Novel Knowledge-Based Transcriptomic Profiling of Lipid Lysophosphatidylinositol-Induced Endothelial Cell Activation

To determine whether pro-inflammatory lipid lysophosphatidylinositols (LPIs) upregulate the expressions of membrane proteins for adhesion/signaling and secretory proteins in human aortic endothelial cell (HAEC) activation, we developed an EC biology knowledge-based transcriptomic formula to profile RNA-Seq data panoramically. We made the following primary findings: first, G protein-coupled receptor 55 (GPR55), the LPI receptor, is expressed in the endothelium of both human and mouse aortas, and is significantly upregulated in hyperlipidemia; second, LPIs upregulate 43 clusters of differentiation (CD) in HAECs, promoting EC activation, innate immune trans-differentiation, and immune/inflammatory responses; 72.1% of LPI-upregulated CDs are not induced in influenza virus-, MERS-CoV virus- and herpes virus-infected human endothelial cells, which hinted the specificity of LPIs in HAEC activation; third, LPIs upregulate six types of 640 secretomic genes (SGs), namely, 216 canonical SGs, 60 caspase-1-gasdermin D (GSDMD) SGs, 117 caspase-4/11-GSDMD SGs, 40 exosome SGs, 179 Human Protein Atlas (HPA)-cytokines, and 28 HPA-chemokines, which make HAECs a large secretory organ for inflammation/immune responses and other functions; fourth, LPIs activate transcriptomic remodeling by upregulating 172 transcription factors (TFs), namely, pro-inflammatory factors NR4A3, FOS, KLF3, and HIF1A; fifth, LPIs upregulate 152 nuclear DNA-encoded mitochondrial (mitoCarta) genes, which alter mitochondrial mechanisms and functions, such as mitochondrial organization, respiration, translation, and transport; sixth, LPIs activate reactive oxygen species (ROS) mechanism by upregulating 18 ROS regulators; finally, utilizing the Cytoscape software, we found that three mechanisms, namely, LPI-upregulated TFs, mitoCarta genes, and ROS regulators, are integrated to promote HAEC activation. Our results provide novel insights into aortic EC activation, formulate an EC biology knowledge-based transcriptomic profile strategy, and identify new targets for the development of therapeutics for cardiovascular diseases, inflammatory conditions, immune diseases, organ transplantation, aging, and cancers.

Association of serum levels of antibodies against ALDOA and FH4 with transient ischemic attack and cerebral infarction

Background Ischemic stroke, including transient ischemic attack (TIA) and acute-phase cerebral infarction (aCI), is a serious health problem in the aging society. Thus, this study aimed to identify TIA and aCI biomarkers. Methods In 19 patients with TIA, candidate antigens recognized by serum IgG autoantibodies were screened using a human aortic endothelial cell cDNA library. Through amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA), serum antibody levels against the candidate antigens were examined in healthy donor (HD), TIA, and aCI cohorts (n = 285, 92, and 529). The plasma antibody levels in the Japan Public Health Center-based Prospective Cohort Study (1991–1993) were also examined. Results The candidate antigens were aldolase A (ALDOA) and fumarate hydratase (FH). In AlphaLISA, patients with TIA or aCI had higher anti-ALDOA antibody (ALDOA-Ab) and anti-FH antibody (FH-Ab) levels than the HDs (P < 0.05). In a multivariate logistic regression analysis, the ALDOA-Ab (odds ratio [OR]: 2.46, P = 0.0050) and FH-Ab (OR: 2.49, P = 0.0037) levels were independent predictors of TIA. According to the case–control study, the ALDOA-Ab (OR: 2.50, P < 0.01) and FH-Ab (OR: 2.60, P < 0.01) levels were associated with aCI risk. In a correlation analysis, both ALDOA-Abs and FH-Abs were well associated with hypertension, coronary heart disease, and habitual smoking. These antibody levels also correlated well with maximum intima–media thickness, which reflects atherosclerotic stenosis. Conclusions ALDOA-Abs and FH-Abs can be novel potential biomarkers for predicting atherosclerotic TIA and aCI.

Ginseng-Sanqi-Chuanxiong (GSC) Extracts Ameliorate Diabetes-Induced Endothelial Cell Senescence through Regulating Mitophagy via the AMPK Pathway

Vascular endothelial senescence induced by high glucose and palmitate (HG/PA) contributes to endothelial dysfunction, which leads to diabetic cardiovascular complications. Reduction of endothelial senescence may attenuate these pathogenic processes. This study is aimed at determining whether Ginseng-Sanqi-Chuanxiong (GSC) extracts, traditional Chinese medicine, can ameliorate human aortic endothelial cell (HAEC) senescence under HG/PA-stressed conditions and further explore the underlying mechanism. We found that GSC extracts significantly increased antisenescent activity by reducing the HG/PA-induced mitochondrial ROS (mtROS) levels in senescent HAECs. GSC extracts also induced cellular mitophagy formation, which mediated the effect of GSC extracts on mtROS reduction. Apart from this, the data showed that GSC extracts stimulated mitophagy via the AMPK pathway, and upon inhibition of AMPK by pharmacological and genetic inhibitors, GSC extract-mediated mitophagy was abolished which further led to reverse the antisenescence effect. Taken together, these data suggest that GSC extracts prevent HG/PA-induced endothelial senescence and mtROS production by mitophagy regulation via the AMPK pathway. Thus, the induction of mitophagy by GSC extracts may provide a novel therapeutic candidate for cardiovascular protection in metabolic syndrome.

Association of serum levels of antibodies against ALDOA and FH4 with transient ischemic attack and cerebral infarction

Background: Ischemic stroke, such as transient ischemic attack (TIA) and acute-phase cerebral infarction (aCI) , are the serious problems in the aging society. Therefore, development of biomarkers for TIA and aCI are attempted. Methods: Candidate antigens recognized by IgG autoantibodies in the serum of 19 TIA patients were screened by a human aortic endothelial cell cDNA library. Serum antibody levels against the antigens were examined by amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in healthy donor (HD), TIA, and aCI cohorts ( n = 285, 92 and 529). The antibody levels in the sera of the Japan Public Health Center-based Prospective Cohort Study (JPHC) from 1991 to 1993 was also examined. Results: Aldolase A, fructose-bisphosphate (ALDOA) and fumarate hydratase (FH) were identified as the candidate antigens. AlphaLISA revealed that the levels of anti-ALDOA antibodies (ALDOA-Abs) and anti-FH antibodies (FH-Abs) were both higher in patients with TIA or aCI than those in HDs ( P < 0.05). The levels of ALDOA-Abs [odds ratio (OR): 2.46, P = 0.0050] and FH-Abs (OR: 2.49, P = 0.0037) were independent predictors of TIA by multivariate logistic regression analysis. The case-control study showed the levels of ALDOA-Abs (OR: 2.50, P < 0.01) and FH-abs (OR: 2.60, P < 0.01) were associated with risk of aCI. Correlation analysis demonstrated that both ALDOA-Abs and FH-Abs were well associated with hypertension, coronary heart disease and habitual smoking. These antibody levels were also correlated well with maximum intima-media thickness, which reflects atherosclerotic stenosis. Conclusions: ALDOA-Abs and FH-Abs can serve as novel potential biomarkers for prediction of atherosclerotic TIA and aCI.

Tumor necrosis factor induces cholesterol accumulation in human arterial endothelial cells through LDL receptor surface redistribution

ABSTRACTTumor necrosis factor alpha (TNFα) and low density lipoprotein (LDL) are important modulators of the atherosclerotic process. Here, the effect of TNFα on confluent primary human aortic endothelial cell (pHAEC) LDL-derived lipids and trafficking were investigated. TNFα promoted up to 2 folds increase in cellular cholesterol and could induce a massive increase in the non-hydrolysable tracer, Dil, by over 200 folds. The lipid increase was associated with increased 125I-LDL surface binding. Further, Dil-LDL cellular association was blocked by excess unlabeled LDL, but not oxidized LDL (oxLDL). Moreover, TNFα-induced Dil and cholesterol increase were enhanced by the endosomal pH-raising agent, chloroquine. Internalization of Dil-LDL was reduced by the scavenger receptor B1 (SR-B1) antagonist, BLT-1, and the LDLR family antagonist, proprotein convertase subtilisin/kexin type 9 (PCSK9), but not receptor associated protein (R AP). Additionally, surface accessible LDLR was higher in TNFα-treated cells by about 30 folds, without a significant change in total LDLR. Correspondingly, specific LDLR antibody blocked Dil-LDL internalization to undetectable levels, and Dil-apoE3-VLDL by 94%, but had no effect on Dil-HDL3 internalization. TNFα did not increase Dil-HDL3 cellular association. Further, ACAT inhibitor reduced cholesteryl esters, but not the total cholesterol increase induced by TNFα. On pHAECs grown on transwell inserts, TNFα did not enhance apical (AP) to basolateral (BL) LDL cholesterol or Dil release. It is concluded that TNFα induces LDLR surface localization and that LDLR does not promote AP to BL LDL transport across pHAECs.

Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis

Emerging data indicate that complement and neutrophils are involved in the maladaptive host immune response that fuels hyper-inflammation and thrombotic microangiopathy increasing the mortality rate in coronavirus disease 2019 (COVID-19). Here, we investigated the interaction between complement and the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 clinical samples, cell-based inhibition studies and NETs/human aortic endothelial cell (HAEC) co-cultures. Increased plasma levels of NETs, TF activity and sC5b-9 were detected in patients. Neutrophils yielded high tissue factor (TF) expression and released NETs carrying functionally active TF. Confirming our ex vivo findings, treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAEC. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. Serum isolated from COVID-19 patients induces complement activation in vitro, which is consistent with high complement activity in clinical samples. Complement inhibition at the level of C3 with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis that reveals the pivotal role of complement and NETs in COVID-19 immmunothrombosis. This study supports emerging strategies against SARS-CoV-2 infection that exploit complement therapeutics or NETosis inhibition.

Association of serum levels of antibodies against ALDOA and FH4 with transient ischemic attack and cerebral infarction

Background and Purpose: Ischemic stroke, such as Transient ischemic attack (TIA) and cerebral infarction (CI) , are the serious problems in the aging society. Therefore, development of biomarkers for TIA and CI is attempted.Methods: Candidate antigens recognized by IgG autoantibodies in the sera of nineteen TIA patients were screened by a human aortic endothelial cell cDNA library. Serum antibody levels against the antigens were examined by amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in healthy donor (HD), TIA, and CI cohorts (n = 285, 92 and 529). A case-control study nested within the Japan Public Health Center-based Prospective Cohort Study (JPHC) was performed.Results: Aldolase A, fructose-bisphosphate (ALDOA) and fumarate hydratase (FH) were identified as the candidate antigens. AlphaLISA revealed that anti-ALDOA and anti-FH antibody levels were both higher in TIA or CI patients than in HDs ( P < 0.0001). The levels of anti-ALDOA [Odds ratio (OR): 2.46, P = 0.005] and anti-FH (OR: 2.49, P = 0.0037) were independent predictors of TIA by multivariate logistic regression analysis, similar results were found in CI. The case-control study showed the levels of anti-ALDOA (OR: 2.50, P < 0.01) and anti-FH (OR: 2.60, P < 0.01) were associated with risk of CI. Spearman’s correlation analysis demonstrated an association between the anti-ALDOA and anti-FH levels and risk factors of ischemic stroke, such as age, smoking habit, coronary heart disease, and hypertension.Conclusions: Anti-ALDOA and anti-FH antibodies can serve as novel potential biomarkers for prediction of TIA and CI.

Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function

Xenogeneic biomaterials contain biologically relevant extracellular matrix (ECM) composition and organization, making them potentially ideal surgical grafts and tissue engineering scaffolds. Defining the effect of ECM niche (e.g., basement membrane vs. non-basement membrane) on repopulating cell phenotype and function has important implications for use of xenogeneic biomaterials, particularly in vascular applications. We aim to understand how serous (i.e., basement membrane) versus fibrous (i.e., non-basement membrane) ECM niche of antigen-removed bovine pericardium (AR-BP) scaffolds influence human aortic endothelial cell (hAEC) adhesion, growth, phenotype, inflammatory response and laminin production. At low and moderate seeding densities hAEC proliferation was significantly increased on the serous side. Similarly, ECM niche modulated cellular morphology, with serous side seeding resulting in a more rounded aspect ratio and intact endothelial layer formation. At moderate seeding densities, hAEC production of human laminin was enhanced following serous seeding. Finally, inflammatory marker and pro-inflammatory cytokine expression decreased following long-term cell growth regardless of seeding side. This work demonstrates that at low and moderate seeding densities AR-BP sidedness significantly impacts endothelial cell growth, morphology, human laminin production, and inflammatory state. These findings suggest that ECM niche has a role in modulating response of repopulating recipient cells toward AR-BP scaffolds for vascular applications.

Delivery of targeted gene therapies using a hybrid cryogel-coated prosthetic vascular graft

ObjectivesThe success of prosthetic vascular grafts in the management of peripheral arterial disease is frequently limited by the development of anastomotic neointimal hyperplasia (ANIH), with the host response to prosthetic grafts beginning soon after implantation. To address this, we combine a platform of polyethylene terephthalate (PET) fabric with an applied cryogel layer containing biologic agents to create a bioactive prosthetic graft system, with the ability to deliver therapeutics targeting modulators of the ANIH-associated transcriptome response, along with antithrombotic agents.MethodsHybrid graft materials were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun (ePET), knitted PET (kPET), or woven PET (wPET). Arg-Gly-Asp (RGD) peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Physical properties such as swelling ratio, pore connectivity, shape recovery, and stiffness were evaluated. Human aortic endothelial cell (HAoEC) adherence was visualized using confocal microscopy after 24 hours and proliferation was evaluated with a resazurin-based assay for 7 days. Confocal microscopy was used to assess delivery of adeno-associated virus (AAV-GFP) after incubation of hybrid grafts with HAoECs. Heparin activity of the materials was measured using an anti-Xa assay.ResultsSEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. Hybrid materials showed a trend towards increased shape recovery, increased stiffness, decreased swelling ratio, and no difference in pore connectivity. HAoECs incorporated, adhered, and proliferated over 7 days on all materials. HAoECs were successfully transduced with AAV-GFP from the hybrid graft materials. Anti-Xa assay confirmed continued activity of heparin from all materials for over 7 days.ConclusionsWe have developed a bioactive prosthetic graft system with a cryogel coating capable of delivering biologic agents with antithrombotic activity. By applying the cryogel and selected agents onto PET prior to graft implantation, this study sets the stage for the system to be individualized and tailored to the patient, with bioengineering and targeted gene therapy strategies dovetailing to create an improved prosthetic graft adaptable to emerging knowledge and technologies.