Endothelial Cell Inflammation Research Articles

Biomimetic Oil-in-Water Nanoemulsions as a Suitable Drug Delivery System to Target Inflamed Endothelial Cells

Biomimetic Oil-in-Water Nanoemulsions as a Suitable Drug Delivery System to Target Inflamed Endothelial Cells

Mesenchymal stromal cells and their secretory products reduce the inflammatory crosstalk between islets and endothelial cells.

Preculturing isolated islets with Mesenchymal Stromal Cells (MSCs) improves their functional survival in vitro and subsequent transplantation outcomes in vivo. The MSC secretory product Annexin A1 (ANXA1) is a key modulator of MSC-mediated improvements in islet function. The current study aims to determine the influence of MSCs and defined MSC secretory products, including ANXA1, on the inflammatory crosstalk between isolated islets and Endothelial Cells (ECs), using in vitro models of the clinically-preferred intraportal islet transplantation niche. Islets were cultured alone, with MSCs, or with MSC secretory products and exposed to pro-inflammatory cytokines. Islet gene expression of C-C Motif Chemokine Ligand 2 (CCL2), C-X-C Motif Chemokine Ligand (CXCL)-10 (CXCL10) and CXCL1 were assessed by RT-qPCR. EC activation was induced with 100 U/ml TNF for 24 h. Islet-EC co-cultures were used to determine the influence of MSCs, or MSC secretory products on the inflammatory crosstalk between isolated islets and ECs. VCAM-1 and ICAM-1 expression were assessed at the mRNA and protein level in ECs, using RT-qPCR and immunofluorescence. MSCs reduce pro-inflammatory cytokine-induced islet CCL2, CXCL10, and CXCL1 gene expression, which is partially mimicked by ANXA1. MSCs and ANXA1 have a similar capacity to reduce TNF-induced EC activation. Isolated islets exacerbate TNF-induced EC activation. Preculturing islets with MSCs reduces islet-exacerbated EC activation. ANXA1 reduces islet-exacerbated EC activation, when present during the islet preculture and islet-EC co-culture period. MSC-derived secretory factors, including ANXA1, may be used in islet transplantation protocols to target donor islet and host EC inflammation at the intraportal niche.

MicroRNA-19a-3p inhibits endothelial dysfunction in atherosclerosis by targeting JCAD

ObjectiveTo examine the influences and mechanisms of MicroRNA-19a-3p (miR-19a-3p) on endothelial dysfunction in atherosclerosis.MethodsAn analysis of miR-19a expression was carried out using the Gene Expression Omnibus (GEO) database. The effect of miR-19a-3p on endothelial function in HUVECs was evaluated by miR-19a-3p overexpression under TNF-α treatment. Luciferase assays were performed to explore the potential target genes. Overexpression of junctional protein associated with coronary artery disease (JCAD) was used to examine the effects of miR-19a-3p on cell adhesion, and proliferation.ResultsMiR-19a-3p expression in endothelial cells decreased after exposure to TNF-α and/or oscillatory flow, consistent with the expression change of miR-19a-3p found in atherosclerotic plaques. Additionally, endothelial cell dysfunction and inflammation were significantly diminished by miR-19a-3p overexpression but markedly exacerbated by miR-19a-3p inhibition. MiR-19a-3p transfection significantly decreased the expression of JCAD by binding to the 3’-UTR of JCAD mRNA. Furthermore, the protective effect of miR-19a-3p against endothelial cell dysfunction and inflammation was achieved by regulating JCAD and was closely linked to the Hippo/YAP signaling pathway.ConclusionMiR-19a-3p expression is a crucial molecular switch in the onset of atherosclerosis and miR-19a-3p overexpression is a possible pharmacological therapeutic strategy for reversing the development of atherosclerosis.

METTL14 derived from exosomes of M1 macrophages promotes high glucose-induced apoptosis, inflammation and oxidative stress in glomerular endothelial cells by mediating PAQR3 m6A modification.

Methyltransferase 14 (METTL14) mediated N6-methyladenine (m6A) RNA methylation and progestin and AdipoQ receptor family member 3 (PAQR3) are reported to be involved in diabetic nephropathy (DN) progression. Here, we explored whether the effects of PAQR3 on DN was associated with METTL14-induced m6A and their relationship with macrophage-related exosomes in DN progression. Human glomerular endothelial cells (GECs) were incubated in high glucose (HG) condition to mimic DN condition in vitro. Exosomes were isolated from M1 macrophages and co-cultured with GECs. qRT-PCR and western blotting detected the levels of genes and proteins. Cell functions were determined using cell counting kit-8 assay and flow cytometry. ELISA analysis detected inflammatory factors, and oxidative stress was evaluated by measuring reactive oxygen species and malondialdehyde. The m6A modification profile was determined by methylated RNA immunoprecipitation assay and the interaction was verified by dual-luciferase reporter assay. HG elevated PAQR3 expression levels in GECs. PAQR3 silencing reversed HG-induced viability arrest, apoptosis, inflammatory response, and oxidative stress. M1 macrophage co-culture could suppress HG-induced GEC injury. PAQR3 was packaged into M1 macrophage-derived exosomes, and M1 macrophages regulated HG-induced GEC injury by secreting PAQR3 into cells via exosomes. Mechanistically, METTL14 induced PAQR3 m6A modification. METTL14 was enriched in M1 macrophage-derived exosomes. METTL14 knockdown in M1 macrophage-derived exosomes protected GEC from HG-induced viability arrest, apoptosis, inflammation and oxidative stress by regulating PAQR3. Exosomal METTL14 derived from M1 macrophages promoted HG-induced apoptosis, inflammation and oxidative stress in GECs by mediating PAQR3 m6A modification.

Platelet-derived exosomes regulate endothelial cell inflammation and M1 macrophage polarization in coronary artery thrombosis via modulating miR-34a-5p expression

As the important factors in coronary artery thrombosis, endothelial injury and M1 macrophage polarization are closely related to the expression of miR-34a-5p. Exosomes in plasma are mainly derived from platelets and play an important role in thrombosis. Based on these facts, this study was conducted to investigate the acting mechanism of platelet-derived exosomes (PLT-exo) in the effects of endothelial injury and M1 macrophage polarization on coronary artery thrombosis. Firstly, rats were divided into the sham-operated group and the coronary microembolization (CME) group, and their plasma-derived exosomes were extracted to detect the expression of miR-34a-5p. Next, the PLT-exo were extracted from healthy volunteers and then co-cultured with ox-LDL-induced endothelial cells and LPS-induced macrophages, respectively. Subsequently, the expression of IL-1β, IL-6, TNF-α, and ICAM-1 in endothelial cells was measured, and the level of markers related to M1 macrophage polarization and Sirt1/NF-κB pathway was detected. Finally, the above indicators were examined again after PLT-exo combined with miR-34a-5p mimic were co-cultured with endothelial cells and macrophages, respectively. The results demonstrated that the expression of miR-34a-5p in the CME group was up-regulated compared with the sham-operated group. In cell experiments, PLT-exo modulated the Sirt1/NF-κB pathway by inhibiting the expression of intracellular miR-34a-5p and down-regulated the expression of IL-1β, IL-6, TNF-α, and ICAM-1 in endothelial cells and M1 macrophage polarization. After the transfection with miR-34a-5p mimic, endothelial cell inflammatory injury and M1 macrophage polarization increased to varying degrees. In conclusion, PLT-exo can alleviate coronary artery thrombosis by reducing endothelial cell inflammation and M1 macrophage polarization via inhibiting miR-34a-5p expression. In contrast, miR-34a-5p overexpression in PLT-exo may exacerbate these pathological injuries in coronary artery thrombosis.

IGFBP7 mediates oxLDL-induced human vascular endothelial cell injury by suppressing the expression of SIRT1

Endothelial cell injury plays an important role in initiating atherosclerotic lesion formation. Insulin-like growth factor binding protein 7 (IGFBP7) is known to modulate the behaviors of tumor-associated endothelial cells. This study was conducted to test whether IGFBP7 is involved in endothelial cell injury during atherosclerosis. Oxidized low-density lipoprotein (oxLDL) treatment was used to mimic atherosclerosis-related endothelial cell apoptosis and inflammation response. Small interfering RNA (siRNA) technology was employed to deplete IGFBP7 expression in human aortic endothelial cells (HAECs). HAECs were exposed to recombinant human IGFBP7 protein to evaluate the function of IGFBP7. Notably, IGFBP7 expression in HAECs was induced by oxLDL treatment. Knockdown of IGFBP7 or treatment with anti-IGFBP7 abolished oxLDL-induced apoptosis and inflammation in HAECs. Moreover, recombinant IGFBP7 (40 ng/mL but not 25 ng/mL) promoted apoptosis and inflammation in HAECs. IGFBP7 co-localized with CD93 on the surface of HAECs. A mechanistic investigation uncovered that IGFBP7 induced endothelial cell injury through interaction with CD93 and reduction of SIRT1 expression via an autocrine manner. Overexpression of SIRT1 rescued IGFBP7-induced phenotype in HAECs. Taken together, IGFBP7 is induced by oxLDL and mediates oxLDL-induced endothelial cell apoptosis and inflammation, likely through downregulation of SIRT1. These observations support a rationale to prevent atherosclerosis by targeting IGFBP7 activity.

Free Fatty Acids and Free Fatty Acid Receptors: Role in Regulating Arterial Function.

The metabolic network's primary sources of free fatty acids (FFAs) are long- and medium-chain fatty acids of triglyceride origin and short-chain fatty acids produced by intestinal microorganisms through dietary fibre fermentation. Recent studies have demonstrated that FFAs not only serve as an energy source for the body's metabolism but also participate in regulating arterial function. Excess FFAs have been shown to lead to endothelial dysfunction, vascular hypertrophy, and vessel wall stiffness, which are important triggers of arterial hypertension and atherosclerosis. Nevertheless, free fatty acid receptors (FFARs) are involved in the regulation of arterial functions, including the proliferation, differentiation, migration, apoptosis, inflammation, and angiogenesis of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). They actively regulate hypertension, endothelial dysfunction, and atherosclerosis. The objective of this review is to examine the roles and heterogeneity of FFAs and FFARs in the regulation of arterial function, with a view to identifying the points of intersection between their actions and providing new insights into the prevention and treatment of diseases associated with arterial dysfunction, as well as the development of targeted drugs.

Macrophage Migration Inhibitory Factor Promotes Thromboinflammation and Predicts Fast Progression of Aortic Stenosis.

Aortic stenosis (AS) is driven by progressive inflammatory and fibrocalcific processes regulated by circulating inflammatory and valve resident endothelial and interstitial cells. The impact of platelets, platelet-derived mediators, and platelet-monocyte interactions on the acceleration of local valvular inflammation and mineralization is presently unknown. We prospectively enrolled 475 consecutive patients with severe symptomatic AS undergoing aortic valve replacement. Clinical workup included repetitive echocardiography, analysis of platelets, monocytes, chemokine profiling, aortic valve tissue samples for immunohistochemistry, and gene expression analysis. The patients were classified as fast-progressive AS by the median ∆Vmax of 0.45 m/s per year determined by echocardiography. Immunohistological aortic valve analysis revealed enhanced cellularity in fast-progressive AS (slow- versus fast-progressive AS; median [interquartile range], 247 [142.3-504] versus 717.5 [360.5-1234]; P<0.001) with less calcification (calcification area, mm2: 33.74 [27.82-41.86] versus 20.54 [13.52-33.41]; P<0.001). MIF (macrophage migration inhibitory factor)-associated gene expression was significantly enhanced in fast-progressive AS accompanied by significantly elevated MIF plasma levels (mean±SEM; 6877±379.1 versus 9959±749.1; P<0.001), increased platelet activation, and decreased intracellular MIF expression indicating enhanced MIF release upon platelet activation (CD62P, %: median [interquartile range], 16.8 [11.58-23.8] versus 20.55 [12.48-32.28], P=0.005; MIF, %: 4.85 [1.48-9.75] versus 2.3 [0.78-5.9], P<0.001). Regression analysis confirmed that MIF-associated biomarkers are strongly associated with an accelerated course of AS. Our findings suggest a key role for platelet-derived MIF and its interplay with circulating and valve resident monocytes/macrophages in local and systemic thromboinflammation during accelerated AS. MIF-based biomarkers predict an accelerated course of AS and represent a novel pharmacological target to attenuate progression of AS.

SARS-CoV-2 S Protein Reduces Cytoprotective Defenses and Promotes Human Endothelial Cell Senescence.

Premature vascular aging and endothelial cell senescence are major risk factors for cardiovascular diseases and atherothrombotic disturbances, which are main complications of both acute and long COVID-19. The S protein of SARS-CoV2, which acts as the receptor binding protein for the viral infection, is able to induce endothelial cells inflammation and it has been found as an isolated element in the circulation and in human tissues reservoirs months after infection. Here, we investigated whether the S protein is able to directly induce endothelial cell senescence and deciphered some of the mechanisms involved. In primary cultures of human umbilical vein endothelial cells (HUVEC), SARS-CoV-2 S protein enhanced in a concentration-dependent manner the cellular content of senescence and DNA damage response markers (senescence-associated-β galactosidase, γH2AX), as well as growth-arrest effectors (p53, p21, p16). In parallel, the S protein reduced the availability of cytoprotective proteins, such as the anti-aging protein klotho, Nrf2 or heme oxygenase-1, and caused functional harm by impairing ex vivo endothelial-dependent vasorelaxation in murine microvessels. These effects were prevented by the pharmacological inhibition of the NLRP3 inflammasome with MCC950. Furthermore, the supplementation with either recombinant klotho or angiotensin-(1-7), equally protected against the pro-senescence, pro-inflammatory and pro-oxidant action of the S protein. Globally, this study proposes novel mechanisms of disease in the context of COVID-19 and its vascular sequelae and provides pharmacological clues in order to prevent such complications.

Quercetin ameliorates atherosclerosis by inhibiting inflammation of vascular endothelial cells via Piezo1 channels

BackgroundNatural antioxidants, exemplified by quercetin (Qu), have been shown to exert a protective effect against atherosclerosis (AS). However, the precise pharmacological mechanisms of Qu also remain elusive. PurposeHere, we aimed to uncover the anti-atherosclerotic mechanisms of Qu. Methods/Study designsThe inflammatory cytokine expression, activity of NLRP3 inflammasome and NF-κB, as well as mechanically activated currents and intracellular calcium levels were measured in endothelial cells (ECs). In addition, to explore whether Qu inhibited atherosclerotic plaque formation via Piezo1 channels, Ldlr−/- and Piezo1 endothelial-specific knockout mice (Piezo1△EC) were established. ResultsOur findings revealed that Qu significantly inhibited Yoda1-evoked calcium response in human umbilical vein endothelial cells (HUVECs), underscoring its role as a selective modulator of Piezo1 channels. Additionally, Qu effectively reduced mechanically activated currents in HUVECs. Moreover, Qu exhibited a substantial inhibitory effect on inflammatory cytokine expression and reduced the activity of NF-κB/NLRP3 in ECs exposed to ox-LDL or mechanical stretch, and these effects remained unaffected after Piezo1 genetic depletion. Furthermore, our study demonstrated that Qu substantially reduced the formation of atherosclerotic plaques, and this effect remained consistent even after Piezo1 genetic depletion. ConclusionThese results collectively provide compelling evidence that Qu ameliorates atherosclerosis by inhibiting the inflammatory response in ECs by targeting Piezo1 channels. In addition, Qu modulated atherosclerosis via inhibiting Piezo1 mediated NFκB/IL-1β and NLRP3/caspase1/ IL-1β axis to suppress the inflammation. Overall, this study reveals the potential mechanisms by which natural antioxidants, such as Qu, protect against atherosclerosis.

Dual-Targeting EGCG/NO-Supplying protein assembled nanoparticles with Multi-Synergistic effects against atherosclerosis

Limited therapeutic options are available to effectively preventing atherosclerosis. Inflammatory endothelial cells, foamy macrophages, and high protease levels contribute to atherosclerotic plaque formation. Studies have shown that catechins effectively scavenge reactive oxygen species (ROS), inhibit monocyte adhesion and reduce cholesterol levels, while nitric oxide (NO) enhances endothelial function. However, due to the poor stability and bioavailability of catechins and the toxicity from the burst release of current synthetic small molecules NO donor, effective deliveryof these bioactive compounds to treat atherosclerosis is still a challenge. Herein, a catechin/protein-based NO donor co-delivery nanosystem was designed for combinatorial anti-atherosclerotic therapy. We engineered a (−)-epigallocatechin-3-gallate (EGCG)/NO-releasing protein co-assembled nanocomplex based on specific catechin-protein interactions. Furthermore, the nanocomplex was surface modified with fucoidan (Fu), a sulfated polysaccharide with anti-inflammatory activity. This nanocomplex exhibits sensitivity to ROS, pH, and enzymes. The Fu-functionalized nanoparticles specifically accumulates in atherosclerotic plaques mediated by P-selectin on inflamed endothelial cells and scavenger receptor A (SR-A) on foamy macrophages. Under environmental stimuli that simulate the condition of plaque, the nanoparticles are readily activated to release EGCG and NO in response to excess ROS and high protease levels, exerting the multi-synergistic anti-atherosclerosic effects on reducing monocyte adhesion, promoting NO production to proliferate endothelial cells, lowering ROS levels, and decreasing the foam cell formation in vitro, and reducing lipid accumulation, plaque size, and inflammatory cytokines release in high-fat diet-induced atherosclerosis model in ApoE−/− mice. The integration of plaques targeting ability and multiple therapeutic functions can provide an advanced therapeutic strategy foratherosclerosistreatment.

Targeted Delivery of Macrophage Membrane Biomimetic Liposomes Through Intranasal Administration for Treatment of Ischemic Stroke.

Ginsenoside Rg3 (Rg3) and Panax notoginseng saponins (PNS) can be used for ischemic stroke treatment, however, the lack of targeting to the ischemic region limits the therapeutic effect. To address this, we leveraged the affinity of macrophage membrane proteins for inflamed brain microvascular endothelial cells to develop a macrophage membrane-cloaked liposome loaded with Rg3 and PNS (MM-Lip-Rg3/PNS), which can precisely target brain lesion region through intranasal administration. MM-Lip-Rg3/PNS was prepared by co-extrusion method and was performed by characterization, stability, surface protein, and morphology. The cellular uptake, immune escape ability, and blood-brain barrier crossing ability of MM-Lip-Rg3/PNS were studied in vitro. The in vivo brain targeting, biodistribution and anti-ischemic efficacy of MM-Lip-Rg3/PNS were evaluated in MACO rats, and we determined the diversity of the nasal brain pathway through the olfactory nerve blockade model in rats. Finally, the pharmacokinetics and brain targeting index of MM-Lip-Rg3/PNS were investigated. Our results indicated that MM-Lip-Rg3/PNS was spherical with a shell-core structure. MM-Lip-Rg3/PNS can avoid mononuclear phagocytosis, actively bind to inflammatory endothelial cells, and have the ability to cross the blood-brain barrier. Moreover, MM-Lip-Rg3/PNS could specifically target ischemic sites, even microglia, increase the cumulative number of drugs in the brain, improve the inflammatory environment of the brain, and reduce the infarct size. By comparing olfactory nerve-blocking rats with normal rats, it was found that there are direct and indirect pathways for nasal entry into the brain. Pharmacokinetics demonstrated that MM-Lip-Rg3/PNS exhibited stronger brain targeting and prolonged drug half-life. MM-Lip-Rg3/PNS might contribute to the accumulation of Rg3 and PNS in the ischemic brain area to improve treatment efficacy. This biomimetic nano-drug delivery system provides a new and promising strategy for the treatment of ischemic stroke.

Advancements in diabetic kidney disease management: integrating innovative therapies and targeted drug development.

Diabetic kidney disease (DKD) is a leading cause of chronic kidney disease and affects approximately 40% of individuals with diabetes . Cases of DKD continue to rise globally as the prevalence of diabetes mellitus increases, with an estimated 415 million people living with diabetes in 2015 and a projected 642 million by 2040. DKD is associated with significant morbidity and mortality, representing 34% and 36% of all chronic kidney disease deaths in men and women, respectively. Common comorbidities including hypertension and ageing-related nephron loss further complicate disease diagnosis and progression. The progression of DKD involves several mechanisms including glomerular endothelial cell dysfunction, inflammation, and fibrosis. Targeting these mechanisms has formed the basis of several therapeutic agents. Renin-angiotensin-aldosterone system (RAAS) blockers, specifically angiotensin receptor blockers (ARBs), demonstrate significant reductions in macroalbuminuria. Sodium-glucose transporter type 2 (SGLT-2) inhibitors demonstrate kidney protection independent of diabetes control while also decreasing the incidence of cardiovascular events. Emerging agents including glucagon-like peptide 1 (GLP-1) agonists, anti-inflammatory agents like bardoxolone, and mineralocorticoid receptor antagonists show promise in mitigating DKD progression. Many novel therapies including monoclonal antibodies CSL346, lixudebart, and tozorakimab; mesenchymal stem/stromal cell infusion; and cannabinoid-1 receptor inverse agonism via INV-202 are currently in clinical trials and present opportunities for further drug development.

Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseases

Accumulating evidence suggests that endothelial cells can be useful therapeutic targets. One of the potential targets is an endothelial cell-specific protein, Roundabout4 (ROBO4). ROBO4 has been shown to ameliorate multiple diseases in mice, including infectious diseases and sepsis. However, its mechanisms are not fully understood. In this study, using RNA-seq analysis, we found that ROBO4 downregulates prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2. Mechanistic analysis reveals that ROBO4 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1) and TNF receptor-associated factor 7 (TRAF7), a ubiquitin E3 ligase. In this complex, ROBO4 enhances IQGAP1 ubiquitination through TRAF7, inhibits prolonged RAC1 activation, and decreases PTGS2 expression in inflammatory endothelial cells. In addition, Robo4-deficiency in mice exacerbates PTGS2-associated inflammatory diseases, including arthritis, edema, and pain. Thus, we reveal the molecular mechanism by which ROBO4 suppresses the inflammatory response and vascular hyperpermeability, highlighting its potential as a promising therapeutic target for inflammatory diseases.

Xa inhibitor edoxaban ameliorates hepatic ischemia-reperfusion injury via PAR-2-ERK 1/2 pathway.

Hepatic ischemia-reperfusion injury causes liver damage during surgery. In hepatic ischemia-reperfusion injury, the blood coagulation cascade is activated, causing microcirculatory incompetence and cellular injury. Coagulation factor Xa (FXa)- protease-activated receptor (PAR)-2 signaling activates inflammatory reactions and the cytoprotective effect of FXa inhibitor in several organs. However, no studies have elucidated the significance of FXa inhibition on hepatic ischemia-reperfusion injury. The present study elucidated the treatment effect of an FXa inhibitor, edoxaban, on hepatic ischemia-reperfusion injury, focusing on FXa-PAR-2 signaling. A 60 min hepatic partial-warm ischemia-reperfusion injury mouse model and a hypoxia-reoxygenation model of hepatic sinusoidal endothelial cells were used. Ischemia-reperfusion injury mice and hepatic sinusoidal endothelial cells were treated and pretreated, respectively with or without edoxaban. They were incubated during hypoxia/reoxygenation in vitro. Cell signaling was evaluated using the PAR-2 knockdown model. In ischemia-reperfusion injury mice, edoxaban treatment significantly attenuated fibrin deposition in the sinusoids and liver histological damage and resulted in both anti-inflammatory and antiapoptotic effects. Hepatic ischemia-reperfusion injury upregulated PAR-2 generation and enhanced extracellular signal-regulated kinase 1/2 (ERK 1/2) activation; however, edoxaban treatment reduced PAR-2 generation and suppressed ERK 1/2 activation in vivo. In the hypoxia/reoxygenation model of sinusoidal endothelial cells, hypoxia/reoxygenation stress increased FXa generation and induced cytotoxic effects. Edoxaban protected sinusoidal endothelial cells from hypoxia/reoxygenation stress and reduced ERK 1/2 activation. PAR-2 knockdown in the sinusoidal endothelial cells ameliorated hypoxia/reoxygenation stress-induced cytotoxicity and suppressed ERK 1/2 phosphorylation. Thus, edoxaban ameliorated hepatic ischemia-reperfusion injury in mice by protecting against micro-thrombosis in sinusoids and suppressing FXa-PAR-2-induced inflammation in the sinusoidal endothelial cells.

Erianin alleviates LPS-induced acute lung injury via antagonizing P-selectin-mediated neutrophil adhesion function

Ethnopharmacological relevanceDendrobium officinale Kimura et Migo, known as “Tiepi Shihu” in traditional Chinese medicine, boasts an extensive history of medicinal use documented in the Chinese Pharmacopoeia. “Shen Nong Ben Cao Jing” records D. officinale as a superior herbal medicine for fortifying “Yin” and invigorating the five viscera. Erianin, a benzidine compound, emerges as a prominent active constituent derived from D. officinale, with the pharmacological efficacy of D. officinale closely linked to the anti-inflammatory properties of erianin. Aim of the studyAcute lung injury (ALI) is a substantial threat to global public health, while P-selectin stands out as a promising novel target for treating acute inflammatory conditions. This investigation aims to explore the therapeutic potential of erianin in ALI treatment and elucidate the underlying mechanisms. Experimental designThe effectiveness of erianin in conferring protection against ALI was investigated through comprehensive histopathological and biochemical analyses of lung tissues and bronchoalveolar lavage fluid (BALF) in an in vivo model of LPS-induced ALI in mice. The impact of erianin on fMLP-induced neutrophil chemotaxis was quantitatively assessed using the Transwell and Zigmond chamber, respectively. To determine the therapeutic target of erianin and elucidate their binding capability, a series of sophisticated assays were employed, including drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and molecular docking analyses. ResultsErianin demonstrated a significant alleviation of LPS-induced acute lung injury, characterized by reduced total cell and neutrophil counts and diminished total protein contents in BALF. Moreover, erianin exhibited a capacity to decrease proinflammatory cytokine production in both lung tissues and BALF. Notably, erianin effectively suppressed the activation of NF-κB signaling in the lung tissues of LPS- challenged mice; however, it did not exhibit in vitro inhibitory effects on inflammation in LPS-induced human pulmonary microvascular endothelial cells (HPMECs). Additionally, erianin blocked the adhesion and rolling of neutrophils on HPMECs. While erianin did not influence endothelial P-selectin expression or cytomembrane translocation, it significantly reduced the ligand affinity between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). ConclusionsErianin inhibits P-selectin-mediated neutrophil adhesion to activated endothelium, thereby alleviating ALI. The present study highlights the potential of erianin as a promising lead for ALI treatment.

PCSK9 induces endothelial cell autophagy by regulating the PI3K/ATK pathway in atherosclerotic coronary heart disease.

Atherosclerosis is a chronic inflammatory disease of the arteries, and its pathogenesis is related to endothelial dysfunction. It has been found that the protein convertase subtilin/kexin9 type (PCSK9) plays an important role in AS, but its specific mechanism is still unclear. In this study, we first cultured human umbilical vein endothelial cells (HUVECs) with 50 or 100μg/ml oxidized low-density lipoprotein (ox-LDL) for 24 hours to establish a coronary atherosclerosis cell model. The results showed that ox-LDL induced HUVEC injury and autophagy and upregulated PCSK9 protein expression in HUVECs in a concentration-dependent manner. Silencing PCSK9 expression with siRNA inhibited ox-LDL-induced HUVEC endothelial dysfunction, inhibited the release of inflammatory factors, promoted HUVEC proliferation and inhibited apoptosis. In addition, ox-LDL increased the expression of LC3B-I and LC3B-II and decreased the expression of p62. However, these processes are reversed by sh-PCSK9. In addition, sh-PCSK9 can inhibit PI3K, AKT and mTOR phosphorylation and promote autophagy. Taken together, our research shows that silencing PCSK9 inhibits the PI3K/ATK/mTOR pathway to activate ox-LDL-induced autophagy in vascular endothelial cells, alleviating endothelial cell injury and inflammation.

Inflammatory subphenotypes previously identified in ARDS are associated with mortality at intensive care unit discharge: a secondary analysis of a prospective observational study

BackgroundIntensive care unit (ICU)-survivors have an increased risk of mortality after discharge compared to the general population. On ICU admission subphenotypes based on the plasma biomarker levels of interleukin-8, protein C and bicarbonate have been identified in patients admitted with acute respiratory distress syndrome (ARDS) that are prognostic of outcome and predictive of treatment response. We hypothesized that if these inflammatory subphenotypes previously identified among ARDS patients are assigned at ICU discharge in a more general critically ill population, they are associated with short- and long-term outcome.MethodsA secondary analysis of a prospective observational cohort study conducted in two Dutch ICUs between 2011 and 2014 was performed. All patients discharged alive from the ICU were at ICU discharge adjudicated to the previously identified inflammatory subphenotypes applying a validated parsimonious model using variables measured median 10.6 h [IQR, 8.0–31.4] prior to ICU discharge. Subphenotype distribution at ICU discharge, clinical characteristics and outcomes were analyzed. As a sensitivity analysis, a latent class analysis (LCA) was executed for subphenotype identification based on plasma protein biomarkers at ICU discharge reflective of coagulation activation, endothelial cell activation and inflammation. Concordance between the subphenotyping strategies was studied.ResultsOf the 8332 patients included in the original cohort, 1483 ICU-survivors had plasma biomarkers available and could be assigned to the inflammatory subphenotypes. At ICU discharge 6% (n = 86) was assigned to the hyperinflammatory and 94% (n = 1397) to the hypoinflammatory subphenotype. Patients assigned to the hyperinflammatory subphenotype were discharged with signs of more severe organ dysfunction (SOFA scores 7 [IQR 5–9] vs. 4 [IQR 2–6], p < 0.001). Mortality was higher in patients assigned to the hyperinflammatory subphenotype (30-day mortality 21% vs. 11%, p = 0.005; one-year mortality 48% vs. 28%, p < 0.001). LCA deemed 2 subphenotypes most suitable. ICU-survivors from class 1 had significantly higher mortality compared to class 2. Patients belonging to the hyperinflammatory subphenotype were mainly in class 1.ConclusionsPatients assigned to the hyperinflammatory subphenotype at ICU discharge showed significantly stronger anomalies in coagulation activation, endothelial cell activation and inflammation pathways implicated in the pathogenesis of critical disease and increased mortality until one-year follow up.

Abstract 1041: Loss Of The ETS Transcription Factor ERG Disrupts Fate-defining Programs In The Aortic Endothelium And Promotes Expansion Of Endothelial Lineage Cells In Atherosclerotic Plaque

Aim: The ETS transcription factor ERG has been identified as a principal regulator of endothelial function through its ability to repress inflammation in endothelial cells (ECs), and loss of ERG induces endothelial to mesenchymal transition (EndMT) in fibrotic disease. To investigate ERG function in atherosclerosis, we employed an EC-specific Erg knockout ( Erg EC - KO ) in PCSK9-overexpression and apoE-deficient murine atherosclerosis models. Methods: Atherosclerosis was induced in 10-week-old Erg EC - KO mice or wild-type counterparts that had a Cre-inducible EC lineage tag ( Cdh5 CreER ; R26-CAG-LSL-Sun1-sfGFP ). Atherosclerosis was modeled by Apoe deletion or AAV8-PCSK9 with 12 weeks of high-cholesterol diet (1.25%). Aortic intimal cells were assessed with flow cytometry. Plaque burden, morphology, and cellularity were characterized with brightfield, Oil Red O, H&amp;E, Movat, and immunofluorescence imaging. Results: Erg EC-KO in PCSK9-overexpression mice led to a 1.6-fold increase in aortic plaque burden and plaque formation in regions that are usually protected (e.g., greater curvature; p≤0.03; n=9-10). Loss of ERG increased elastin breaks (1.7-fold; p=0.02; n=9) and decreased fibrous cap thickness (0.76-fold; p=0.02; n=9-10) in aortic arch cross sections. Aortic intimal digests from Erg EC- KO mice contained increased Sun1-sfGFP + EC lineage cells (1.9-fold; p=0.01; n=4), which had reduced CD31 positivity (0.79-fold; p=0.03; n=4). Notably, a 5.3-fold increase in EC lineage cells was observed in plaques from brachiocephalic artery cross sections (p=0.0001; n=6-15), which had hallmarks of EndMT (e.g., 31.5-fold increase in GFP + ACTA2 + cells, p=0.02, n=3-5). Increased aortic plaque burden was recapitulated in apoE-deficient E rg EC- KO mice (2.1-fold; p=0.02; n=4-8). Conclusion: Endothelial Erg KO results in increased plaque burden and altered plaque topography in murine atherosclerosis. Loss of ERG induces EndMT and marked ingression of ECs from the luminal endothelium to the plaque interior. These findings highlight loss of ERG as a novel mechanism by which intimal ECs acquire mesenchymal identity in atherosclerosis and implicate EC identity loss as a driver of EC migration into plaques, which is associated with a vulnerable morphology.

Abstract 3106: Targeted Mrna Delivery To The Inflamed Lung For The Treatment Of Ards

Acute respiratory distress syndrome (ARDS), caused by widespread endothelial barrier disruption and uncontrolled cytokine storm, is the major feared complication of pandemic influenza and COVID-19. Currently, there are few pharmacological treatments that directly target ARDS. We have previously discovered that reduction of Krüppel-like Factor 2 (KLF2, a transcription factor) in inflamed endothelium leads to ARDS induced by LPS, influenza, and high-tidal volume ventilation. Recently, we detected a significant reduction of KLF2 in lung autopsies from COVID-19 patients and demonstrated the anti-viral function of endothelial KLF2. Here, we devised a VCAM1-targeting nanomedicine strategy to restore KLF2 in inflamed endothelial cells. We engineered a rationally designed liposome which possesses potent mRNA delivery and transfection capacity. This VCAM1-targeting liposome selectively delivered functional KLF2 mRNA to inflamed vascular endothelium and significantly reduced mouse ARDS induced by influenza virus H1N1 and SARS-CoV-2, by calming local cytokine storm and promoting endothelium health. Moreover, we demonstrated that this targeted liposome serves as a broad platform to effectively deliver various mRNA molecules to targeted tissues of interest by displaying different targeting motifs. Our results demonstrate that this KLF2 mRNA encapsulated, VCAM1-targeting liposome is a promising mRNA therapeutic strategy to treat ARDS and vascular diseases.