separated with semi-permeable membranes that allow exchange of soluble factors. Once assembled, the upper channel was loaded with female (XX) human umbilical vein endothelial cells. Collagen hydrogels were injected into the tissue layer and the devices were cultured on a rocker to induce endothelial barrier alignment via application of an average shear stress of 5 dynes/cm2. We optimized rocking conditions by assessing endothelial layer alignment, junctional organization, and permeability. TNBC cells and XX mammary carcinoma fibroblasts were added to the hydrogel to model the effects of TME-derived signals on endothelial barrier permeability. 40 kDa FITC- Dextran was chosen to assay permeability as it can show leakage due to active disruption of the barrier. Staining for F-actin and ZO-1 allowed for quantification of junctional organization. Sex hormone modulation of proinflammatory gene expression was assessed by RT-qPCR. The presence of TNBC cells significantly increased transport of 40kDa FITC-Dextran across the endothelial barrier, disrupted intercellular junction quality, and induced the expression of inflammatory adhesion molecules including ICAM-1. Our results demonstrate that sex hormones modulate TNBC-associated changes in endothelial cell bioenergetics, junctional organization, and macromolecular barrier permeability. Ongoing work will investigate the effects of age-matched XX monocyte derived macrophages on barrier function in the model. This organ chip platform provides an assay for measuring altered vascular inflammation and permeability in the TNBC microenvironment and may provide a venue for investigating vascular-normalizing effects of various candidate therapeutics. Citation Format: Delia Carlino, Ashley Martier, Yasmin Maurice, G. Wills Kpeli, Matthew Burow, Mark Mondrinos. Investigation of sex hormone effects in an organ chip model of endothelial barrier dysfunction in the triple negative breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6791.

Decreased proteasome function increases oxidative stress in the early stage of pressure ulcer development

The aging process in the elderly results in heightened skin fragility associated with various disorders, including pressure ulcers (PUs). Despite the high incidence of PUs in the elderly population, there is a limited body of research specifically examining the impact of aging on the development of pressure ulcers. Therefore, investigating age-related physiological abnormalities is essential to elucidate the pathogenesis of PUs. Ischemia-reperfusion (I/R) injury and the subsequent oxidative stress caused by reactive oxygen species (ROS) play essential roles in the early stage of PUs. In this study, we used a mouse model of proteasomal dysfunction with an age-related phenotype to examine the role of proteasome activity in cutaneous I/R injury in vivo. Decreased proteasome function did not affect the expression of inflammatory cytokines and adhesion molecules in the I/R area in transgenic mice; however, proteasome inhibition increased oxidative stress that was not attenuated by activation of the oxidative stress response mediated by NF-E2-related factor 2 (Nrf2). In dermal fibroblasts (FCs) subjected to hypoxia-reoxygenation (H/R), proteasome inhibition induced oxidative stress and ROS production, and Nrf2 activation did not adequately upregulate antioxidant enzyme expression, possibly leading to antioxidant/oxidant imbalance. The free radical scavenger edaravone had protective effects against I/R injury in vivo and decreased oxidative stress in FCs treated with a proteasome inhibitor and subjected to H/R in vitro. The results suggest that the age-related decline in proteasome activity promotes cutaneous I/R injury-induced oxidative stress, and free radical scavengers may exert protective effects by preventing oxidative stress in the early stage of PUs.