Subphenotypes of self-reported symptoms and outcomes in long COVID: a prospective cohort study with latent class analysis.

OBJECTIVE: To characterise subphenotypes of self-reported symptoms and outcomes (SRSOs) in postacute sequelae of COVID-19 (PASC). DESIGN: Prospective, observational cohort study of subjects with PASC. SETTING: Academic tertiary centre from five clinical referral sources. PARTICIPANTS: Adults with COVID-19 ≥20 days before enrolment and presence of any new self-reported symptoms following COVID-19. EXPOSURES: We collected data on clinical variables and SRSOs via structured telephone interviews and performed standardised assessments with validated clinical numerical scales to capture psychological symptoms, neurocognitive functioning and cardiopulmonary function. We collected saliva and stool samples for quantification of SARS-CoV-2 RNA via quantitative PCR. OUTCOMES MEASURES: Description of PASC SRSOs burden and duration, derivation of distinct PASC subphenotypes via latent class analysis (LCA) and relationship with viral load. RESULTS: We analysed baseline data for 214 individuals with a study visit at a median of 197.5 days after COVID-19 diagnosis. Participants reported ever having a median of 9/16 symptoms (IQR 6-11) after acute COVID-19, with muscle-aches, dyspnoea and headache being the most common. Fatigue, cognitive impairment and dyspnoea were experienced for a longer time. Participants had a lower burden of active symptoms (median 3 (1-6)) than those ever experienced (p<0.001). Unsupervised LCA of symptoms revealed three clinically active PASC subphenotypes: a high burden constitutional symptoms (21.9%), a persistent loss/change of smell and taste (20.6%) and a minimal residual symptoms subphenotype (57.5%). Subphenotype assignments were strongly associated with self-assessments of global health, recovery and PASC impact on employment (p<0.001) as well as referral source for enrolment. Viral persistence (5.6% saliva and 1% stool samples positive) did not explain SRSOs or subphenotypes. CONCLUSIONS: We identified three distinct PASC subphenotypes. We highlight that although most symptoms progressively resolve, specific PASC subpopulations are impacted by either high burden of constitutional symptoms or persistent olfactory/gustatory dysfunction, requiring prospective identification and targeted preventive or therapeutic interventions.

Impact of antiretroviral therapy during acute or early HIV infection on virologic and immunologic outcomes: results from a multinational clinical trial.

OBJECTIVE: To assess how antiretroviral therapy (ART) initiation during acute or early HIV infection (AEHI) affects the viral reservoir and host immune responses. DESIGN: Single-arm trial of ART initiation during AEHI at 30 sites in the Americas, Africa, and Asia. METHODS: HIV DNA was measured at week 48 of ART in 5 million CD4 + T cells by sensitive qPCR assays targeting HIV gag and pol . Peripheral blood mononuclear cells were stimulated with potential HIV T cell epitope peptide pools consisting of env , gag , nef, and pol peptides and stained for expression of CD3, CD4, CD8, and intracellular cytokines/chemokines. RESULTS: From 2017 to 2019, 188 participants initiated ART during Fiebig stages I ( n  = 6), II ( n  = 43), III ( n  = 56), IV ( n  = 23), and V ( n  = 60). Median age was 27 years (interquartile range 23-38), 27 (14%) participants were female, and 180 (97%) cisgender. Among 154 virally suppressed participants at week 48, 100% had detectable HIV gag or pol DNA. Participants treated during Fiebig I had the lowest HIV DNA levels ( P  < 0.001). Week 48 HIV DNA mostly did not correlate with concurrent CD4 + or CD8 + T cell HIV-specific immune responses (rho range -0.11 to +0.19, all P  > 0.025). At week 48, the magnitude, but not polyfunctionality, of HIV-specific T cell responses was moderately reduced among participants who initiated ART earliest. CONCLUSION: Earlier ART initiation during AEHI reduced but did not eliminate the persistence of HIV-infected cells in blood. These findings explain the rapid viral rebound observed after ART cessation in early-treated individuals with undetectable HIV DNA by less sensitive methods.

Noninvasive diagnosis of secondary infections in COVID-19 by sequencing of plasma microbial cell-free DNA.

Secondary infection (SI) diagnosis in severe COVID-19 remains challenging. We correlated metagenomic sequencing of plasma microbial cell-free DNA (mcfDNA-Seq) with clinical SI assessment, immune response, and outcomes. We classified 42 COVID-19 inpatients as microbiologically confirmed-SI (Micro-SI, n = 8), clinically diagnosed-SI (Clinical-SI, n = 13, i.e., empiric antimicrobials), or no-clinical-suspicion-for-SI (No-Suspected-SI, n = 21). McfDNA-Seq was successful in 73% of samples. McfDNA detection was higher in Micro-SI (94%) compared to Clinical-SI (57%, p = 0.03), and unexpectedly high in No-Suspected-SI (83%), similar to Micro-SI. We detected culture-concordant mcfDNA species in 81% of Micro-SI samples. McfDNA correlated with LRT 16S rRNA bacterial burden (r = 0.74, p = 0.02), and biomarkers (white blood cell count, IL-6, IL-8, SPD, all p < 0.05). McfDNA levels were predictive of worse 90-day survival (hazard ratio 1.30 [1.02-1.64] for each log10 mcfDNA, p = 0.03). High mcfDNA levels in COVID-19 patients without clinical SI suspicion may suggest SI under-diagnosis. McfDNA-Seq offers a non-invasive diagnostic tool for pathogen identification, with prognostic value on clinical outcomes.

TIM-3 signaling contributes to the suppressive capacity of Tregs from people with HIV on antiretroviral therapy.

TIM-3 expression is increased on peripheral regulatory T cells (Tregs) of virally suppressed persons with HIV-1 on antiretroviral therapy (PWH-ART). However, the relevance of TIM-3 expression in this setting is unclear. We used flow cytometry to evaluate the suppressive phenotype and signaling pathways in peripheral TIM-3- vs TIM-3+ Tregs in PWH-ART. TIM-3+ Tregs showed increased expression of IL-10 compared with persons without HIV-1. In addition, TIM-3+ Tregs displayed elevated signaling and activation, relative to TIM-3- Tregs from the same PWH-ART. Dramatically, TIM-3 blockade restrained the in vitro suppressive capacity of peripheral Tregs. Therefore, our data demonstrate not only that TIM-3 expression by Tregs is associated with an immunosuppressive response among PWH-ART, but also that TIM-3 contributes directly to the enhanced suppressive activity of Tregs in this setting.

Rapid Emergence of Potentially Transmissible Severe Acute Respiratory Syndrome Coronavirus 2 With Resistance to Combination Monoclonal Antibody Therapy.

Prolonged coronavirus disease 2019 may generate new viral variants. We report an immunocompromised patient treated with monoclonal antibodies who experienced rebound of viral RNA and emergence of an antibody-resistant (>1000-fold) variant containing 5 mutations in the spike gene. The mutant virus was isolated from respiratory secretions, suggesting the potential for secondary transmission.

Therapy With Allogeneic Severe Acute Respiratory Syndrome Coronavirus-2-Specific T Cells for Persistent Coronavirus Disease 2019 in Immunocompromised Patients.

We administered severe acute respiratory syndrome coronavirus-2 viral-specific T cells (VSTs) under emergency investigational new drug applications to 6 immunocompromised patients with persistent coronavirus disease 2019 (COVID-19) and characterized clinical and virologic responses. Three patients had partial responses after failing other therapies but then died. Two patients completely recovered, but the role of VSTs in recovery was unclear due to concomitant use of other antivirals. One patient had not responded to 2 courses of remdesivir and experienced sustained recovery after VST administration. The use of VSTs in immunocompromised patients with persistent COVID-19 requires further study.

Plasma SARS-CoV-2 RNA Levels as a Biomarker of Lower Respiratory Tract SARS-CoV-2 Infection in Critically Ill Patients With COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in serially collected samples from mechanically ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (n = 33, r = 0.83, P < 10-9) and then declined in parallel in available serial samples except in nonsurvivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early after ICU admission.

Contribution of Coronavirus-Specific Immunoglobulin G Responses to Complement Overactivation in Patients with Severe Coronavirus Disease 2019.

BACKGROUND: Excessive complement activation has been implicated in the pathogenesis of coronavirus disease 2019 (COVID-19), but the mechanisms leading to this response remain unclear. METHODS: We measured plasma levels of key complement markers, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antibodies against SARS-CoV-2 and seasonal human common cold coronaviruses (CCCs) in hospitalized patients with COVID-19 of moderate (n = 18) and critical severity (n = 37) and in healthy controls (n = 10). RESULTS: We confirmed that complement activation is systemically increased in patients with COVID-19 and is associated with a worse disease outcome. We showed that plasma levels of C1q and circulating immune complexes were markedly increased in patients with severe COVID-19 and correlated with higher immunoglobulin (Ig) G titers, greater complement activation, and higher disease severity score. Additional analyses showed that the classical pathway was the main arm responsible for augmented complement activation in severe patients. In addition, we demonstrated that a rapid IgG response to SARS-CoV-2 and an anamnestic IgG response to the nucleoprotein of the CCCs were strongly correlated with circulating immune complex levels, complement activation, and disease severity. CONCLUSIONS: These findings indicate that early, nonneutralizing IgG responses may play a key role in complement overactivation in severe COVID-19. Our work underscores the urgent need to develop therapeutic strategies to modify complement overactivation in patients with COVID-19.

Plasma SARS-CoV-2 RNA levels as a biomarker of lower respiratory tract SARS-CoV-2 infection in critically ill patients with COVID-19.

Plasma SARS-CoV-2 viral RNA (vRNA) levels are predictive of COVID-19 outcomes in hospitalized patients, but whether plasma vRNA reflects lower respiratory tract (LRT) vRNA levels is unclear. We compared plasma and LRT vRNA levels in simultaneously collected longitudinal samples from mechanically-ventilated patients with COVID-19. LRT and plasma vRNA levels were strongly correlated at first sampling (r=0.83, p<10 -8 ) and then declined in parallel except in non-survivors who exhibited delayed vRNA clearance in LRT samples. Plasma vRNA measurement may offer a practical surrogate of LRT vRNA burden in critically ill patients, especially early in severe disease.

Severe Acute Respiratory Syndrome Coronavirus 2 Viremia Is Associated With Coronavirus Disease 2019 Severity and Predicts Clinical Outcomes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA) is detected in the bloodstream of some patients with coronavirus disease 2019 (COVID-19), but it is not clear whether this RNAemia reflects viremia (ie, virus particles) and how it relates to host immune responses and outcomes. METHODS: SARS-CoV-2 vRNA was quantified in plasma samples from observational cohorts of 51 COVID-19 patients including 9 outpatients, 19 hospitalized (non-intensive care unit [ICU]), and 23 ICU patients. vRNA levels were compared with cross-sectional indices of COVID-19 severity and prospective clinical outcomes. We used multiple imaging methods to visualize virions in plasma. RESULTS: SARS-CoV-2 vRNA was detected in plasma of 100%, 52.6%, and 11.1% of ICU, non-ICU, and outpatients, respectively. Virions were detected in plasma pellets using electron tomography and immunostaining. Plasma vRNA levels were significantly higher in ICU > non-ICU > outpatients (P < .0001); for inpatients, plasma vRNA levels were strongly associated with higher World Health Organization (WHO) score at admission (P = .01), maximum WHO score (P = .002), and discharge disposition (P = .004). A plasma vRNA level >6000 copies/mL was strongly associated with mortality (hazard ratio, 10.7). Levels of vRNA were significantly associated with several inflammatory biomarkers (P < .01) but not with plasma neutralizing antibody titers (P = .8). CONCLUSIONS: Visualization of virus particles in plasma indicates that SARS-CoV-2 RNAemia is due, at least in part, to viremia. The levels of SARS-CoV-2 RNAemia correlate strongly with disease severity, patient outcome, and specific inflammatory biomarkers but not with neutralizing antibody titers.

Insertional activation of STAT3 and LCK by HIV-1 proviruses in T cell lymphomas.

Retroviruses cause cancers in animals by integrating in or near oncogenes. Although HIV-1 infection increases the risk of cancer, most of the risk is associated with immunodeficiency and coinfection by oncogenic virus (Epstein-Barr virus, Kaposi sarcoma herpesvirus, and human papillomavirus). HIV-1 proviruses integrated in some oncogenes cause clonal expansion of infected T cells in vivo; however, the infected cells are not transformed, and it is generally believed that HIV-1 does not cause cancer directly. We show that HIV-1 proviruses integrated in the first introns of signal transducer and activator of transcription 3 (STAT3) and lymphocyte-specific protein tyrosine kinase (LCK) can play an important role in the development of T cell lymphomas. The development of these cancers appears to be a multistep process involving additional nonviral mutations, which could help explain why T cell lymphomas are rare in persons with HIV-1 infection.

Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR.

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

Fatty Acids Rich Extract From Clerodendrum volubile Suppresses Cell Migration; Abates Oxidative Stress; and Regulates Cell Cycle Progression in Glioblastoma Multiforme (U87 MG) Cells.

Glioblastoma multiforme (GBM) is a malignant primary type of brain cancer with high proliferation and metastasis rates due to involvement of the microglial cell. It is resistant against available chemotherapy. Many strategic protocols have been developed but prognosis and patient life has not improved substantially. In this study, the anti-metastatic and antioxidant effect of fatty acids from Clerodendrum volubile leaves were investigated in U87-MG (Human Glioblastoma Multiforme) cell lines. The extracted fatty acids were incubated with U87-MG cells for 48 h. The anti-proliferative effect was determined by MTT assay, while apoptosis and cell cycle were analyzed with BD FACSCalibur. The transwell assay protocol was utilized in the analysis of cell migration and invasion. The treated cell lines were also assessed for reduced glutathione (GSH) level, catalase, superoxide dismutase (SOD) and lipid peroxidation. The fatty acid extract showed significant inhibitory activity on cell proliferation and cell cycle progression, mitigated oxidative stress, and suppressed migration and invasion in U-87 MG cell lines. These results give credence to the therapeutic potential of this plant against cancer, especially GBM.

Sirtuin 1 regulates cardiac electrical activity by deacetylating the cardiac sodium channel.

The voltage-gated cardiac Na+ channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na+ current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.

Sirtuin1-regulated lysine acetylation of p66Shc governs diabetes-induced vascular oxidative stress and endothelial dysfunction.

The 66-kDa Src homology 2 domain-containing protein (p66Shc) is a master regulator of reactive oxygen species (ROS). It is expressed in many tissues where it contributes to organ dysfunction by promoting oxidative stress. In the vasculature, p66Shc-induced ROS engenders endothelial dysfunction. Here we show that p66Shc is a direct target of the Sirtuin1 lysine deacetylase (Sirt1), and Sirt1-regulated acetylation of p66Shc governs its capacity to induce ROS. Using diabetes as an oxidative stimulus, we demonstrate that p66Shc is acetylated under high glucose conditions and is deacetylated by Sirt1 on lysine 81. High glucose-stimulated lysine acetylation of p66Shc facilitates its phosphorylation on serine 36 and translocation to the mitochondria, where it promotes hydrogen peroxide production. Endothelium-specific transgenic and global knockin mice expressing p66Shc that is not acetylatable on lysine 81 are protected from diabetic oxidative stress and vascular endothelial dysfunction. These findings show that p66Shc is a target of Sirt1, uncover a unique Sirt1-regulated lysine acetylation-dependent mechanism that governs the oxidative function of p66Shc, and demonstrate the importance of p66Shc lysine acetylation in vascular oxidative stress and diabetic vascular pathophysiology.

Canonical Wnt signaling induces vascular endothelial dysfunction via p66Shc-regulated reactive oxygen species.

OBJECTIVE: Reactive oxygen species regulate canonical Wnt signaling. However, the role of the redox regulatory protein p66(Shc) in the canonical Wnt pathway is not known. We investigated whether p66(Shc) is essential for canonical Wnt signaling in the endothelium and determined whether the canonical Wnt pathway induces vascular endothelial dysfunction via p66(Shc)-mediated oxidative stress. APPROACH AND RESULTS: The canonical Wnt ligand Wnt3a induced phosphorylation (activation) of p66(Shc) in endothelial cells. Wnt3a-stimulated dephosphorylation of ß-catenin, and ß-catenin-dependent transcription, was inhibited by knockdown of p66(Shc). Exogenous H2O2-induced ß-catenin dephosphorylation was also mediated by p66(Shc). Moreover, p66(Shc) overexpression dephosphorylated ß-catenin and increased ß-catenin-dependent transcription, independent of Wnt3a ligand. P66(Shc)-induced ß-catenin dephosphorylation was inhibited by antioxidants N-acetyl cysteine and catalase. Wnt3a upregulated endothelial NADPH oxidase-4, and ß-catenin dephosphorylation was suppressed by knocking down NADPH oxidase-4 and by antioxidants. Wnt3a increased H2O2 levels in endothelial cells and impaired endothelium-dependent vasorelaxation in mouse aortas, both of which were rescued by p66(Shc) knockdown. P66(Shc) knockdown also inhibited adhesion of monocytes to Wnt3a-stimulated endothelial cells. Furthermore, constitutively active ß-catenin expression in the endothelium increased vascular reactive oxygen species and impaired endothelium-dependent vasorelaxation. In vivo, high-fat diet feeding-induced endothelial dysfunction in mice was associated with increased endothelial Wnt3a, dephosphorylated ß-catenin, and phosphorylated p66(Shc). High-fat diet-induced dephosphorylation of endothelial ß-catenin was diminished in mice in which p66(Shc) was knocked down. CONCLUSIONS: p66(Shc) plays a vital part in canonical Wnt signaling in the endothelium and mediates Wnt3a-stimulated endothelial oxidative stress and dysfunction.

Redox factor-1 activates endothelial SIRTUIN1 through reduction of conserved cysteine sulfhydryls in its deacetylase domain.

Apurinic/Apyrmidinic Endonuclease 1/Redox Factor-1 (APE1/Ref-1) is a reductant which is important for vascular homeostasis. SIRTUIN1 (SIRT1) is a lysine deacetylase that also promotes endothelium-dependent vasorelaxation. We asked if APE1/Ref-1 governs the redox state and activity of SIRT1, and whether SIRT1 mediates the effect of APE1/Ref-1 on endothelium-dependent vascular function. APE1/Ref-1 maintains sulfhydryl (thiol) groups of cysteine residues in SIRT1 in the reduced form and promotes endothelial SIRT1 activity. APE1/Ref-1 stimulates SIRT1 activity by targeting highly conserved vicinal thiols 371 and 374 which form a zinc tetra-thiolate motif in the deacetylase domain of SIRT1. Cysteine residues in the N-terminal redox domain of APE1/Ref-1 are essential for reducing SIRT1 and stimulating its activity. APE1/Ref-1 protects endothelial SIRT1 from hydrogen peroxide-induced oxidation of sulfhydryls and from inactivation. APE1/Ref-1 also promotes lysine deacetylation of the SIRT1 target endothelial nitric oxide synthase (eNOS). SIRT1 mutated at cysteines 371 and 374, which renders it non-reducible by APE1/Ref-1, prevents lysine deacetylation of eNOS by APE1/Ref-1. SIRT1 free thiol (reduced sulfhydryl) content and deacetylase activity are diminished in all examined tissues of APE1/Ref-1(+/-) mice, including the vasculature. Overexpression of SIRT1 in aortas of APE1/Ref-1(+/-) mice restores endothelium-dependent vasorelaxation and bioavailable nitric oxide (NO) to levels similar to those observed in wild-type mice. Thus, APE1/Ref-1, by maintaining functionally important cysteine sulfhydryls in SIRT1 in the reduced form, promotes endothelial SIRT1 activity. This reductive activation of endothelial SIRT1 by APE1/Ref-1 mediates the effect of APE1/Ref-1 on eNOS acetylation, promoting endothelium-derived NO and endothelium-dependent vasorelaxation.

Histone and DNA methylation-mediated epigenetic downregulation of endothelial Kruppel-like factor 2 by low-density lipoprotein cholesterol.

OBJECTIVE: Low-density lipoprotein (LDL) cholesterol induces endothelial dysfunction and is a major modifiable risk factor for coronary heart disease. Endothelial Kruppel-like Factor 2 (KLF2) is a transcription factor that is vital to endothelium-dependent vascular homeostasis. The purpose of this study is to determine whether and how LDL affects endothelial KLF2 expression. APPROACH AND RESULTS: LDL downregulates KLF2 expression and promoter activity in endothelial cells. LDL-induced decrease in KLF2 parallels changes in endothelial KLF2 target genes thrombomodulin, endothelial NO synthase, and plasminogen activator inhibitor-1. Pharmacological inhibition of DNA methyltransferases or knockdown of DNA methyltransferase 1 prevents downregulation of endothelial KLF2 by LDL. LDL induces endothelial DNA methyltransferase 1 expression and DNA methyltransferase activity. LDL stimulates binding of the DNA methyl-CpG-binding protein-2 and histone methyltransferase enhancer of zeste homolog 2, whereas decreases binding of the KLF2 transcriptional activator myocyte enhancing factor-2, to the KLF2 promoter in endothelial cells. Knockdown of myocyte enhancing factor-2, or mutation of the myocyte enhancing factor-2 site in the KLF2 promoter, abrogates LDL-induced downregulation of endothelial KLF2 and thrombomodulin, and KLF2 promoter activity. Similarly, knockdown of enhancer of zeste homolog 2 negates LDL-induced downregulation of KLF2 and thrombomodulin in endothelial cells. Finally, overexpression of KLF2 rescues LDL-induced clotting of platelet-rich plasma on endothelial cells. CONCLUSIONS: LDL represses endothelial KLF2 expression via DNA and histone methylation. Downregulation of KLF2 by LDL leads to a dysfunctional, hypercoagulable endothelium.

Epigenetic upregulation of p66shc mediates low-density lipoprotein cholesterol-induced endothelial cell dysfunction.

Hypercholesterolemia characterized by elevation of low-density lipoprotein (LDL) cholesterol is a major risk factor for atherosclerotic vascular disease. p66shc mediates hypercholesterolemia-induced endothelial dysfunction and atheromatous plaque formation. We asked if LDL upregulates endothelial p66shc via changes in the epigenome and examined the role of p66shc in LDL-stimulated endothelial cell dysfunction. Human LDL stimulates human p66shc promoter activity and p66shc expression in human endothelial cells. LDL leads to hypomethylation of two CpG dinucleotides and acetylation of histone 3 in the human p66shc promoter. These two CpG dinucleotides mediate LDL-stimulated p66shc promoter activity. Inhibition or knock down of DNA methyltransferases negates LDL-induced endothelial p66shc expression. p66shc mediates LDL-stimulated increase in expression of endothelial intercellular adhesion molecule-1 (ICAM1) and decrease in expression of thrombomodulin (TM). Mirroring these changes in ICAM1 and TM expression, p66shc mediates LDL-stimulated adhesion of monocytes to endothelial cells and plasma coagulation on endothelial cells. These findings indicate that LDL cholesterol upregulates human endothelial p66shc expression via hypomethylation of CpG dinucleotides in the p66shc promoter. Moreover, they show that LDL-stimulated p66shc expression mediates a dysfunctional endothelial cell surface, with proadhesive and procoagulant features.

Homocysteine promotes human endothelial cell dysfunction via site-specific epigenetic regulation of p66shc.

AIMS: Hyperhomocysteinaemia is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial dysfunction. Homocysteine modulates cellular methylation reactions. P66shc is a protein that promotes oxidative stress whose expression is governed by promoter methylation. We asked if homocysteine induces endothelial p66shc expression via hypomethylation of CpG dinucleotides in the p66shc promoter, and whether p66shc mediates homocysteine-stimulated endothelial cell dysfunction. METHODS AND RESULTS: Homocysteine stimulates p66shc transcription in human endothelial cells and hypomethylates specific CpG dinucleotides in the human p66shc promoter. Knockdown of p66shc inhibits the increase in reactive oxygen species, and decrease in nitric oxide, elicited by homocysteine in endothelial cells and prevents homocysteine-induced up-regulation of endothelial intercellular adhesion molecule-1. In addition, knockdown of p66shc mitigates homocysteine-induced adhesion of monocytes to endothelial cells. Inhibition of DNA methyltransferase activity or knockdown of DNA methyltransferase 3b abrogates homocysteine-induced up-regulation of p66shc. Comparison of plasma homocysteine in humans with coronary artery disease shows a significant difference between those with highest and lowest p66shc promoter CpG methylation in peripheral blood leucocytes. CONCLUSION: Homocysteine up-regulates human p66shc expression via hypomethylation of specific CpG dinucleotides in the p66shc promoter, and this mechanism is important in homocysteine-induced endothelial cell dysfunction.