The relationship between serum astroglial and neuronal markers and AQP4 and MOG autoantibodies.

BACKGROUND: Certain demyelinating disorders, such as neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) exhibit serum autoantibodies against aquaporin-4 (αAQP4) and myelin oligodendrocyte glycoprotein (αMOG). The variability of the autoantibody presentation warrants further research into subtyping each case. METHODS: To elucidate the relationship between astroglial and neuronal protein concentrations in the peripheral circulation with occurrence of these autoantibodies, 86 serum samples were analyzed using immunoassays. The protein concentration of glial fibrillary acidic protein (GFAP), neurofilament light chain (NFL) and tau protein was measured in 3 groups of subcategories of suspected NMOSD: αAQP4 positive (n = 20), αMOG positive (n = 32) and αMOG/αAQP4 seronegative (n = 34). Kruskal-Wallis analysis, univariate predictor analysis, and multivariate logistic regression with ROC curves were performed. RESULTS: GFAP and NFL concentrations were significantly elevated in the αAQP4 positive group (p = 0.003; p = 0.042, respectively), and tau was elevated in the αMOG/αAQP4 seronegative group (p < 0.001). A logistic regression model to classify serostatus was able to separate αAQP4 seropositivity using GFAP + tau, and αMOG seropositivity using tau. The areas under the ROC curves (AUCs) were 0.77 and 0.72, respectively. Finally, a combined seropositivity versus negative status logistic regression model was generated, with AUC = 0.80. CONCLUSION: The 3 markers can univariately and multivariately classify with moderate accuracy the samples with seropositivity and seronegativity for αAQP4 and αMOG.

Clinical evaluation of a novel plasma pTau217 electrochemiluminescence immunoassay in Alzheimer's disease.

A growing literature suggests that plasma levels of tau phosphorylated at amino acid 217 (pTau217) performs similarly to cerebrospinal fluid (CSF) biomarkers and PET imaging to detect amyloid pathology and to provide diagnostic and prognostic information in Alzheimer's disease (AD), but a significant limiting factor thus far has been a lack of widely available immunoassays. We evaluated a novel pTau217 S-PLEX® assay developed by Meso Scale Discovery (MSD; Rockville, MD) in plasma from 131 individuals with AD confirmed by CSF biomarkers and controls. Technical performance of the assay was excellent with an LLOQ of 1.84 pg/mL and intra/interplate CVs of 5.5% (0.3-15.0%) and 5.7% (range 0.3-13.4%), respectively. The pTau217 plasma assay differentiated AD and controls with an AUC of 0.98 (95% CI 0.96-1.0) and pTau217 levels were 3.9-fold higher in individuals with AD. This performance was significantly better than what was observed for plasma pTau181, performed in parallel, and comparable to published data on existing pTau217 assays. While further clinical validation and head-to-head comparisons are needed to fully establish the role for the novel pTau217 S-PLEX assay, these data demonstrate the utility of the assay to detect AD pathology.

HEG1 Protects Against Atherosclerosis by Regulating Stable Flow-Induced KLF2/4 Expression in Endothelial Cells.

BACKGROUND: Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. METHODS: Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow-induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA-mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1iECKO) mice. To determine the role of HEG1 in atherosclerosis, HEG1iECKO and littermate-control mice were injected with an adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. RESULTS: S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow-induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow-induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. CONCLUSIONS: Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.

The relationship between serum astroglial and neuronal markers and AQP4 and MOG autoantibodies.

Background: Certain demyelinating disorders, such as neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) exhibit serum autoantibodies against aquaporin-4 (αAQP4) and myelin oligodendrocyte glycoprotein (αMOG). The variability of the autoantibody presentation warrants further research into subtyping each case. Methods: To elucidate the relationship between astroglial and neuronal protein concentrations in the peripheral circulation with occurrence of these autoantibodies, 86 serum samples were analyzed using immunoassays. The protein concentration of glial fibrillary acidic protein (GFAP), neurofilament light chain (NFL) and tau protein was measured in 3 groups of subcategories of suspected NMOSD: αAQP4 positive (n = 20), αMOG positive (n = 32) and αMOG/αAQP4 seronegative (n = 34). Kruskal-Wallis analysis, univariate predictor analysis, and multivariate logistic regression with ROC curves were performed. Results: GFAP and NFL concentrations were significantly elevated in the αAQP4 positive group (p = 0.003; p = 0.042, respectively), and tau was elevated in the αMOG/αAQP4 seronegative group (p < 0.001). A logistic regression model to classify serostatus was able to separate αAQP4 seropositivity using GFAP + tau, and αMOG seropositivity using tau. The areas under the ROC curves (AUCs) were 0.77 and 0.72, respectively. Finally, a combined seropositivity versus negative status logistic regression model was generated, with AUC = 0.80. Conclusion: The 3 markers can univariately and multivariately classify with moderate accuracy the samples with seropositivity and seronegativity for αAQP4 and αMOG.

Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells.

Atherosclerosis is a chronic inflammatory disease and occurs preferentially in arterial regions exposed to disturbed blood flow (d-flow) while the stable flow (s-flow) regions are spared. D-flow induces endothelial inflammation and atherosclerosis by regulating endothelial gene expression partly through the flow-sensitive transcription factors (FSTFs). Most FSTFs, including the well-known Kruppel-like factors KLF2 and KLF4, have been identified from in vitro studies using cultured endothelial cells (ECs). Since many flow-sensitive genes and pathways are lost or dysregulated in ECs during culture, we hypothesized that many important FSTFs in ECs in vivo have not been identified. We tested the hypothesis by analyzing our recent gene array and single-cell RNA sequencing (scRNAseq) and chromatin accessibility sequencing (scATACseq) datasets generated using the mouse partial carotid ligation model. From the analyses, we identified 30 FSTFs, including the expected KLF2/4 and novel FSTFs. They were further validated in mouse arteries in vivo and cultured human aortic ECs (HAECs). These results revealed 8 FSTFs, SOX4, SOX13, SIX2, ZBTB46, CEBPß, NFIL3, KLF2, and KLF4, that are conserved in mice and humans in vivo and in vitro. We selected SOX13 for further studies because of its robust flow-sensitive regulation, preferential expression in ECs, and unknown flow-dependent function. We found that siRNA-mediated knockdown of SOX13 increased endothelial inflammatory responses even under the unidirectional laminar shear stress (ULS, mimicking s-flow) condition. To understand the underlying mechanisms, we conducted an RNAseq study in HAECs treated with SOX13 siRNA under shear conditions (ULS vs. oscillatory shear mimicking d-flow). We found 94 downregulated and 40 upregulated genes that changed in a shear- and SOX13-dependent manner. Several cytokines, including CXCL10 and CCL5, were the most strongly upregulated genes in HAECs treated with SOX13 siRNA. The robust induction of CXCL10 and CCL5 was further validated by qPCR and ELISA in HAECs. Moreover, the treatment of HAECs with Met-CCL5, a specific CCL5 receptor antagonist, prevented the endothelial inflammation responses induced by siSOX13. In addition, SOX13 overexpression prevented the endothelial inflammation responses. In summary, SOX13 is a novel conserved FSTF, which represses the expression of pro-inflammatory chemokines in ECs under s-flow. Reduction of endothelial SOX13 triggers chemokine expression and inflammatory responses, a major proatherogenic pathway.

Characterization of Poldip2 knockout mice: Avoiding incorrect gene targeting.

POLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from three limitations: perinatal lethality in homozygotes, constitutive Poldip2 inactivation and inadvertent downregulation of the adjacent Tmem199 gene. To overcome these limitations, we developed a new conditional floxed Poldip2 model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication around Poldip2 with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxed Poldip2 mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects of Poldip2 ablation in the vasculature, RNA-seq and RT-qPCR experiments were performed in constitutive knockout arteries. Results show that POLDIP2 inactivation affects multiple cellular processes and provide new opportunities for future in-depth study of its functions.

Atorvastatin and blood flow regulate expression of distinctive sets of genes in mouse carotid artery endothelium.

Hypercholesterolemia is a well-known pro-atherogenic risk factor and statin is the most effective anti-atherogenic drug that lowers blood cholesterol levels. However, despite systemic hypercholesterolemia, atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while the stable flow (s-flow) regions are spared. Given their predominant effects on endothelial function and atherosclerosis, we tested whether (1) statin and flow regulate the same or independent sets of genes and (2) statin can rescue d-flow-regulated genes in mouse artery endothelial cells in vivo. To test the hypotheses, C57BL/6 J mice (8-week-old male, n=5 per group) were pre-treated with atorvastatin (10mg/kg/day, Orally) or vehicle for 5 days. Thereafter, partial carotid ligation (PCL) surgery to induce d-flow in the left carotid artery (LCA) was performed, and statin or vehicle treatment was continued. The contralateral right carotid artery (RCA) remained exposed to s-flow to be used as the control. Two days or 2 weeks post-PCL surgery, endothelial-enriched RNAs from the LCAs and RCAs were collected and subjected to microarray gene expression analysis. Statin treatment in the s-flow condition (RCA+statin versus RCA+vehicle) altered the expression of 667 genes at 2-day and 187 genes at 2-week timepoint, respectively (P<0.05, fold change (FC)≥±1.5). Interestingly, statin treatment in the d-flow condition (LCA+statin versus LCA+vehicle) affected a limited number of genes: 113 and 75 differentially expressed genes at 2-day and 2-week timepoint, respectively (P<0.05, FC≥±1.5). In contrast, d-flow in the vehicle groups (LCA+vehicle versus RCA+vehicle) differentially regulated 4061 genes at 2-day and 3169 genes at 2-week timepoint, respectively (P<0.05, FC≥±1.5). Moreover, statin treatment did not reduce the number of flow-sensitive genes (LCA+statin versus RCA+statin) compared to the vehicle groups: 1825 genes at 2-day and 3788 genes at 2-week, respectively, were differentially regulated (P<0.05, FC≥±1.5). These results revealed that both statin and d-flow regulate expression of hundreds or thousands of arterial endothelial genes, respectively, in vivo. Further, statin and d-flow regulate independent sets of endothelial genes. Importantly, statin treatment did not reverse d-flow-regulated genes except for a small number of genes. These results suggest that both statin and flow play important independent roles in atherosclerosis development and highlight the need to consider their therapeutic implications for both.

Delivery of siRNA to Endothelial Cells In Vivo Using Lysine/Histidine Oligopeptide-Modified Poly(ß-amino ester) Nanoparticles.

PURPOSE: Endothelial cell (EC) dysfunction underlies the pathology of multiple disease conditions including cardiovascular and pulmonary diseases. Dysfunctional ECs have a distinctive gene expression profile compared to healthy ECs. RNAi therapy is a powerful therapeutic approach that can be used to silence multiple genes of interests simultaneously. However, the delivery of RNAi to ECs in vivo continues to be a major challenge. Here, we optimized a polymer formulation based on poly(ß-amino ester)s (pBAEs) to deliver siRNA to vascular ECs. METHODS: We developed a library of bioinspired oligopeptide-modified pBAE nanoparticles (NPs) with different physicochemical proprieties and screened them for cellular uptake and efficacy of RNAi delivery in vitro using ECs, vascular smooth muscle cells, and THP-1 monocytes. From the screening, the lysine-/histidine-oligopeptide modified pBAE (C6-KH) NP was selected and further tested ex vivo using mouse aorta and in mice to determine efficiency of siRNA delivery in vivo. RESULTS: The in vitro screening study showed that C6-KH was most efficient in delivering siRNA to ECs. Ex vivo study showed that C6-KH nanoparticles containing siRNAs accumulated in the endothelial layer of mouse aortas. In vivo study showed that C6-KH nanoparticles carrying siICAM2 injected via tail-vein in mice significantly reduced ICAM2 level in the artery endothelium (55%), lung (52%), and kidney (31%), but not in the liver, heart, and thymus, indicating a tissue-specific delivery pattern. CONCLUSIONS: We demonstrate that C6-KH pBAE can used for delivery of siRNAs to the artery endothelium and lung, while minimizing potential side or toxic effects in the liver and heart.