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Myeloid fatty acid metabolism activates neighboring hematopoietic stem cells to promote heart failure with preserved ejection fraction
No full textBACKGROUND: Despite the high morbidity and mortality of heart failure with preserved ejection fraction (HFpEF), treatment options remain limited. The HFpEF syndrome is associated with a high comorbidity burden, including high prevalence of obesity and hypertension. Although inflammation is implicated to play a key role in HFpEF pathophysiology, underlying causal mechanisms remain unclear. METHODS: Comparing patient samples and animal models, we defined the innate immune response during HFpEF in situ and through flow cytometry and single-cell RNA sequencing. After identifying transcriptional and cell signatures, we implemented a high-fat diet and hypertensive model of HFpEF and tested roles for myeloid and hematopoietic stem cells during HFpEF. Contributions of macrophage metabolism were also evaluated, including through mass spectrometry and carbon labeling. Primary macrophages were studied ex vivo to gain insight into complementary cell-intrinsic mechanisms. RESULTS: Here we report evidence that patients with cardiometabolic HFpEF exhibit elevated peripheral blood hematopoietic stem cells. This phenotype was conserved across species in a murine mode of high-fat diet and hypertension. Hematopoietic stem cell proliferation was coupled to striking remodeling of the peripheral hematopoietic stem cell niche and expression of the macrophage adhesion molecule Vcam1. This could be partially inhibited by sodium-glucose cotransporter-2 inhibitors and explained by elevated fatty acid metabolism in macrophage mitochondria, which in turn remodeled the Vcam1 promoter to enhance its expression. CONCLUSIONS: These findings identify a significant new stem cell signature of cardiometabolic HFpEF and support a role for myeloid maladaptive fatty acid metabolism in the promotion of systemic inflammation and cardiac diastolic dysfunction
Circulating extracellular vesicles as putative mediators of cardiovascular disease in paediatric chronic kidney disease
Get PDFCardiovascular disease (CVD) is the leading cause of mortality in chronic kidney disease (CKD). However, the pathogenesis of CVD in CKD remains incompletely understood. Endothelial extracellular vesicles (EC-EVs) have previously been associated with CVD. We hypothesized that CKD alters EV release and cargo, subsequently promoting vascular remodelling. We recruited 94 children with CKD, including patients after kidney transplantation and healthy donors, and performed EV phenotyping and functional EV analyses in the absence of age-related comorbidities. Plasma EC-EVs were increased in haemodialysis patients and decreased after kidney transplantation. Thirty microRNAs were less abundant in total CKD plasma EVs with predicted importance in angiogenesis and smooth muscle cell proliferation. In vitro, CKD plasma EVs induced transcriptomic changes in angiogenesis pathways and functionally impaired angiogenic properties, migration and proliferation in ECs. High shear stress, as generated by arterio-venous fistulas, and uremic toxins were considered as potential drivers of EV release, but only the combination increased EV generation from venous ECs. The resulting EVs recapitulated miRNA changes observed in CKD in vivo. In conclusion, CKD results in the release of EVs with altered miRNA profiles and anti-angiogenic properties, which may mediate vascular pathology in children with CKD. EVs and their miRNA cargo may represent future therapeutic targets to attenuate CVD in CKD
Endothelial but not systemic ferroptosis inhibition protects from antineutrophil cytoplasmic antibody-induced crescentic glomerulonephritis
Get PDFAntineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic autoimmune diseases featuring small blood vessel inflammation and organ damage, including necrotizing crescentic glomerulonephritis (NCGN). Persistent vascular inflammation leads to endothelial and kidney cell necrosis. Ferroptosis is a regulated cell death pathway executed by reactive oxygen species and iron-dependent lipid peroxidation culminating in cell membrane rupture. Here we show that ANCA-activated neutrophils induced endothelial cell (EC) death in vitro that was prevented by ferroptosis inhibition with Ferrostatin-1, Liproxstatin-1 and small inhibiting RNA against the enzyme AcylCoA Synthetase Long Chain Family Member 4 (ACSL4). In contrast, neither necroptosis nor apoptosis inhibition affected EC death. Moreover, both ferroptosis inhibitors alleviated lipid peroxide accumulation in EC. Increased lipid peroxidation was detected in kidney sections of AAV mice by immunohistochemistry. We generated MPO(-/-) ACSL4(flox) Tie2-Cre(+) mice lacking ACSL4 specifically in EC (ACSL4(ΔEC)) to study the significance of endothelial ferroptosis in vivo. ACSL4(ΔEC) chimeric mice, but not control mice (ACSL4(WT)), were protected from NCGN in a MPO-AAV bone-marrow transplantation model. These data establish that EC ferroptosis contributes to ANCA-induced glomerulonephritis. However, systemic pharmacological ferroptosis inhibition with Ferrostatin-1 or Liproxstatin-1 did not protect from NCGN in a murine AAV model. Ferrostatin-1 treatment both directly activated T cell proliferation and indirectly myeloid-mediated T cell proliferation and polarization in vitro. Conceivably, both effects may cancel the beneficial effect of endothelial ferroptosis inhibition. Mechanistically, we describe the importance of EC ferroptosis for the development of AAV. However, the lack of protection with systemic pharmacological ferroptosis inhibition should discourage clinicians from evaluating this treatment strategy in clinical AAV studies
SEC24C deficiency causes trafficking and glycosylation abnormalities in an epileptic encephalopathy with cataracts and dyserythropoeisis
Get PDFAs a major component of intracellular trafficking, the coat protein complex II (COPII) is indispensable for cellular function during embryonic development and throughout life. The 4 SEC24 proteins (A–D) are essential COPII components involved in cargo selection and packaging. A human disorder corresponding to alterations of SEC24 function is currently known only for SEC24D. Here, we reported that biallelic loss of SEC24C leads to a syndrome characterized by primary microcephaly, brain anomalies, epilepsy, hearing loss, liver dysfunction, anemia, and cataracts in an extended consanguineous family with 4 affected individuals. We showed that knockout of sec24C in zebrafish recapitulated important aspects of the human phenotype. SEC24C-deficient fibroblasts displayed alterations in the expression of several COPII components as well as impaired anterograde trafficking to the Golgi, indicating a severe impact on COPII function. Transcriptome analysis revealed that SEC24C deficiency also affected the proteasome and autophagy pathways. Moreover, a shift in the N-glycosylation pattern and deregulation of the N-glycosylation pathway suggested a possible secondary alteration of protein glycosylation, linking the described disorder with the congenital disorders of glycosylation
Senescence-associated lineage-aberrant plasticity evokes T-cell-mediated tumor control
Get PDFCellular senescence is a stress-inducible state switch relevant in aging, tumorigenesis and cancer therapy. Beyond a lasting arrest, senescent cells are characterized by profound chromatin remodeling and transcriptional reprogramming. We show here myeloid-skewed aberrant lineage plasticity and its immunological ramifications in therapy-induced senescence (TIS) of primary human and murine B-cell lymphoma. We find myeloid transcription factor (TF) networks, specifically AP-1-, C/EBPβ- and PU.1-governed transcriptional programs, enriched in TIS but not in equally chemotherapy-exposed senescence-incapable cancer cells. Dependent on these master TF, TIS lymphoma cells adopt a lineage-promiscuous state with properties of monocytic-dendritic cell (DC) differentiation. TIS lymphoma cells are preferentially lysed by T-cells in vitro, and mice harboring DC-skewed Eμ-myc lymphoma experience significantly longer tumor-free survival. Consistently, superior long-term outcome is also achieved in diffuse large B-cell lymphoma patients with high expression of a TIS-related DC signature. In essence, these data demonstrate a therapeutically exploitable, prognostically favorable immunogenic role of senescence-dependent aberrant myeloid plasticity in B-cell lymphoma
Carboxyhemoglobin and methemoglobin as biomarkers of hemolysis and mortality in acute respiratory distress syndrome treated by veno-venous extracorporeal membrane oxygenation
No full textBACKGROUND: Critically ill patients who receive circulatory or respiratory assist using extracorporeal membrane oxygenation (ECMO) may develop hemolysis, which can complicate the delivery of supportive care and be a potential risk factor for increased morbidity and mortality. Clinically, hemolysis is often identified using laboratory markers such as cell-free hemoglobin (CFH) and haptoglobin (Hp). However, such measurements require photometry or enzyme-linked immunosorbent assay (ELISA) and are labor intensive. In contrast, metabolic downstream products of CFH, such as carboxyhemoglobin (CO-Hb) and methemoglobin (Met-Hb), can be regularly monitored via arterial blood gas analyses in the intensive care unit (ICU). We hypothesized that CO-Hb and Met-Hb values measured during ECMO would correlate with the presence of hemolytic events as measured by CFH values exceeding 50mg/dl. We further hypothesized that CO-Hb and Met-Hb levels would correlate with peri-ECMO mortality. METHODS: Retrospective analysis of 435 patients with acute respiratory distress syndrome (ARDS) and veno-venous ECMO admitted to a tertiary ARDS referral center. Plasma concentrations of CO-Hb and Met-Hb were correlated with hemolytic events. Cutoff values of mean CO-Hb (mCO-Hb) and mean Met-Hb (mMet-Hb) associated with increased ICU mortality were calculated with recursive binary partitioning. Single and multivariable regression models for HE and ICU mortality were trained and compared. RESULTS: Simple and multivariable models including potential confounders identified associations between Met-Hb and hemolytic events (adj. odds ratio [OR] 2.99 [95% confidence interval {CI}, 2.19-4.10], P < .001). A cutoff value with 90% specificity of a hemolytic event was estimated for Met-Hb (1.55%). Both, mean CO-Hb (OR 2.03 [95% CI, 1.60-2.61], P < .001) and Met-Hb (2.78 [1.59-5.09], P < .001) were associated with ICU mortality. Cutoff values for mortality were 2% for mean CO-Hb and 1.25% for mean Met-Hb. The multivariable regression model for mortality including the continuous markers mCO-Hb and mMet-Hb produced an area under the curve (AUC) of 0.803. CONCLUSIONS: In patients with ARDS and ECMO, Met-Hb plasma concentrations were independently associated with hemolytic events. Both, mean CO-Hb and Met-Hb levels were associated with ICU mortality. These markers and their associated cutoff values might serve as a risk indicator in clinical practice
Blood biomarkers confirm subjective cognitive decline (SCD) as a distinct molecular and clinical stage within the NIA-AA framework of Alzheimer´s disease
Get PDFSubjective cognitive decline (SCD) is proposed as an indicator of transitional disease stage 2 in the Alzheimer’s disease (AD) continuum. However, molecular and particularly longitudinal fluid biomarker data for this stage are still limited. This study aimed to determine whether blood-based biomarkers in amyloid-positive individuals with SCD (A + SCD) support the notion of stage 2 as a distinct stage between stages 1 and 3 of AD and to identify those at high risk for clinical progression. In a prospective multicenter study (DELCODE) involving 457 participants across the AD continuum, we analyzed plasma phospho-tau 181 (p181) and neurofilament light chain (NfL) and assessed their association with longitudinal cognition, hippocampal atrophy, and AD clinical stage transition. The results showed that baseline plasma p181 levels were elevated and increased more rapidly in A + SCD individuals compared to amyloid-positive cognitively unimpaired (A + CU) individuals (stage 1). NfL levels rose across A + CU, A + SCD, and amyloid-positive mild cognitive impairment (A + MCI, stage 3). In A + SCD, but not in A + CU, higher p181 levels predicted cognitive decline (PACC5) and transition to MCI. In conclusion, plasma p181 provides molecular biomarker evidence supporting A + SCD as a pre-dementia AD stage (stage 2) distinct from A + CU (stage 1) and helps identify individuals at risk for cognitive decline early in the AD continuum
An integrated landscape of mRNA and protein isoforms
Get PDFAlternative splicing and proteolytic processing expand the functional diversity of the human proteome by generating distinct protein isoforms from a single gene. However, the extent to which transcript isoforms give rise to distinct protein products remains unclear, in part due to technical limitations in proteomic workflows. Here, we combine full-length mRNA sequencing with SDS-PAGE-based protein fractionation and quantitative mass spectrometry to construct an integrated landscape of mRNA and protein isoforms in human RPE-1 cells. To overcome the inherent ambiguity of bottom-up proteomics in isoform detection, we developed IsoFrac, a computational pipeline that resolves protein isoforms based on their migration profiles across gel fractions. This approach enabled the identification of ~45,000 full-length transcripts, ~32,000 ORFs, and ~16,000 distinct protein isoforms. Comparative analyses revealed widespread translation of alternative transcripts and uncovered proteolytic processing as a major, underappreciated source of proteome complexity. Our results establish a scalable framework for isoform-resolved proteogenomics and provide a reference resource for studying the molecular diversity encoded by the human genome
High-quality peptide evidence for annotating noncanonical open reading frames as human proteins
Get PDFA major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of noncanonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed
Multimodal longitudinal optical imaging reveals optic neuritis preceding retinal pathology in experimental autoimmune encephalomyelitis
Get PDFBACKGROUND AND OBJECTIVES: Inflammatory demyelinating diseases of the CNS, chief among them multiple sclerosis (MS), are a major cause of disability in young adults. Early manifestations of MS commonly involve visual dysfunction, which is often caused by optic neuritis and is accompanied by quantifiable structural changes of the anterior visual pathway. Retinal optical coherence tomography (OCT) has emerged as an important tool for clinical assessment of these structural alterations, but the underlying pathobiological mechanisms and temporal dynamics are yet poorly understood at a cellular level. METHODS: Using the experimental autoimmune encephalomyelitis (EAE) model of MS in fluorescent reporter mouse strains for neuronal function and innate immune cells, we use a unique combination of retinal intravital 2-photon microscopy (2PM) and OCT. In this fashion, we elucidate the spatiotemporal interplay of functional and structural retinal changes over the course of 1 month after EAE induction, with histopathologic imaging validating main results. RESULTS: While all mice display histologic signs of optic neuritis early after EAE induction and independently of motor symptom severity, retinal signs of neuronal stress and parenchymal immune activation spike well after clinical peak of disease, with no signs of lasting structural damage appearing within 1 month after EAE induction. Thus, local retinal endpoints appear to be functions of downstream axonal damage rather than of immediate immune activation directed at the retina. However, as early as 1 week after EAE induction, retinal 2PM can detect recruitment of perivascular immune cells towards the optic nerve (ON), providing the earliest sign of disease activity in otherwise clinically inconspicuous mice. DISCUSSION: Our work identifies the recruitment of CX3CR1+ cells to the ON as an early sign of disease underlining the importance of combined structural and functional retinal imaging for the spatiotemporal characterization of neuroinflammatory and neurodegenerative processes. It further proposes retinal phagocyte orientation, morphology, and abundance as potential surrogate markers for neurodegenerative activity