MitoPlex: A targeted multiple reaction monitoring assay for quantification of a curated set of mitochondrial proteins.

Mitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. The organelles play an especially important role in the maintenance of cardiac homeostasis; their inability to generate ATP following impairment due to ischemic damage has been directly linked to organ failure. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondrial DNA, or relative quantification by untargeted mass spectrometry. Here, we present a high throughput, reproducible and quantitative mass spectrometry multiple reaction monitoring based assay of 37 proteins critical to central carbon chain metabolism and overall mitochondrial function termed 'MitoPlex'. We coupled this protein multiplex with a parallel analysis of the central carbon chain metabolites (219 metabolite assay) extracted in tandem from the same sample, be it cells or tissue. In tests of its biological applicability in cells and tissues, "MitoPlex plus metabolites" indicated profound effects of HMG-CoA Reductase inhibition (e.g., statin treatment) on mitochondria of i) differentiating C2C12 skeletal myoblasts, as well as a clear opposite trend of statins to promote mitochondrial protein expression and metabolism in heart and liver, while suppressing mitochondrial protein and ii) aspects of metabolism in the skeletal muscle obtained from C57Bl6 mice. Our results not only reveal new insights into the metabolic effect of statins in skeletal muscle, but present a new high throughput, reliable MS-based tool to study mitochondrial dynamics in both cell culture and in vivo models.

Identification of Putative Early Atherosclerosis Biomarkers by Unsupervised Deconvolution of Heterogeneous Vascular Proteomes.

Coronary artery disease remains a leading cause of death in industrialized nations, and early detection of disease is a critical intervention target to effectively treat patients and manage risk. Proteomic analysis of mixed tissue homogenates may obscure subtle protein changes that occur uniquely in underlying tissue subtypes. The unsupervised 'convex analysis of mixtures' (CAM) tool has previously been shown to effectively segregate cellular subtypes from mixed expression data. In this study, we hypothesized that CAM would identify proteomic information specifically informative to early atherosclerosis lesion involvement that could lead to potential markers of early disease detection. We quantified the proteome of 99 paired abdominal aorta (AA) and left anterior descending coronary artery (LAD) specimens (N = 198 specimens total) acquired during autopsy of young adults free of diagnosed cardiac disease. The CAM tool was then used to segregate protein subsets uniquely associated with different underlying tissue types, yielding markers of normal and fibrous plaque (FP) tissues in LAD and AA (N = 62 lesions markers). CAM-derived FP marker expression was validated against pathologist estimated luminal surface involvement of FP, as well as in an orthogonal cohort of "pure" fibrous plaque, fatty streak, and normal vascular specimens. A targeted mass spectrometry (MS) assay quantified 39 of 62 CAM-FP markers in plasma from women with angiographically verified coronary artery disease (CAD, N = 46) or free from apparent CAD (control, N = 40). Elastic net variable selection with logistic regression reduced this list to 10 proteins capable of classifying CAD status in this cohort with <6% misclassification error, and a mean area under the receiver operating characteristic curve of 0.992 (confidence interval 0.968-0.998) after cross validation. The proteomics-CAM workflow identified lesion-specific molecular biomarker candidates by distilling the most representative molecules from heterogeneous tissue types.

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP.

Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7-3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4-3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5ß (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP.

Human iPSC-Derived Blood-Brain Barrier Chips Enable Disease Modeling and Personalized Medicine Applications.

The blood-brain barrier (BBB) tightly regulates the entry of solutes from blood into the brain and is disrupted in several neurological diseases. Using Organ-Chip technology, we created an entirely human BBB-Chip with induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (iBMECs), astrocytes, and neurons. The iBMECs formed a tight monolayer that expressed markers specific to brain vasculature. The BBB-Chip exhibited physiologically relevant transendothelial electrical resistance and accurately predicted blood-to-brain permeability of pharmacologics. Upon perfusing the vascular lumen with whole blood, the microengineered capillary wall protected neural cells from plasma-induced toxicity. Patient-derived iPSCs from individuals with neurological diseases predicted disease-specific lack of transporters and disruption of barrier integrity. By combining Organ-Chip technology and human iPSC-derived tissue, we have created a neurovascular unit that recapitulates complex BBB functions, provides a platform for modeling inheritable neurological disorders, and advances drug screening, as well as personalized medicine.

In Vitro and In Vivo Proteomic Comparison of Human Neural Progenitor Cell-Induced Photoreceptor Survival.

Retinal degenerative diseases lead to blindness with few treatments. Various cell-based therapies are aimed to slow the progression of vision loss by preserving light-sensing photoreceptor cells. A subretinal injection of human neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) rat model of retinal degeneration has aided in photoreceptor survival, though the mechanisms are mainly unknown. Identifying the retinal proteomic changes that occur following hNPC treatment leads to better understanding of neuroprotection. To mimic the retinal environment following hNPC injection, a co-culture system of retinas and hNPCs is developed. Less cell death occurs in RCS retinal tissue co-cultured with hNPCs than in retinas cultured alone, suggesting that hNPCs provide retinal protection in vitro. Comparison of ex vivo and in vivo retinas identifies nuclear factor (erythroid-derived 2)-like 2 (NRF2) mediated oxidative response signaling as an hNPC-induced pathway. This is the first study to compare proteomic changes following treatment with hNPCs in both an ex vivo and in vivo environment, further allowing the use of ex vivo modeling for mechanisms of retinal preservation. Elucidation of the protein changes in the retina following hNPC treatment may lead to the discovery of mechanisms of photoreceptor survival and its therapeutic for clinical applications.

Proteomic Architecture of Human Coronary and Aortic Atherosclerosis.

BACKGOUND: The inability to detect premature atherosclerosis significantly hinders implementation of personalized therapy to prevent coronary heart disease. A comprehensive understanding of arterial protein networks and how they change in early atherosclerosis could identify new biomarkers for disease detection and improved therapeutic targets. METHODS: Here we describe the human arterial proteome and proteomic features strongly associated with early atherosclerosis based on mass spectrometry analysis of coronary artery and aortic specimens from 100 autopsied young adults (200 arterial specimens). Convex analysis of mixtures, differential dependent network modeling, and bioinformatic analyses defined the composition, network rewiring, and likely regulatory features of the protein networks associated with early atherosclerosis and how they vary across 2 anatomic distributions. RESULTS: The data document significant differences in mitochondrial protein abundance between coronary and aortic samples (coronary>>aortic), and between atherosclerotic and normal tissues (atherosclerotic<

Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier.

Inactivating mutations in the thyroid hormone (TH) transporter Monocarboxylate transporter 8 (MCT8) cause severe psychomotor retardation in children. Animal models do not reflect the biology of the human disease. Using patient-specific induced pluripotent stem cells (iPSCs), we generated MCT8-deficient neural cells that showed normal TH-dependent neuronal properties and maturation. However, the blood-brain barrier (BBB) controls TH entry into the brain, and reduced TH availability to neural cells could instead underlie the diseased phenotype. To test potential BBB involvement, we generated an iPSC-based BBB model of MCT8 deficiency, and we found that MCT8 was necessary for polarized influx of the active form of TH across the BBB. We also found that a candidate drug did not appreciably cross the mutant BBB. Our results therefore clarify the underlying physiological basis of this disorder, and they suggest that circumventing the diseased BBB to deliver active TH to the brain could be a viable therapeutic strategy.

Association between chronic organochlorine exposure and immunotoxicity in the round stingray (Urobatis halleri).

Chronic organochlorine (OC) exposure has been shown to cause immune impairment in numerous vertebrate species. To determine if elasmobranchs exhibited compromised immunity due to high OC contamination along the coastal mainland of southern California, innate immune function was compared in round stingrays (Urobatis halleri) collected from the mainland and Santa Catalina Island. Proliferation and phagocytosis of peripheral blood, splenic, and epigonal leukocytes were assessed. Percent phagocytosis and mean fluorescence intensity (MFI) were evaluated by quantifying % leukocytes positive for, and relative amounts of ingested fluorescent E. coli BioParticles. Total cell proliferation differed between sites, with mainland rays having a higher cell concentration in whole blood. ∑PCB load explained significantly higher % phagocytosis in blood of mainland rays, while ∑PCB and ∑pesticide loads described increased splenic % phagocytosis and MFI in the mainland population. Data provides evidence of strong OC-correlated immunostimulation; however, other site-specific environmental variables may be contributing to the observed effects.

Macrophage molecular signaling and inflammatory responses during ingestion of atherogenic lipoproteins are modulated by complement protein C1q.

BACKGROUND AND AIMS: This study investigated the effect of innate immune protein C1q on macrophage programmed responses during the ingestion of atherogenic lipoproteins. C1q plays a dual role in atherosclerosis where activation of complement by C1q is known to drive inflammation and promote disease progression. However, C1q is atheroprotective in early disease using mouse models. Our previous studies have highlighted a non-complement associated role for C1q in polarizing macrophages towards an M2-like anti-inflammatory phenotype during ingestion of targets such as atherogenic lipoproteins. This study aims to investigate the molecular mechanisms involved. METHODS: We investigated the molecular signaling mechanisms involved in macrophage polarization using an unbiased examination of gene expression profiles in human monocyte derived macrophages ingesting oxidized or acetylated low density lipoproteins in the presence or absence of C1q. RESULTS: Expression of genes involved in Janus kinase and signal transducer and activator of transcription (JAK-STAT) signaling, peroxisome proliferator activating receptor (PPAR) signaling and toll-like receptor (TLR) signaling were modulated by C1q in this screen. C1q was also shown to significantly suppress JAK-STAT pathway activation (a maximum 55% ± 13% reduction, p = 0.044) and increase transcriptional activation of PPARs (a maximum 229% ± 54% increase, p = 0.0002), consistent with an M2-like polarized response. These pathways were regulated in macrophages by C1q bound to different types of modified atherogenic lipoprotein and led to a reduction in the release of pro-inflammatory cytokine IL-6. CONCLUSIONS: This study identifies potential molecular mechanisms for the beneficial role C1q plays in early atherosclerosis.

Complement protein C1q promotes macrophage anti-inflammatory M2-like polarization during the clearance of atherogenic lipoproteins.

OBJECTIVE: Innate immune protein C1q plays a dual role in the chronic inflammatory disease of atherosclerosis. Complement activation via C1q exacerbates pathology in the atherosclerotic lesion in later stages of the disease. However, in early stages of disease C1q is protective. We hypothesize that complement-independent activities of C1q are involved in reprogramming macrophage inflammatory polarization. METHODS: The influence of C1q on macrophage inflammatory responses during clearance of oxLDL was examined. Changes in cytokines at the gene and protein level were measured by quantitative PCR and ELISA assay. RESULTS: C1q modulated cytokine expression in Raw264.7 macrophages during ingestion of oxLDL. Levels of pro-inflammatory cytokines IL-1ß and IL-6 were downregulated by C1q, whereas levels of the anti-inflammatory cytokine IL-10 were increased. In addition, data from an NFκB-luciferase gene reporter assay suggest that C1q suppresses activation of NFκB during lipoprotein clearance in macrophages, providing one mechanism by which C1q downregulates pro-inflammatory cytokine production. CONCLUSIONS: C1q-polarization of macrophages toward an anti-inflammatory (M2-like) phenotype may be important in dampening inflammation in the early atherosclerotic lesion. Further investigation of molecular pathways targeted by C1q may provide novel therapeutic targets for this disease.