RNAseq profiling of blood from patients with coronary artery disease: Signature of a T cell imbalance.

Background: Cardiovascular disease had a global prevalence of 523 million cases and 18.6 million deaths in 2019. The current standard for diagnosing coronary artery disease (CAD) is coronary angiography either by invasive catheterization (ICA) or computed tomography (CTA). Prior studies employed single-molecule, amplification-independent RNA sequencing of whole blood to identify an RNA signature in patients with angiographically confirmed CAD. The present studies employed Illumina RNAseq and network co-expression analysis to identify systematic changes underlying CAD. Methods: Whole blood RNA was depleted of ribosomal RNA (rRNA) and analyzed by Illumina total RNA sequencing (RNAseq) to identify transcripts associated with CAD in 177 patients presenting for elective invasive coronary catheterization. The resulting transcript counts were compared between groups to identify differentially expressed genes (DEGs) and to identify patterns of changes through whole genome co-expression network analysis (WGCNA). Results: The correlation between Illumina amplified RNAseq and the prior SeqLL unamplified RNAseq was quite strong (r = 0.87), but there was only 9 % overlap in the DEGs identified. Consistent with the prior RNAseq, the majority (93 %) of DEGs were down-regulated ~1.7-fold in patients with moderate to severe CAD (>20 % stenosis). DEGs were predominantly related to T cells, consistent with known reductions in Tregs in CAD. Network analysis did not identify pre-existing modules with a strong association with CAD, but patterns of T cell dysregulation were evident. DEGs were enriched for transcripts associated with ciliary and synaptic transcripts, consistent with changes in the immune synapse of developing T cells. Conclusions: These studies confirm and extend a novel mRNA signature of a Treg-like defect in CAD. The pattern of changes is consistent with stress-related changes in the maturation of T and Treg cells, possibly due to changes in the immune synapse.

Diagnostic accuracy of novel mRNA blood biomarkers of infection to predict outcomes in emergency department patients with undifferentiated abdominal pain.

Abdominal pain represents greater than 20% of US Emergency Department (ED) visits due to a wide range of illnesses. There are currently no reliable blood biomarkers to predict serious outcomes in patients with abdominal pain. Our previous studies have identified three mRNA transcripts related to innate immune activation: alkaline phosphatase (ALPL), interleukin-8 receptor-ß (IL8RB), and defensin-1 (DEFA1) as promising candidates to detect an intra-abdominal infection. The objective of this study was to evaluate the accuracy of these mRNA biomarkers to predict likely infection, hospitalization and surgery in Emergency Department patients with undifferentiated abdominal pain. We prospectively enrolled Emergency Department patients with undifferentiated abdominal pain who received an abdominal CT scan as part of their evaluation. Clinical outcomes were abstracted from the CT scan and medical records. mRNA biomarker levels were calculated independent of the clinical outcomes and their accuracy was assessed to predict infectious diagnoses, surgery and hospital admission. 89 patients were enrolled; 21 underwent surgery; 47 underwent hospital admission; and, no deaths were observed within 30 days. In identifying which cases were likely infectious, mRNA biomarkers' AUC values were: ALPL, 0.83; DEFA1 0.51; IL8RB, 0.74; and ALPL + IL8RB, 0.79. In predicting which Emergency Department patients would receive surgery, the AUC values were: ALPL, 0.75; DEFA1, 0.58; IL8RB, 0.75; and ALPL + IL8RB, 0.76. In predicting hospital admission, the AUC values were: ALPL, 0.78; DEFA1, 0.52; IL8RB, 0.74; and, ALPL + IL8RB, 0.77. For predicting surgery, ALPL + IL8RB's positive likelihood ratio (LR) was 3.97; negative LR (NLR) was 0.70. For predicting hospital admission, the same marker's positive LR was 2.80 with an NLR of 0.45. Where the primary cause for admission was a potentially infectious disorder, 33 of 34 cases (97%) had positive RNA scores. In a pragmatic, prospective diagnostic accuracy trial in Emergency Department patients with undifferentiated abdominal pain, mRNA biomarkers showed good accuracy to identify patients with potential infection, as well as those needing surgery or hospital admission.

RNA Sequencing in COVID-19 patients identifies neutrophil activation biomarkers as a promising diagnostic platform for infections.

Infection with the SARS-CoV2 virus can vary from asymptomatic, or flu-like with moderate disease, up to critically severe. Severe disease, termed COVID-19, involves acute respiratory deterioration that is frequently fatal. To understand the highly variable presentation, and identify biomarkers for disease severity, blood RNA from COVID-19 patient in an intensive care unit was analyzed by whole transcriptome RNA sequencing. Both SARS-CoV2 infection and the severity of COVID-19 syndrome were associated with up to 25-fold increased expression of neutrophil-related transcripts, such as neutrophil defensin 1 (DEFA1), and 3-5-fold reductions in T cell related transcripts such as the T cell receptor (TCR). The DEFA1 RNA level detected SARS-CoV2 viremia with 95.5% sensitivity, when viremia was measured by ddPCR of whole blood RNA. Purified CD15+ neutrophils from COVID-19 patients were increased in abundance and showed striking increases in nuclear DNA staining by DAPI. Concurrently, they showed >10-fold higher elastase activity than normal controls, and correcting for their increased abundance, still showed 5-fold higher elastase activity per cell. Despite higher CD15+ neutrophil elastase activity, elastase activity was extremely low in plasma from the same patients. Collectively, the data supports the model that increased neutrophil and decreased T cell activity is associated with increased COVID-19 severity, and suggests that blood DEFA1 RNA levels and neutrophil elastase activity, both involved in neutrophil extracellular traps (NETs), may be informative biomarkers of host immune activity after viral infection.

RNAseq analysis of treatment-dependent signaling changes during inflammation in a mouse cutaneous wound healing model.

BACKGROUND: Despite proven therapeutic effects in inflammatory conditions, the specific mechanisms of phytochemical therapies are not well understood. The transcriptome effects of Traumeel (Tr14), a multicomponent natural product, and diclofenac, a non-selective cyclooxygenase (COX) inhibitor, were compared in a mouse cutaneous wound healing model to identify both known and novel pathways for the anti-inflammatory effect of plant-derived natural products. METHODS: Skin samples from abraded mice were analyzed by single-molecule, amplification-free RNAseq transcript profiling at 7 points between 12 and 192 h after injury. Immediately after injury, the wounds were treated with either diclofenac, Tr14, or placebo control (n = 7 per group/time). RNAseq levels were compared between treatment and control at each time point using a systems biology approach. RESULTS: At early time points (12-36 h), both control and Tr14-treated wounds showed marked increase in the inducible COX2 enzyme mRNA, while diclofenac-treated wounds did not. Tr14, in contrast, modulated lipoxygenase transcripts, especially ALOX12/15, and phospholipases involved in arachidonate metabolism. Notably, Tr14 modulated a group of cell-type specific markers, including the T cell receptor, that could be explained by an overarching effect on the type of cells that were recruited into the wound tissue. CONCLUSIONS: Tr14 and diclofenac had very different effects on the COX/LOX synthetic pathway after cutaneous wounding. Tr14 allowed normal autoinduction of COX2 mRNA, but suppressed mRNA levels for key enzymes in the leukotriene synthetic pathway. Tr14 appeared to have a broad 'phytocellular' effect on the wound transcriptome by altering the balance of cell types present in the wound.

RNA sequencing of blood in coronary artery disease: involvement of regulatory T cell imbalance.

BACKGROUND: Cardiovascular disease had a global prevalence of 523 million cases and 18.6 million deaths in 2019. The current standard for diagnosing coronary artery disease (CAD) is coronary angiography. Surprisingly, despite well-established clinical indications, up to 40% of the one million invasive cardiac catheterizations return a result of 'no blockage'. The present studies employed RNA sequencing of whole blood to identify an RNA signature in patients with angiographically confirmed CAD. METHODS: Whole blood RNA was depleted of ribosomal RNA (rRNA) and analyzed by single-molecule sequencing of RNA (RNAseq) to identify transcripts associated with CAD (TRACs) in a discovery group of 96 patients presenting for elective coronary catheterization. The resulting transcript counts were compared between groups to identify differentially expressed genes (DEGs). RESULTS: Surprisingly, 98% of DEGs/TRACs were down-regulated ~ 1.7-fold in patients with mild to severe CAD (> 20% stenosis). The TRACs were independent of comorbid risk factors for CAD, such as sex, hypertension, and smoking. Bioinformatic analysis identified an enrichment in transcripts such as FoxP1, ICOSLG, IKZF4/Eos, SMYD3, TRIM28, and TCF3/E2A that are likely markers of regulatory T cells (Treg), consistent with known reductions in Tregs in CAD. A validation cohort of 80 patients confirmed the overall pattern (92% down-regulation) and supported many of the Treg-related changes. TRACs were enriched for transcripts associated with stress granules, which sequester RNAs, and ciliary and synaptic transcripts, possibly consistent with changes in the immune synapse of developing T cells. CONCLUSIONS: These studies identify a novel mRNA signature of a Treg-like defect in CAD patients and provides a blueprint for a diagnostic test for CAD. The pattern of changes is consistent with stress-related changes in the maturation of T and Treg cells, possibly due to changes in the immune synapse.

COVID-19 infection among emergency department healthcare providers in a large tertiary academic medical center following the peak of the pandemic.

The COVID-19 pandemic has spread through the US during the last few months exposing healthcare providers to possible infection. Here we report testing of emergency department (ED) healthcare providers (HCP) for exposure to COVID-19 through lateral flow point of care (POC) and lab-based enzyme-linked immunosorbent assay (ELISA), and RTq-PCR for evidence of acute infection. 138 ED HCP were tested between May 26th (approximately one month after the peak of COVID-19 first wave of cases) and June 14th. Enrolled ED HCP represented about 70% of the total ED HCP workforce during the study period. Subjects were tested with a POC COVID-19 antibody test, and standard ELISA performed by a university-based research lab. Subjects also provided a mid-turbinate swab and a saliva specimen for RTq-PCR. All subjects provided demographic information, past medical history, information about personal protective equipment (PPE) use, COVID-19 symptoms, as well as potential COVID-19 exposures during the previous 4 weeks, both in the ED, and outside the clinical setting. None of the HCP had positive RT-PCR results; 7 HCP (5%) had positive IgG for COVID-19; there was strong agreement between the lab-based ELISA (reference test) and the POC Ab test (P ≤ 0.0001). For the POC Ab test there were no false negatives and only one false positive among the 138 participants. There was no significant difference in demographic/ethnic variables, past medical history, hours worked in the ED, PPE use, or concerning exposures between seropositive and seronegative individuals. Moreover, there was no significant difference in reported symptoms between the two groups during the previous four weeks. The rate of COVID-19 seroconversion in our ED was 5% during the month following the pandemic's first wave. Based on questionnaire responses, differences in demographics/ethnicity, medical history, COVID-19 exposures, and PPE use were not associated with ED HCP having been infected with SARS-CoV-2. In the setting of our limited cohort of subjects the COVID-19 POC Ab test performed comparably to the ELISA lab-based standard.

Deep Sequencing Transcriptome Analysis of Murine Wound Healing: Effects of a Multicomponent, Multitarget Natural Product Therapy-Tr14.

Wound healing involves an orchestrated response that engages multiple processes, such as hemostasis, cellular migration, extracellular matrix synthesis, and in particular, inflammation. Using a murine model of cutaneous wound repair, the transcriptome was mapped from 12 h to 8 days post-injury, and in response to a multicomponent, multi-target natural product, Tr14. Using single-molecule RNA sequencing (RNA-seq), there were clear temporal changes in known transcripts related to wound healing pathways, and additional novel transcripts of both coding and non-coding genes. Tr14 treatment modulated >100 transcripts related to key wound repair pathways, such as response to wounding, wound contraction, and cytokine response. The results provide the most precise and comprehensive characterization to date of the transcriptome's response to skin damage, repair, and multicomponent natural product therapy. By understanding the wound repair process, and the effects of natural products, it should be possible to intervene more effectively in diseases involving aberrant repair.

Acute appendicitis: transcript profiling of blood identifies promising biomarkers and potential underlying processes.

BACKGROUND: The diagnosis of acute appendicitis can be surprisingly difficult without computed tomography, which carries significant radiation exposure. Circulating blood cells may carry informative changes in their RNA expression profile that would signal internal infection or inflammation of the appendix. METHODS: Genome-wide expression profiling was applied to whole blood RNA of acute appendicitis patients versus patients with other abdominal disorders, in order to identify biomarkers of appendicitis. From a large cohort of emergency patients, a discovery set of patients with surgically confirmed appendicitis, or abdominal pain from other causes, was identified. RNA from whole blood was profiled by microarrays, and RNA levels were filtered by a combined fold-change (>2) and p value (<0.05). A separate set of patients, including patients with respiratory infections, was used to validate a partial least squares discriminant (PLSD) prediction model. RESULTS: Transcript profiling identified 37 differentially expressed genes (DEG) in appendicitis versus abdominal pain patients. The DEG list contained 3 major ontologies: infection-related, inflammation-related, and ribosomal processing. Appendicitis patients had lower level of neutrophil defensin mRNA (DEFA1,3), but higher levels of alkaline phosphatase (ALPL) and interleukin-8 receptor-ß (CXCR2/IL8RB), which was confirmed in a larger cohort of 60 patients using droplet digital PCR (ddPCR). CONCLUSIONS: Patients with acute appendicitis have detectable changes in the mRNA expression levels of factors related to neutrophil innate defense systems. The low defensin mRNA levels suggest that appendicitis patient's immune cells are not directly activated by pathogens, but are primed by diffusible factors in the microenvironment of the infection. The detected biomarkers are consistent with prior evidence that biofilm-forming bacteria in the appendix may be an important factor in appendicitis.

Metataxonomic and Metagenomic Approaches vs. Culture-Based Techniques for Clinical Pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

BACKGROUND: Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. HYPOTHESIS: Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. METHODS AND RESULTS: Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. CONCLUSIONS: HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.

Single-molecule long-read 16S sequencing to characterize the lung microbiome from mechanically ventilated patients with suspected pneumonia.

In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles.

Clinical PathoScope: rapid alignment and filtration for accurate pathogen identification in clinical samples using unassembled sequencing data.

BACKGROUND: The use of sequencing technologies to investigate the microbiome of a sample can positively impact patient healthcare by providing therapeutic targets for personalized disease treatment. However, these samples contain genomic sequences from various sources that complicate the identification of pathogens. RESULTS: Here we present Clinical PathoScope, a pipeline to rapidly and accurately remove host contamination, isolate microbial reads, and identify potential disease-causing pathogens. We have accomplished three essential tasks in the development of Clinical PathoScope. First, we developed an optimized framework for pathogen identification using a computational subtraction methodology in concordance with read trimming and ambiguous read reassignment. Second, we have demonstrated the ability of our approach to identify multiple pathogens in a single clinical sample, accurately identify pathogens at the subspecies level, and determine the nearest phylogenetic neighbor of novel or highly mutated pathogens using real clinical sequencing data. Finally, we have shown that Clinical PathoScope outperforms previously published pathogen identification methods with regard to computational speed, sensitivity, and specificity. CONCLUSIONS: Clinical PathoScope is the only pathogen identification method currently available that can identify multiple pathogens from mixed samples and distinguish between very closely related species and strains in samples with very few reads per pathogen. Furthermore, Clinical PathoScope does not rely on genome assembly and thus can more rapidly complete the analysis of a clinical sample when compared with current assembly-based methods. Clinical PathoScope is freely available at: http://sourceforge.net/projects/pathoscope/.

Engaging the next generation of healthcare professionals in genomics: planning for the future.

There is broad agreement that healthcare professionals require fundamental training in genomics to keep pace with scientific advancement. Strong models that promote effective genomic education, however, are lacking. Furthermore, curricula at many institutions are now straining to adapt to the integration of additional material on next-generation sequencing and the bioethical and legal issues that will accompany clinical genomic testing. This article advocates for core competencies focused on job function, which will best prepare providers to be end-users of healthcare information. In addition, it argues in favor of online and blended learning models that incorporate student genotyping and specific training in the ethical, legal and social issues raised by genomic testing.

Genomic profiling reveals the potential role of TCL1A and MDR1 deficiency in chemotherapy-induced cardiotoxicity.

BACKGROUND: Anthracyclines, such as doxorubicin (Adriamycin), are highly effective chemotherapeutic agents, but are well known to cause myocardial dysfunction and life-threatening congestive heart failure (CHF) in some patients. METHODS: To generate new hypotheses about its etiology, genome-wide transcript analysis was performed on whole blood RNA from women that received doxorubicin-based chemotherapy and either did, or did not develop CHF, as defined by ejection fractions (EF)≤40%. Women with non-ischemic cardiomyopathy unrelated to chemotherapy were compared to breast cancer patients prior to chemo with normal EF to identify heart failure-related transcripts in women not receiving chemotherapy. Byproducts of oxidative stress in plasma were measured in a subset of patients. RESULTS: The results indicate that patients treated with doxorubicin showed sustained elevations in oxidative byproducts in plasma. At the RNA level, women who exhibited low EFs after chemotherapy had 260 transcripts that differed >2-fold (p<0.05) compared to women who received chemo but maintained normal EFs. Most of these transcripts (201) were not altered in non-chemotherapy patients with low EFs. Pathway analysis of the differentially expressed genes indicated enrichment in apoptosis-related transcripts. Notably, women with chemo-induced low EFs had a 4.8-fold decrease in T-cell leukemia/lymphoma 1A (TCL1A) transcripts. TCL1A is expressed in both cardiac and skeletal muscle, and is a known co-activator for AKT, one of the major pro-survival factors for cardiomyocytes. Further, women who developed low EFs had a 2-fold lower level of ABCB1 transcript, encoding the multidrug resistance protein 1 (MDR1), which is an efflux pump for doxorubicin, potentially leading to higher cardiac levels of drug. In vitro studies confirmed that inhibition of MDR1 by verapamil in rat H9C2 cardiomyocytes increased their susceptibility to doxorubicin-induced toxicity. CONCLUSIONS: It is proposed that chemo-induced cardiomyopathy may be due to a reduction in TCL1A levels, thereby causing increased apoptotic sensitivity, and leading to reduced cardiac MDR1 levels, causing higher cardiac levels of doxorubicin and intracellular free radicals. If so, screening for TCL1A and MDR1 SNPs or expression level in blood, might identify women at greatest risk of chemo-induced heart failure.

Aspirin insensitive thrombophilia: transcript profiling of blood identifies platelet abnormalities and HLA restriction.

Aspirin is the most widely used antiplatelet agent because it is safe, efficient, and inexpensive. However, a significant subset of patients does not exhibit a full inhibition of platelet aggregation, termed 'aspirin resistance' (AR). Several major studies have observed that AR patients have a 4-fold increased risk of myocardial infarction (MI), stroke, and other thrombotic events. Arachidonic acid-stimulated whole blood aggregation was tested in 132 adults at risk for ischemic events, and identified an inadequate response to aspirin therapy in 9 patients (6.8%). Expression profiling of blood RNA by microarray was used to generate new hypotheses about the etiology of AR. Among the differentially expressed genes, there were decreases in several known platelet transcripts, including clusterin (CLU), glycoproteins IIb/IIIa (ITGA2B/3), lipocalin (LCN2), lactoferrin (LTF), and the thrombopoetin receptor (MPL), but with increased mRNA for the T-cell Th1 chemokine CXCL10. There was a strong association of AR with expression of HLA-DRB4 and HLA-DQA1. Similar HLA changes have been linked to autoimmune disorders, particularly antiphospholipid syndrome (APS), in which autoantibodies to phospholipid/protein complexes can trigger platelet activation. Consistent with APS, AR patients exhibited a 30% reduction in platelet counts. Follow-up testing for autoimmune antibodies observed only borderline titers in AR patients. Overall, these results suggest that AR may be related to changes in platelet gene expression creating a hyperreactive platelet, despite antiplatelet therapy. Future studies will focus on determining the protein levels of these differential transcripts in platelets, and the possible involvement of HLA restriction as a contributing factor.

Transforming growth factor-ß and atherosclerosis: interwoven atherogenic and atheroprotective aspects.

Age-related progression of cardiovascular disease is by far the largest health problem in the US and involves vascular damage, progressive vascular fibrosis and the accumulation of lipid-rich atherosclerotic lesions. Advanced lesions can restrict flow to key organs and can trigger occlusive thrombosis resulting in a stroke or myocardial infarction. Transforming growth factor-beta (TGF-ß) is a major orchestrator of the fibroproliferative response to tissue damage. In the early stages of repair, TGF-ß is released from platelets and activated from matrix reservoirs; it then stimulates the chemotaxis of repair cells, modulates immunity and inflammation and induces matrix production. At later stages, it negatively regulates fibrosis through its strong antiproliferative and apoptotic effects on fibrotic cells. In advanced lesions, TGF-ß might be important in arterial calcification, commonly referred to as "hardening of the arteries". Because TGF-ß can signal through multiple pathways, namely the SMADs, a MAPK pathway and the Rho/ROCK pathways, selective defects in TGF-ß signaling can disrupt otherwise coordinated pathways of tissue regeneration. TGF-ß is known to control cell proliferation, cell migration, matrix synthesis, wound contraction, calcification and the immune response, all being major components of the atherosclerotic process. However, many of the effects of TGF-ß are essential to normal tissue repair and thus, TGF-ß is often thought to be "atheroprotective". The present review attempts to parse systematically the known effects of TGF-ß on both the major risk factors for atherosclerosis and to isolate the role of TGF-ß in the many component pathways involved in atherogenesis.

Apoptotic role of IKK in T-ALL therapeutic response.

Despite considerable progress in the treatment of T cell acute lymphoblastic leukemia (T-ALL), it is still the highest risk malignancy among ALL. The outcome of relapsed patients remains dismal. The pro-survival role of NOTCH1 and NFκB in T-ALL is well documented; also, both factors were reported to be predictive of relapse. The NOTCH1 signaling pathway, commonly activated in T-ALL, was shown to enhance the transcriptional function of NFκB via several mechanisms. Thus, pharmacological inhibition of NOTCH1-NFκB signaling was suggested to be incorporated into existing T-ALL treatment protocols. However, conventional chemotherapy is based on activation of various types of stress, such as DNA damage, mitotic perturbations or endoplasmic reticulum overload. NFκB is frequently activated in response to stress and, depending on yet unknown mechanisms, it either protects cells from the drug action or mediates apoptosis. Here, we report that T-ALL cells respond to NFκB inhibition in opposite ways depending on whether they were treated with a stress-inducing chemotherapeutic agent or not. Moreover, we found that NOTCH1 enhances NFκB apoptotic function in the stressed cells. The data argue for further studies of NFκB status in T-ALL patients on different treatment protocols and the impact of activating NOTCH1 mutations on treatment response.

Apolipoprotein L6, induced in atherosclerotic lesions, promotes apoptosis and blocks Beclin 1-dependent autophagy in atherosclerotic cells.

Inflammatory cytokine-regulated apoptosis and autophagy play pivotal roles in plaque rupture and thrombosis of atherosclerotic lesions. However, the molecular interplay between apoptosis and autophagy in vascular cells has not been investigated. Our prior study showed that human apolipoprotein L6 (ApoL6), a pro-apoptotic BH3-only member of the Bcl-2 family, was one of the downstream targets of interferon-γ (INFγ), which sensitizes atherosclerotic lesion-derived cells (LDCs) to Fas-induced apoptosis. To investigate whether ApoL6 plays a causal role in atherosclerotic apoptosis and autophagy, in this study, we demonstrate that IFNγ treatment itself strongly induces ApoL6, and ApoL6 is highly expressed and partially co-localized with activated caspase 3 in activated smooth muscle cells in atherosclerotic lesions. In addition, overexpression of ApoL6 promotes reactive oxygen species (ROS) generation, caspase activation, and subsequent apoptosis, which can be blocked by pan caspase inhibitor and ROS scavenger. Knockdown of ApoL6 expression by siApoL6 suppresses INFγ- and Fas-mediated apoptosis. Further, ApoL6 binds Bcl-X(L), one of the most abundant anti-death proteins in LDCs. Interestingly, forced ApoL6 expression in LDCs induces degradation of Beclin 1, accumulation of p62, and subsequent attenuation of LC3-II formation and translocation and thus autophagy, whereas siApoL6 treatment reverts the phenotype. Taken together, our results suggest that ApoL6 regulates both apoptosis and autophagy in SMCs. IFNγ-initiated, ApoL6-induced apoptosis in vascular cells may be an important factor causing plaque instability and a potential therapeutic target for treating atherosclerosis and cardiovascular disease.