Inflammatory Vascular Injury Research Articles

Repeated low-intensity noise exposure exacerbates age-related hearing loss via RAGE signaling pathway.

Repeated low-intensity noise exposure is prevalent in industrialized societies. It has long been considered risk-free until recent evidence suggests that the temporary threshold shift (TTS) induced by such exposure might be a high-risk factor for hearing loss. This study was conducted to further investigate the manner in which repeated low-intensity noise exposure contributed to hearing damage. Two-month-old C57BL/6 J mice were exposed to white noise at 96 dB SPL for 8 h per day over 7 days to induce TTS. Auditory brainstem response (ABR) was monitored to assess changes in hearing thresholds, tracking the effects of noise exposure until the mice reached 12 months of age. Our results indicated that noise-exposed mice exhibited accelerated age-related hearing loss spanning from high to low frequencies. Proteomics analysis revealed an upregulation in the receptor for the advanced glycation end-products (RAGE) signaling pathway, which was associated with an activated inflammatory response, vascular injury, and mitochondrial and synaptic dysfunction. Further analysis confirmed increased levels of inflammatory cytokines in the cochlear lymph fluid and significant macrophages infiltration in the cochlear lateral wall, accompanied by hyperpermeability of the blood-labyrinth barrier. Additionally, degenerated mitochondria in the outer hair cells and decreased synaptic ribbons in the inner hair cells were also observed. These pathological changes indicated that noise exposure damages the cochlear cellular components, increasing the cochlear susceptibility to age-related stress. Our findings suggest that TTS caused by repeated low-intensity noise exposure correlates with a severe sensorineural hearing loss during aging; targeting the RAGE signaling pathway could be a promising strategy to mitigate damage from low-intensity noise and slow down the progression of age-related hearing loss.

Abstract 4139195: Tipping Point Analysis Identifies C-terminal src kinase-Collagen 22 Regulation by Inflammation in the Pathogenesis of Early-Stage Pulmonary Arterial Hypertension

Inflammatory injury to pulmonary artery endothelial cells (PAECs) is a key pathogenetic event that drives fibrotic vascular remodeling in pulmonary arterial hypertension (PAH), a progressive disease that lacks early stage-specific therapeutic targets. In an inflammatory rodent model of PAH, we established early-stage disease based on abnormalities in right ventricular (RV) afterload, pulmonary vascular resistance, and RV cardiomyocyte phenotype that were evident without substantial elevation in pulmonary artery pressure. We performed RNA-Seq on PAEC mRNA isolated ex vivo from control, early-stage, and advanced-stage PAH rats and deployed a ‘tipping point’ methodology to identify key pulmonary endothelial protein-protein interactions (PPIs) that regulate the transition from control to early-stage PAH in silico . Building on prior data on the intersection between glucose and proline signaling in fibrosis, we interrogated our early-stage PAH network for convergent PPIs involving metabolism and fibrosis endotypes. C-terminal src kinase (Csk), a conserved inhibitor of Src, was predicted to regulate PAEC fibrosis and was increased in early-stage PAH. In human PAECs, inflammatory stimuli affected markers of Src activation as well as collagen 22 (Col22A1). We observed that pulmonary arterial Col22A1 expression increased progressively across all PAH stages in vivo and that arterial Col22A1 expression is upregulated in human PAH. Further, although inflammation upregulated total Csk mRNA and protein expression in HPAECs, it decreased phosphorylation of Src at tyrosine 527 (pTyr527Src), a Csk-dependent brake on Src kinase activity. Targeting Src through small molecule inhibition or adenovirus overexpression of Csk to increase pTyr527Src, in turn, attenuated inflammation-induced Col22A1 in vitro . Taken together, these data suggest that acquired inhibition of Csk is a novel molecular mechanism by which inflammation induces pulmonary endothelial dysfunction, Src activation, and fibrosis. In early-stage PAH, disrupted Csk inhibition of Src may induce transcriptional upregulation of Csk as a negative feedback response to Src activation. These data suggest that the Csk-Src-Col22A1 axis is a novel target for advancing early-stage therapeutics in patients with PAH subtypes characterized by inflammatory vascular injury.

Correlations between coagulation abnormalities and inflammatory markers in trauma-induced coagulopathy.

In multiple trauma patients, the occurrence of trauma-induced coagulopathy (TIC) is closely associated with tissue damage and coagulation function abnormalities in the pathophysiological process. This study established a multiple trauma and shock model in Sprague-Dawley (SD) rats and comprehensively utilized histological staining and radiographic imaging techniques to observe injuries in the intestine, liver, skeletal muscles, and bones. Monitoring activated partial thromboplastin time (APTT), platelet (PLT) count, respiratory rate, blood pressure, and other physiological indicators revealed time-dependent alterations in coagulation function and physiological indicators. Enzyme-linked immunosorbent assay (ELISA) measurements of inflammatory factors Tumor Necrosis Factor-alpha (TNF-α), Interleukin-6 (IL-6) and vascular endothelial injury marker (Syndecan-1) were also conducted. Experimental results demonstrated significant changes in tissue structure after multiple traumas, although widespread necrosis or hemorrhagic lesions were not observed. There were time-dependent alterations in coagulation function and physiological indicators. ELISA measurements showed a strong positive correlation between the significant decrease in PLT count and the increase in TNF-α and IL-6 concentrations. The study provides crucial information for the early diagnosis and treatment of TIC. The findings suggest that structured monitoring of coagulation and inflammatory indicators can help in understanding the pathophysiological changes and aid in the management of TIC in multiple trauma patients.

Effect of Dendrobii Officinalis Caulis water extract on hyperviscosity rats based on TLR4/NF-κB/ICAM-1 signaling pathway

This study aims to reveal the effect and mechanism of Dendrobii Officinalis Caulis water extract on the rat model of hyperviscosity induced by a high-sugar, high-salt, and high-fat diet. Thirty-six male SD rats were randomized into normal, model, Compound Danshen Tablets(0.5 g·kg~(-1)), and low-, medium-, and high-dose(0.25, 0.5, and 1.0 g·kg~(-1), respectively) Dendrobii Officinalis Caulis water extract groups. Except the normal group, the remaining groups were fed with a high-salt and high-fat diet and received regular gavage of sucrose solution(20 g·kg~(-1)) for 12 consecutive weeks. The modeling was accompanied by gavage of corresponding drugs. During the experimental period, general signs(such as grip strength, automatic activity, vertigo time, and pain threshold), blood rheology, and blood flow in the tail microcirculation were measured. Flow cytometry was employed to detect the positive cells of intercellular cell adhesion molecule 1(ICAM-1/CD54) and vascular cell adhesion molecule 1(VCAM-1/CD106) in the peripheral blood and the Ca~(2+) fluorescence intensity. Enzyme-linked immunosorbent assay was employed to measure the levels of ICAM-1, VCAM-1, platelet endothelial cell adhesion molecule 1(PECAM-1/CD31), endothelin 1(ET-1), thromboxane A2(TXA2), prostaglandin I2(PGI2), nitric oxide(NO), interleukin-1β(IL-1β), C-reactive protein(CRP), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), and lipopolysaccharide(LPS). Hematoxylin-eosin, Masson, and Gomori's aldehyde-fuchsin staining were used to observe the pathological and morphological changes in rat colon and aorta. Fluorescence quantitative PCR was conducted to determine the mRNA levels of Toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88), nuclear factor-kappaB(NF-κB), ICAM-1, and VCAM-1 in the aortic tissue. Western blot was employed to determine the protein levels of occludin and claudin-1 in the colon tissue and ICAM-1, VCAM-1, and IL-6 in the aortic tissue. Immunohistochemistry and immunofluorescence were employed to detect the expression of TLR4 and NF-κB in the aortic tissue. The results showed that Dendrobii Officinalis Caulis water extract significantly improved the general signs, reduced the whole blood viscosity, whole blood reducing viscosity, and whole blood flow resistance, increased the blood flow of tail microcirculation, reduced the rates of CD54, CD106-positive cells in peripheral blood, and attenuated the blood flow obstruction in the rat model of hyperviscosity. The water extract significantly lowered the serum levels of ICAM-1, VCAM-1, PECAM-1, and ET-1, elevated the PGI2 and NO level, and alleviated vascular endothelial injury. In addition, the water extract decreased the content of IL-1β, TNF-α, IL-6, and CRP in the serum and reduced the vascular inflammatory injury. Moreover, the Dendrobii Officinalis Caulis water extract improved the morphology of colon and aortic tissues, promoted the expression of occludin and claudin-1 in the colon, and down-regulated the mRNA levels of TLR4, MyD88, NF-κB, ICAM-1, and VCAM-1 as well as the protein levels of TLR4, NF-κB, and IL-6 in the aortic tissue. In conclusion, the results suggest that the Dendrobii Officinalis Caulis water extract may regulate the endothelial inflammatory injury via the TLR4/NF-κB/ICAM-1 pathway, thereby ameliorating hyperviscosity.

Endothelial RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of β-catenin and ILK signaling pathways after inflammatory vascular injury

Endothelial repair is essential for restoring tissue fluid homeostasis following lung injury. R-spondin3 (RSPO3), a secreted protein mainly produced by endothelial cells (ECs), has shown its protective effect on endothelium. However, the specific mechanisms remain unknown. To explore whether and how RSPO3 regulates endothelial regeneration after inflammatory vascular injury, the role of RSPO3 in sepsis-induced pulmonary endothelial injury was investigated in EC-specific RSPO3 knockdown, inducible EC-specific RSPO3 deletion mice, EC-specific RSPO3 overexpression mice, systemic RSPO3-administration mice, in isolated mouse lung vascular endothelial cells (MLVECs), and in plasma from septic patients. Here we show that plasma RSPO3 levels are decreased in septic patients and correlated with endothelial injury markers and PaO2/FiO2 index. Both pulmonary EC-specific knockdown of RSPO3 and inducible EC-specific RSPO3 deletion inhibit pulmonary ECs proliferation and exacerbate ECs injury, whereas intra-pulmonary EC-specific RSPO3 overexpression promotes endothelial recovery and attenuates ECs injury during endotoxemia. We show that RSPO3 mediates pulmonary endothelial regeneration by a LGR4-dependent manner. Except for β-catenin, integrin-linked kinase (ILK)/Akt is also identified as a novel downstream effector of RSPO3/LGR4 signaling. These results conclude that EC-derived RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of β-catenin and ILK signaling pathways after inflammatory vascular injury.

M2 Macrophage‐Derived Exosomes Inhibiting Neutrophil Extracellular Traps for Ischemic Stroke Therapy

AbstractNeutrophil extracellular traps (NETs) are highly associated with inflammatory response and vascular injury after ischemic stroke. As the primary degrader of NETs, DNase 1 is limited by easy deactivation and low efficiency of crossing the blood‐brain barrier (BBB). Here, CD206+ M2‐like macrophages‐derived exosomal (M2exo) system is developed for DNase 1 delivery to achieve enhanced ischemic stroke therapy. The nanoplatform can cross the BBB through transcytosis of exosomes, subsequently clearing NETs by DNase 1 to inhibit inflammatory factors release and prevent vascular injury. Moreover, M2exo induces the polarization of M1 microglia to M2 phenotype, alleviating neuroinflammation via producing anti‐inflammatory cytokines. This nanoplatform exhibits significant efficiency in reducing brain infarct area, improving long‐term neurologic outcomes, and promoting BBB remodeling. The mechanism for the synergistic effect from M2exo and DNase 1 is unveiled at the genetic level through transcriptome analysis. This work provides a paradigm for improving the effectiveness of ischemic stroke therapy.

The mechanism of intravenous immunoglobulin (IVIG) in vascular endothelial injury in kawasaki disease based on neutrophil extracellular traps

This study explores the role of Neutrophil extracellular traps (NETs) in kawasaki disease (KD)-induced vascular inflammatory injury and the protective effect and mechanism of IVIG on vascular endothelial damage. A total of 37 children diagnosed with KD and admitted to Dongguan maternal and Child Health Care Hospital between March 2020 and June 2022 were included in the study. The children were divided into different groups based on their treatment and the presence or absence of coronary artery damage: IVIG treatment group (KDIVIG group), subgroup with coronary artery damage (KDCAL group), and subgroup without coronary artery damage (KDNCAL group), and a Control group consisting of 9 children who underwent surgical treatment. Flow cytometry was used to detect the proportion of neutrophils and the number of NETs in peripheral blood. It was found that the proportion of neutrophils in the peripheral blood of the acute KD group significantly increased with the presence of NETs. RT-PCR and ELISA detection showed that the levels of inflammatory factors TNF-α, IL-6 and CitH3 were abnormally elevated in this acute KD group, and the CAL group exhibited higher proportions of neutrophils and NETs-related markers compared to the NCAL group, while the IVIG group had significantly decreased proportions of neutrophils. PMA culture of neutrophils induced an increase expression of NETs marker protein, the content of NETs cfDNA increased. NETs culture could promote the secretion of TNF-α, whereas IVIG cultured cells inhibited the secretion of TNF-α. Finally, HCAEC cells were cultured with different levels of TNF-α, and the function of HCAEC cells was assessed using CCK8, scratch assay and flow cytometry. The high expression of TNF-α in the NETs group inhibited the proliferation and migration of HUVEC cells and enhanced their apoptosis. In contrast, the IVIG culture group exhibited similar effects to the TNF-α monoclonal antibody, as it inhibited HUVEC cell apoptosis and improved their viability by reducing TNF-α expression. Total protein was extracted from the cells using nano-magnetic beads, and RT-PCR and western blot detection indicated that the increase of TNF-α expression could increase the phosphorylation of NF-κB and and the expression of MMP-9. However, when TNF-α was inhibited by IVIG and TNF-α monoclonal antibody culture, the activity of NF-κB/MMP-9 athway was decreased. Therefore, IVIG may inhibit the production of NETs in KD children, thereby reducing TNF-α/NF-NF-κB/MMP-9 mediated inflammatory response process and protecting the function of vascular endothelial cells.

Artesunate inhibits macrophage-like phenotype switching of vascular smooth muscle cells and attenuates vascular inflammatory injury in atherosclerosis via NLRP3

Inflammation is one of the main pathogenic factors of atherosclerosis (AS), and the phenotypic transformation of macrophages in human vascular smooth muscle cells (HVSMCs) contributes to the inflammatory injury of blood vessels and the formation of atherosclerotic plaques. Artesunate reportedly exerts anti-inflammatory activity against AS. Herein, we aimed to explore the artesunate-mediated anti-inflammatory and HVSMC phenotypic switch effects against AS and elucidate potential underlying mechanisms. In vitro, artesunate decreased expression of NLRP3, caspase-1, and interleukin (IL)− 1β. Artesunate significantly inhibited low-density lipoprotein (LDL) expression in HVSMCs and macrophages. In vivo, artesunate reduced atherosclerotic plaque formation in high-fat diet (HFD)-fed ApoE-/- mice, as well as decreased NLRP3 and CD68 expression in atherosclerotic plaques. Artesunate decreased serum levels of triglycerides and increased high-density lipoprotein levels in HFD-med mice; however, serum levels of total cholesterol and LDL were unaltered. Treatment with artesunate substantially increased α-smooth muscle actin expression in aortic tissues while inhibiting expression levels of NLRP3, IL-1β, heparinase, matrix metalloproteinase 9, and Krüppel-like factor 4 (KLF4). Collectively, our findings suggest that artesunate-mediated effects may involve inhibition of the ERK1/2/NF-κB/IL-1β pathway in HVSMCs via the downregulation of NLRP3 expression. Thus, artesunate could serve as a novel strategy to treat AS by inhibiting AS plaque formation and suppressing macrophage-like phenotype switching of HVSMCs.

Protective effects of 18β-glycyrrhetinic acid on Pasteurella multocida-induced vascular inflammatory injury in mice.

Pasteurella multocida (Pm) is a widespread zoonotic pathogen with the ability to infect wild animals, livestock, and humans. Pm infection can cause haemorrhagic pneumonia, indicating that the pathogenesis involves serious vascular injury and inflammation. 18β-Glycyrrhetinic acid (GA) has cardiovascular protective and anti-inflammatory effects, but its effect on vascular injury caused by Pm infection is not clear. This study focused on the protective effects of GA on Pm-induced vascular inflammatory injury in mice. The results showed that GA intervention significantly improved the survival rate and the changes in haematological and biochemical parameters caused by Pm infection in mice. Haematoxylin and eosin staining revealed that GA delayed the progression of vascular injury, including abnormalities in elastic fibres, local rupture of the vascular intima, and inflammatory cell infiltration in response to Pm infection. The immunohistochemical results showed that after the GA intervention, the vascular inflammatory response in Pm-infected mice was alleviated. These protective effects may be related to the reduced expression of poly (ADP-ribose) polymerase-1, high mobility group box 1, interleukin-1β, and interleukin-18 in vascular tissue by GA. These findings suggest that GA inhibits the activation of inflammation to protect vascular injury in vivo. Hence, GA exhibits therapeutic potential in the treatment of vascular injury.

Controllable Adaptive Molybdate-Oligosaccharide Nanoparticles Regulate M2 Macrophage Mitochondrial Function and Promote Angiogenesis via PI3K/HIF-1α/VEGF Pathway to Accelerate Diabetic Wound Healing.

The complex wound environment of diabetic wounds leads to poor treatment efficacy, and the inflammatory disorders and vascular injury are the primary causes of death in such patients. Herein, a sprayable, controllable adaptive, pH-responsive nanosystem of molybdate and oligosaccharide (CMO) is specially developed as an immunomodulatory and angiogenesis-promotion material for diabetic wound healing. CMO exhibited pH-responsive release of Mo2+ and oligosaccharide (COS), specifically in response to the alkalescent environment observed in diabetic wounds. CMO provide an anti-inflammatory environment by promoting M2 polarization through significantly stimulating macrophage mitochondrial function. Specifically, CMO with a certain concentration reduce reactive oxygen species (ROS) and tumor necrosis factor α (TNF-α) expression, and upregulated mitochondrial membrane potential (MMP), superoxide dismutase (SOD), and interleukin 10 (IL-10) expression in macrophages. Moreover, CMO facilitate angiogenesis via upregulating the PI3K/HIF-1α/VEGF pathway-a critical process for the formation of new blood vessels that supply nutrients and oxygen to the healing tissue. Remarkably, CMO promote cell viability and migration of endothelial cells, and enhance the expression of angiogenic genes. In vitro and in vivo studies suggest this simple but powerful nanosystem targeting mitochondrial function has the potential to become an effective treatment for diabetic wound healing.

Low hemoglobin levels are associated with Bowman’s capsule rupture and peritubular capillaritis in ANCA-associated renal vasculitis: a link of vascular injury to anemia?

BackgroundAnemia in anti-neutrophil cytoplasmic antibody (ANCA)-associated renal vasculitis is a severe complication that predicts renal survival. We here conducted correlative analyses to evaluate correlations of low hemoglobin levels and histopathological characteristics in ANCA-associated renal vasculitis.MethodsFifty-two patients with biopsy-proven ANCA-associated renal vasculitis observed between 2015 and 2020 were retrospectively evaluated. Spearman’s correlation was performed to assess correlations, and statistical evaluation was performed by simple and stepwise multivariable regression.ResultsRegarding laboratory anemia parameters, no significant association with serum hemoglobin levels was observed. Serum hemoglobin levels were associated with the estimated glomerular filtration rate in the total cohort (β = 0.539, p < 0.001), and in the MPO-ANCA subgroup (β = 0.679, p = 0.008). Among tubulointerstitial lesions, decreased serum hemoglobin levels correlated with peritubular capillaritis in the whole cohort (β = − 0.358, p = 0.013), and was suggested in the MPO-ANCA subgroup (p = 0.029, r = − 0.446). Regarding glomerular lesions, the prevalence of necrotic glomeruli significantly associated with low serum hemoglobin levels in PR3-ANCA (β = − 0.424, p = 0.028). In the total cohort, a significant correlation between decreased serum hemoglobin levels and the occurrence of diffuse Bowman’s capsule rupture was identified (β = − 0.374, p = 0.014), which was implied in the MPO-ANCA subgroup (p = 0.013, r = − 0.546; p = 0.0288, slope = − 16.65).ConclusionPeritubular capillaritis and Bowman’s capsule rupture correlate with low hemoglobin levels; this may indicate that histopathological lesions are linked with inflammatory vascular injury and relative erythropoietin deficiency in ANCA-associated renal vasculitis.Graphical abstract

Alterations in mitochondrial protein glycosylation in myocardial ischaemia reperfusion injury

The alterations in mitochondrial protein glycosylation in myocardial ischaemia reperfusion (I/R) injury are still unclear. Therefore, based on a lectin microarray and liquid chromatograph-mass spectrometer/mass spectrometer (LC‒MS/MS) technology combined with a bioinformatics analysis, we studied the changes in mitochondrial protein glycosylation during I/R injury. This study revealed significant differences in mitochondrial glycoprotein during I/R injury. Compared with the sham operation group, the model group, which underwent ischaemia for 30 min, showed a high expression of glycan structures recognized by lectins, such as WFA, PTL-I, LTL, GSL-I, SBA and SNA, and a low expression of glycan structures recognized by ConA, VVA and RCA120. The model group, which underwent ischaemia for 45 min, showed a high expression of glycan structures recognized by LTL and SNA and a low expression of glycan structures recognized by ECA. Further analysis showed that the Siaα2-6Gal/N-acetylgalactosamine (GalNAc) structures recognized by SNA were significantly increased. In total, 91 differential proteins were identified by LC‒MS/MS, and 8 hub genes were screened by Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and protein interaction analyses. Compared with the Gene Expression Omnibus (GEO) database genes, two differential genes, Pros1 and Vtn, were obtained. Pros1 is a key regulator of the inflammatory response and vascular injury response. The Vtn gene variant is associated with the risk of myocardial infarction. This study is expected to provide a new method for the treatment of I/R injury and could provide new ideas for the postoperative prognosis of patients.

Proline and glucose metabolic reprogramming supports vascular endothelial and medial biomass in pulmonary arterial hypertension.

In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-Seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all aa and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest that elevated glucose and proline avidity underlie increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.

Microscopic polyangiitis plasma-derived exosomal miR-1287-5p induces endothelial inflammatory injury and neutrophil adhesion by targeting CBL.

An inflammatory environment around the vessel wall caused by leukocyte infiltration is one of the characteristic histopathological features of microscopic polyangiitis (MPA); however, the pathogenic mechanisms are not fully understood. Studies have found that circulating microRNA (miRNA) can be used as potential biomarkers for the diagnosis and classification of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), and the E3 ubiquitin ligase casitas B-lineage lymphoma (CBL) seems to be associated with inflammation. In addition, evidence indicates that miRNA can be tracked into exosomes and transferred into recipient cells to mediate the process of vascular endothelial injury. Herein, we aimed to identify the profiles of exosomal miRNA, and determine the effect of exosomal miR-1287-5p and its target gene CBL on vascular endothelial cells in MPA. We isolated plasma exosomes from patients with MPA (MPA-exo) and healthy controls (HC-exo) by ultracentrifugation and conducted exosome small-RNA sequencing to screen differential miRNA expression in MPA-exo (n=3) compared to HC-exo (n=3). We measured the expression levels of miR-1303, miR-1287-5p, and miR-129-1-3p using quantitative reverse transcription-polymerase chain reaction (qRT-PCR, n=6) and performed dual luciferase reporter gene assays to confirm the downstream target gene of miR-1287-5p. In addition, we treated human umbilical vein endothelial cell (HUVEC) with MPA-exo, or transfected them with miR-1287-5p mimic/inhibitor or with CBL-siRNA/CBL-siRNA+ miR-1287-5p inhibitor. After cell culture, we evaluated the effects on vascular endothelial cells by examining the mRNA levels of IL-6, IL-8, MCP-1, ICAM-1 and E-selectin using qRT-PCR and performed neutrophil adhesion assay with haematoxylin staining. Transmission electron microscopy, Western blot and nanoparticle tracking analysis showed that we successfully purified exosomes and MPA-exo could be absorbed into HUVEC. We screened a total of 1,077 miRNA by sequencing and observed a high abundance of miR-1287-5p in the exosomes obtained from MPA plasma. The dual luciferase reporter assay identified CBL as a downstream target gene of miR-1287-5p, and the results revealed that MPA-exo decreased CBL protein expression in HUVEC. In addition, treatment with MPA-exo, up-regulating miR-1287-5p or silencing of CBL in HUVEC significantly increased the mRNA expression of inflammatory factors (including IL-6, IL-8, and MCP-1) and adhesion molecules (including ICAM-1 and E-selection) and promoted the adhesion of neutrophils to HUVEC. However, down-regulating miR-1287-5p had the opposite effect. Our study revealed that MPA-exo was involved in the intercellular transfer of miR-1287-5p and subsequently promote the development of acute endothelial injury in MPA. MiR-1287-5p and CBL agonists may be promising therapeutic approach for MPA-induced vascular inflammatory injury.

Abstract 15339: Er-Resident S1pr1 in Lung Endothelium Regulates Innate Immune Function and Mediates Inflammatory Lung Injury

Endothelial-cell surface localized sphingosine 1 phosphate receptor 1 (S1PR1) is known to promote anti-inflammatory and barrier enhancing niche upon ligating S1P. Recently we showed that S1P and TNFα, later being a well-known inflammatory agonist, phosphorylate S1PR1 at tyrosine 143 (Y 143 ) which functions as an endoplasmic reticulum (ER) import signal (Anwar et al, 2021). ER-retained S1PR1 instructs barrier disruptive signaling but the mechanism remains unclear. Here, we generated S1PR1 knock-in mice using CRISPR-Cas9 strategy to edit endogenous S1PR1 into Y 143 D-S1PR1 (phospho mimicking) or Y 143 F-S1PR1 (phosphodefective) to test the hypothesis that ER-localized S1PR1 subverts EC from anti-inflammatory to pro-inflammatory EC leading to vascular injury. Because EC constitutes about 50% of cells in the lungs, we assessed if knock-in of Y 143 D-S1PR1 impaired lung homeostasis. We show that editing of S1PR1 into Y 143 D- or Y 143 F-S1PR1 did not alter total S1PR1 expression. Interestingly, Y 143 D-S1PR1 knock-in mice showed marked vascular leak at homeostasis along with increased neutrophil influx and inflammatory cytokine generation including TNFα, IL1ß and MiP2 as compared to Y 143 F-S1PR1 or WT mice. We next challenge these mice with intratracheal LPS. LPS-induced non-resolvable vascular inflammatory injury in Y 143 D-S1PR1 mice. Surprisingly, Y 143 F-S1PR1 knock-in mice did not develop vascular inflammatory injury. Furthermore, NFκB activity, a predominant transcription factor inducing inflammatory EC phenotype, was increased in EC transducing Y 143 D-S1PR1 mutant as compared to WT. However, TNFα failed to induce NFkB activity in EC transducing Y 143 F-S1PR1 mutant. Together, these results show that ER-resident S1PR1 program endothelial niche into immune-active niche by activating NFkB pathway leading to irreparable lung injury. Further experiments are being done to assess epigenetic changes (ATACseq and ChIP-Seq) in EC to address the concept the ER-resident S1PR1 controls the fate of immune cells in the lungs. We believe that understanding how ER-resident S1PR1 programs EC into inflammatory phenotype would allow development of new targets for treating the inflammatory vascular diseases including lung injury, ARDS, and COVID-19

Onion and Apple Functional Ingredients Intake Improves Antioxidant and Inflammatory Status and Vascular Injury in Obese Zucker Rats.

The objective of this study was to investigate the effects of onion and apple functional ingredients in homozygous (fa/fa) obese Zucker rats. Rodents were fed three diets: standard diet [obese control (OC) group], standard diet containing 10% onion [obese onion 10% (OO) group] and standard diet containing 10% apple [obese apple 10% (OA) group] for 8 weeks. Food intake and body weight gain were higher in obese than in lean rats. Food efficiency was lower in OO and AO groups compared with OC group. Within the obese groups, total cholesterol, LDL-cholesterol, triacylglycerols, glucose, insulin and triglyceride-glucose index were lower in OO group than in OC group, and HDL-cholesterol was higher in OO group than in OC group. In general, antioxidant activity (ABTS•+ and FRAP), antioxidant enzyme activities (CAT, SOD, GPx), GSH/GSSG ratio, nitrate/nitrite and GLP-1 increased in OO and OA groups compared with OC. Oxidative stress biomarkers, namely protein carbonyls, 8-hydroxy-2′-deoxyguanosine, 8-epi-prostaglandin F2α, inflammatory and vascular injury biomarkers (PAI-1, TIMP-1, VEGF, sICAM-1, sE-Selectin, MCP-1) and leptin, were lower in OO and OA groups than in OC group. Endothelial impairment was partially reversed, and superoxide content and gene expression of NLRP3, NFKβ1 and COX2 decreased, in OO and OA groups with respect to OC group. The study demonstrates that high pressure-processed onion and apple functional ingredients administration to obese Zucker rats causes beneficial effects on metabolic health, in particular through improving food efficiency ratio; exerting pronounced lipid-lowering effects; reducing glycemia, insulinemia, and biomarkers of hepatic injury (ALT, AST); improving antioxidant, oxidative stress, inflammatory and vascular injury biomarkers, metabolic hormones, and endothelial function; and decreasing proinflammatory gene expression of NLRP3, NFKβ1 and COX2.

Berberine alleviates NLRP3 inflammasome induced endothelial junction dysfunction through Ca2+ signalling in inflammatory vascular injury

BackgroundBerberine has received rising attention for its application in cardiovascular disease because of its relationship with inflammation. The endothelial NLRP3 inflammasome triggers inflammatory vascular injury which would lead to cardiovascular disease. Endothelial calcium signalling plays a crucial role in both the activation of NLRP3 inflammasome and endothelial cells dysfunction. However, the efficacy of BBR on the endothelial NLRP3 inflammasome in inflammatory vascular injury remains unknown. PurposeIn this study, we focused on the NLRP3 pathway to determine whether BBR regulates endothelial junction function in inflammatory vascular injury. MethodsThe integrity of the junction proteins VE-cadherin (VEC) and zonula occludens‐1 (ZO‐1) detected by immunofluorescence and immunoblotting was used to determine the therapeutic effect of BBR (50, 100, or 200 mg/kg/day) in LPS (100 μg/kg/day)-induced inflammatory vascular injury in mice and mouse microvascular endothelial cells (MECs) treated with LPS (1 μLPS ) and ATP (5 mM). Endothelial permeability was assessed by FITC-labelled dextran and trans-endothelial electrical resistance (TEER) in vitro. The assembly and activation of NLRP3 inflammasomes were detected by western blotting and immunofluorescence. Pharmacophore-based virtual molecular docking studies and calcium imaging analyses were used to determine the interaction of BBR with the ATP-gated Ca2+ channel P2X7R (purinergic P2X receptor 7) in the context of inflammatory vascular injury. ResultsBBR recovered the expression of ZO‐1 and VEC and inhibited endothelial NLRP3 inflammasome activation in coronary microvascular endothelium and in MECs. These results suggested a crucial role of the NLRP3 inflammasome in BBR-regulated endothelial integrity. Further analysis demonstrated that BBR treatment suppressed the binding of TXNIP (thioredoxin interacting protein) with NLRP3. Intriguingly, eliminating extracellular Ca2+ showed a similar effect as BBR. Virtual docking analysis indicated that R574 of P2X7R is a potential target for BBR binding. Ca2+ imaging showed that BBR inhibited the Ca2+ influx in response to ATP, supporting the potential interaction of BBR with P2X7R. ConclusionsThese findings suggest that BBR exhibits potential and specific therapeutic value by targeting calcium signals and the endothelial NLRP3 inflammasome in inflammatory vascular injury.

The Involvement of CXC Motif Chemokine Ligand 10 (CXCL10) and Its Related Chemokines in the Pathogenesis of Coronary Artery Disease and in the COVID-19 Vaccination: A Narrative Review.

Coronary artery disease (CAD) and coronary heart disease (CHD) constitute two of the leading causes of death in Europe, USA and the rest of the world. According to the latest reports of the Iranian National Health Ministry, CAD is the main cause of death in Iranian patients with an age over 35 years despite a significant reduction in mortality due to early interventional treatments in the context of an acute coronary syndrome (ACS). Inflammation plays a fundamental role in coronary atherogenesis, atherosclerotic plaque formation, acute coronary thrombosis and CAD establishment. Chemokines are well-recognized mediators of inflammation involved in several bio-functions such as leucocyte migration in response to inflammatory signals and oxidative vascular injury. Different chemokines serve as chemo-attractants for a wide variety of cell types including immune cells. CXC motif chemokine ligand 10 (CXCL10), also known as interferon gamma-induced protein 10 (IP-10/CXLC10), is a chemokine with inflammatory features whereas CXC chemokine receptor 3 (CXCR3) serves as a shared receptor for CXCL9, 10 and 11. These chemokines mediate immune responses through the activation and recruitment of leukocytes, eosinophils, monocytes and natural killer (NK) cells. CXCL10, interleukin (IL-15) and interferon (IFN-g) are increased after a COVID-19 vaccination with a BNT162b2 mRNA (Pfizer/BioNTech) vaccine and are enriched by tumor necrosis factor alpha (TNF-α) and IL-6 after the second vaccination. The aim of the present study is the presentation of the elucidation of the crucial role of CXCL10 in the patho-physiology and pathogenesis of CAD and in identifying markers associated with the vaccination resulting in antibody development.

Omega-3 fatty acids differentially reduced expression of neutrophil degranulation-associated proteins in endothelial cells during IL-6 exposure

Abstract Background Neutrophil degranulation contributes to atherogenesis and tissue injury. Mixed omega-3 fatty acid (n3-FA) formulations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have failed to reduce CV events compared to EPA only (REDUCE-IT), but the mechanisms are not understood. Purpose The purpose of this study was to compare the effects of EPA and DHA on expression of proteins linked to neutrophil degranulation in the vascular endothelium in vitro. Methods Human umbilical vein endothelial cells (HUVECs) were pretreated with EPA or DHA at equimolar levels (10 μM) for 2 h, then challenged with IL-6 at 12 ng/ml for 24 h. Proteomic analysis was performed using LC/MS to measure relative protein expression. Only significant (p&amp;lt;0.05) changes between treatment groups &amp;gt;1-fold were analyzed. Results In the Reactome “neutrophil degranulation” pathway, EPA and DHA downregulated 27 and 14 proteins, respectively, (p=9.97E-9 and 5.30E-4, respectively) relative to IL-6 alone. There were 12 protein changes common to both n3-FAs, including heme oxygenase-2 and ferritin light chain. EPA downregulated 15 proteins unlike DHA, including peroxiredoxin-6 and mitogen-activated protein kinase-1 (MAPK1). A combined 21 proteins downregulated by EPA and DHA versus IL-6 were upregulated by IL-6 alone relative to vehicle. EPA also increased expression of Rho-associated protein kinase-1 (ROCK-1), a protein downregulated by IL-6 alone and unaffected by DHA. Conclusions EPA and DHA differentially modulated expression of proteins linked to neutrophil degranulation. The distinct effects of EPA on protein expression may contribute to reduced inflammation in vascular injury compared to DHA. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amarin Pharma Inc., Elucida Research

Inflammation and its associations with aortic stiffness, coronary artery disease and peripheral artery disease in different ethnic groups: The HELIUS Study.

Backgroundevidence shows important ethnic differences in vascular dysfunction rates; however, the mechanisms driving these differences remain unclear. One potential factor is the ethnic differences in the role of inflammation in vascular injury. We tested the hypothesis that low-grade inflammation is unequally associated with vascular dysfunction in different ethnic groups.Methodswe included 5698 participants (similar-sized Dutch, African Surinamese, South-Asian Surinamese, Ghanaians, Turkish, and Moroccans) of the HELIUS study (the Netherlands) conducted between 2011 and 2015. Logistic regression was used to examine the associations of Z-score inflammatory biomarker concentration (high sensitivity C-reactive protein [hs-CRP], fibrinogen, and d-dimer) with vascular dysfunction (aortic stiffness, coronary artery disease [CAD], and peripheral artery disease [PAD]), with adjustments for age, sex, smoking (pack-years), BMI, hypertension, HbA1c, total cholesterol, and statin useFindingsin the fully adjusted models, higher Z-score hs-CRP was positively associated with CAD in Dutch [OR 1·63, (95% CI 1·21–2·18)] and PAD in South Asians [1·25(1·03–1·53)], respectively. Higher Z-score fibrinogen was positively associated with CAD in African Surinamese [1·28(1·03–1·59)] while higher Z-score d-dimer was positively associated with PAD in Moroccans [1·39(1·01–1·93)]. Higher Z-score hs-CRP [0·71(0·54–0·94)] and fibrinogen [0·75(0·58–0·97)] concentrations were negatively associated with PAD in African Surinamese.Interpretationour study shows that inflammatory biomarkers are unequally associated with vascular dysfunction in different ethnic groups. These observations provide opportunities for future studies aimed at assessing the predictive roles of inflammation on vascular disease in different ethnic groups.