Proteomics-Based Metabolic Modeling Reveals That Fatty Acid Oxidation (FAO) Controls Endothelial Cell (EC) Permeability

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability.Endothelial cells (ECs)¹ line the inner layer of the blood vessel wall and constitute a barrier between blood and surrounding tissue. As such, a tight regulation of EC permeability is crucial to maintain vessel functionality and avoid excessive extravasation of fluid and plasma proteins (1). Increased endothelial permeability is typical in inflammatory states and a hallmark of diseases such thrombosis, atherosclerosis, and cancer (2, 3). Because of their unique localization, ECs are constantly exposed to oxygen and nutrients that fuel cell metabolism and whose levels vary in physiological and pathological conditions. Yet, how cell metabolism regulates endothelial permeability remains incompletely understood.Previous studies have reported that EC cultures use glucose as predominant source of energy by producing lactate through glycolysis. However, also fatty acids and glutamine contribute to ATP and metabolic intermediate production (4–7). Recent in vivo studies have shown that glycolysis is necessary for EC proliferation and motility in physiological and pathological angiogenesis (4, 8). Moreover, the peroxisome proliferator-activated receptor gamma coactivator 1-α, which can activate oxidative phosphorylation, blocks EC sprouting in diabetes (9). The intriguing information emerging from these studies is that key metabolic pathways, such as glycolysis and oxidative phosphorylation in the mitochondria, play an important role in ECs and that they are actively involved in the regulation of key cell functions.Mitochondrial fatty acid oxidation (FAO) is the process that converts fatty acids (FAs) into acetyl-CoA, which fuels the tricarboxylic acid cycle (TCAc) and generates reducing factors for producing ATP via oxidative phosphorylation. Cells can incorporate FAs from the culture media or can generate FAs from the hydrolysis of triglycerides or through de novo synthesis. FAs, then, can access the mitochondria according to their length; whereas short and medium-chain FAs (up to 12 carbon atoms) diffuse through the mitochondrial membrane, long-chain FAs (with 13–21 carbon atoms) are actively transported by the carnitine O-palmitoyl transferase (CPT) proteins, which are rate-limiting enzymes for this pathway (10). Previous work suggested that FAO is poorly utilized by EC cultures (4), however, under certain stress conditions such as glucose deprivation, FAO becomes a major source of energy (7). Although it is striking to note how cells can adapt and remodel their metabolism, the role of key FAO enzymes in the control of EC functions is still largely unclear.Because of the complexity of the cell metabolome, global-scale metabolomic studies for in depth and quantitative analysis of metabolic fluxes are still challenging and computational models have provided invaluable help to better understand cell metabolism. Among them, the integrative metabolic analysis tool (iMAT), which integrates gene expression data with genome-scale metabolic network model (GSMM), has been successfully used to predict enzyme metabolic flux in several model systems and diseases (11, 12). Because gene expression and protein levels do not always correlate, and because enzymes levels do not necessarily reflect their enzymatic activity or the flux of the reaction that they are involved in, iMAT uses expression data as cue for the likelihood, but not final determinant, of enzyme activity. Modern MS technology and robust approaches for protein quantification, such as stable-isotope labeling with amino acids in cell culture (SILAC) (13) and advanced label-free algorithms (14), allow global comparative proteomic analysis and accurate measurements of protein and post-translational modification levels (15). We reasoned that the integration of quantitative MS-proteomic data into GSMM could contribute to the study of cell metabolism. Moreover, metabolic changes trigger activation of protein kinases (16, 17) to rapidly remodel the intracellular signaling and enable cells to adapt to these sudden alterations. Protein phosphorylation therefore plays an important role in regulating cell response to metabolic alteration and may hide information on cellular pathways and functions controlled by specific metabolic activities. MS-based proteomic approaches therefore offer an additional opportunity to investigate in an unbiased manner the interplay between cell metabolism and cell function (18).We have previously shown (19) that when human primary ECs are cultured for 1 day on the three-dimensional matrix matrigel and assemble into a complex network, a simplified model that recapitulates some aspects of vascular network assembly in vivo (20), the levels of metabolic enzymes are profoundly regulated. This result suggested an interplay between cell metabolism and EC behavior. Here we investigate further this aspect. Integrating label-free quantitative MS-proteomics, predictive metabolic modeling and metabolomics we discovered increased FAO when ECs are assembled into a fully formed network. Moreover, by inhibiting CPT1 pharmacologically, we elucidated that FAO is a central regulator of EC permeability in vitro and blood vessel stability in vivo. Thus, proteomics significantly contributes to the study of cell metabolism and here we identified FAO as a promising target for therapeutic intervention for the control of pathological vascular permeability.     

Modeling cancer metabolism on a genome scale

Cancer cells have fundamentally altered cellular metabolism that is associated with their tumorigenicity and malignancy. In addition to the widely studied Warburg effect, several new key metabolic alterations in cancer have been established over the last decade, leading to the recognition that altered tumor metabolism is one of the hallmarks of cancer. Deciphering the full scope and functional implications of the dysregulated metabolism in cancer requires both the advancement of a variety of omics measurements and the advancement of computational approaches for the analysis and contextualization of the accumulated data. Encouragingly, while the metabolic network is highly interconnected and complex, it is at the same time probably the best characterized cellular network. Following, this review discusses the challenges that genome‐scale modeling of cancer metabolism has been facing. We survey several recent studies demonstrating the first strides that have been done, testifying to the value of this approach in portraying a network‐level view of the cancer metabolism and in identifying novel drug targets and biomarkers. Finally, we outline a few new steps that may further advance this field.     

Gain and loss of photosynthetic membranes during plastid differentiation in the shoot apex of Arabidopsis

Chloroplasts of higher plants develop from proplastids, which are undifferentiated plastids that lack photosynthetic (thylakoid) membranes. In flowering plants, the proplastid-chloroplast transition takes place at the shoot apex, which consists of the shoot apical meristem (SAM) and the flanking leaf primordia. It has been believed that the SAM contains only proplastids and that these become chloroplasts only in the primordial leaves. Here, we show that plastids of the SAM are neither homogeneous nor necessarily null. Rather, their developmental state varies with the specific region and/or layer of the SAM in which they are found. Plastids throughout the L1 and L3 layers of the SAM possess fairly developed thylakoid networks. However, many of these plastids eventually lose their thylakoids during leaf maturation. By contrast, plastids at the central, stem cell-harboring region of the L2 layer of the SAM lack thylakoid membranes; these appear only at the periphery, near the leaf primordia. Thus, plastids in the SAM undergo distinct differentiation processes that, depending on their lineage and position, lead to either development or loss of thylakoid membranes. These processes continue along the course of leaf maturation.     

Intracellular delivery of purine nucleoside phosphorylase (PNP) fused to protein transduction domain corrects PNP deficiency in vitro

Purine nucleoside phosphorylase (PNP) is an intracellular enzyme crucial for purine degradation. PNP defects result in metabolic abnormalities and fatal T cell immunodeficiency. Protein transduction domains (PTD) transfer molecules across biological membranes. We hypothesized that fusion of PTD to PNP (PTD-PNP) would be an effective method for treating PNP deficiency. We find that PTD-PNP rapidly enters PNP-deficient lymphocytes and increases intracellular enzyme activity for 96 h. Similar to endogenous PNP, PTD-PNP is predominantly distributed in the cytoplasm. PTD-PNP improve viability and correct abnormal functions of PNP-deficient T lymphocytes including their response to stimulation and IL-2 secretion. Intracellular transduction protects PTD-PNP from antibody neutralization and from elimination, which may also provide significant in vivo therapeutic advantages to PNP. In conclusion, PTD fusion is an attractive method for extended PNP intracellular enzyme replacement therapy for PNP-deficient patients as well as for the intracellular delivery of other proteins.     

An ELISA method for the detection and quantification of human heparanase

Heparanase is a mammalian endo-beta-D-glucuronidase that cleaves heparan sulfate side chains at a limited number of sites. Heparanase enzymatic activity is thought to participate in degradation and remodeling of the extracellular matrix and to facilitate cell invasion associated with tumor metastasis, angiogenesis, and inflammation. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase upregulation was detected in an increasing number of primary human tumors, correlating, in some cases, with poor postoperative survival and increased tumor vascularity. The present study was undertaken to develop a highly sensitive ELISA suitable for the determination and quantification of human heparanase in tissue extracts and body fluids. The assay preferentially detects the 8+50 kDa active heparanase heterodimer vs. the latent 65 kDa proenzyme and correlates with immunoblot analysis of heparanase containing samples. It detects heparanase at concentrations as low as 200 pg/ml and is suitable for quantification of heparanase in tissue extracts and urine.     

Sleep-Disordered Breathing and Mortality: A Prospective Cohort Study

Background

Sleep-disordered breathing is a common condition associated with adverse health outcomes including hypertension and cardiovascular disease. The overall objective of this study was to determine whether sleep-disordered breathing and its sequelae of intermittent hypoxemia and recurrent arousals are associated with mortality in a community sample of adults aged 40 years or older.

Methods and Findings

We prospectively examined whether sleep-disordered breathing was associated with an increased risk of death from any cause in 6,441 men and women participating in the Sleep Heart Health Study. Sleep-disordered breathing was assessed with the apnea–hypopnea index (AHI) based on an in-home polysomnogram. Survival analysis and proportional hazards regression models were used to calculate hazard ratios for mortality after adjusting for age, sex, race, smoking status, body mass index, and prevalent medical conditions. The average follow-up period for the cohort was 8.2 y during which 1,047 participants (587 men and 460 women) died. Compared to those without sleep-disordered breathing (AHI: <5 events/h), the fully adjusted hazard ratios for all-cause mortality in those with mild (AHI: 5.0–14.9 events/h), moderate (AHI: 15.0–29.9 events/h), and severe (AHI: ≥30.0 events/h) sleep-disordered breathing were 0.93 (95% CI: 0.80–1.08), 1.17 (95% CI: 0.97–1.42), and 1.46 (95% CI: 1.14–1.86), respectively. Stratified analyses by sex and age showed that the increased risk of death associated with severe sleep-disordered breathing was statistically significant in men aged 40–70 y (hazard ratio: 2.09; 95% CI: 1.31–3.33). Measures of sleep-related intermittent hypoxemia, but not sleep fragmentation, were independently associated with all-cause mortality. Coronary artery disease–related mortality associated with sleep-disordered breathing showed a pattern of association similar to all-cause mortality.

Conclusions

Sleep-disordered breathing is associated with all-cause mortality and specifically that due to coronary artery disease, particularly in men aged 40–70 y with severe sleep-disordered breathing. Please see later in the article for the Editors'' Summary     

Oligonucleotides are potent antioxidants acting primarily through metal ion chelation

We report on a rather unknown feature of oligonucleotides, namely, their potent antioxidant activity. Previously, we showed that nucleotides are potent antioxidants in Fe^II/Cu^I/II–H₂O₂ systems. Here, we explored the potential of 2′-deoxyoligonucleotides as inhibitors of the Fe^II/Cu^I/II-induced ·OH formation from H₂O₂. The oligonucleotides [d(A)_5,7,20; d(T)₂₀; (2′-OMe-A)₅] proved to be highly potent antioxidants with IC₅₀ values of 5–17 or 48–85 μM in inhibiting Fe^II/Cu^I- or Cu^II-induced H₂O₂ decomposition, respectively, thus representing a 40–215-fold increase in potency as compared with Trolox, a standard antioxidant. The antioxidant activity is only weakly dependent on the oligonucleotides’ length or base identity. We analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry and ¹H-NMR spectroscopy the composition of the d(A)₅ solution exposed to the aforementioned oxidative conditions for 4 min or 24 h. We concluded that the primary (rapid) inhibition mechanism by oligonucleotides is metal ion chelation and the secondary (slow) mechanism is radical scavenging. We characterized the Cu^I–d(A)₅ and Cu^II–d(A)₇ complexes by ¹H-NMR and ³¹P-NMR or frozen-solution ESR spectroscopy, respectively. Cu^I is probably coordinated to d(A)₅ via N1 and N7 of two adenine residues and possibly also via two phosphate/bridging water molecules. The ESR data suggest Cu^II chelation through two nitrogen atoms of the adenine bases and two oxygen atoms (phosphates or water molecules). We conclude that oligonucleotides at micromolar concentrations prevent Fe^II/Cu^I/II-induced oxidative damage, primarily through metal ion chelation. Furthermore, we propose the use of a short, metabolically stable oligonucleotide, (2′-OMe-A)₅, as a highly potent and relatively long lived (t _1/2 ~ 20 h) antioxidant.     

An ancient autosomal haplotype bearing a rare achromatopsia-causing founder mutation is shared among Arab Muslims and Oriental Jews

Numerous cultural aspects, mainly based on historical records, suggest a common origin of the Middle-Eastern Arab Muslim and Jewish populations. This is supported, to some extent, by Y-chromosome haplogroup analysis of Middle-Eastern and European samples. Up to date, no genomic regions that are shared among Arab Muslim and Jewish chromosomes and are unique to these populations have been reported. Here, we report of a rare achromatopsia-causing CNGA3 mutation (c.1585G>A) presents in both Arab Muslim and Oriental Jewish patients. A haplotype analysis of c.1585G>A-bearing chromosomes from Middle Eastern and European origins revealed a shared Muslim–Jewish haplotype, which is different from those detected in European patients, indicating a recurrent mutation stratified by a Jewish–Muslim founder effect. Comprehensive whole-genome haplotype analysis using 250 K single nucleotide polymorphism arrays revealed a large homozygous region of ~11 Mbp shared by both Arab Muslim and Oriental Jewish chromosomes. A subsequent microsatellite analysis of a 21.5 cM interval including CNGA3 and the adjacent chromosome 2 centromere revealed a unique and extremely rare haplotype associated with the c.1585G>A mutation. The age of the shared c.1585G>A mutation was calculated using the microsatellite genotyping data to be about 200 generations ago. A similar analysis of mutation age based on the Arab Muslim data alone showed that the mutation was unlikely to be the product of a recent gene flow event. The data present here demonstrate a large (11 Mbp) genomic region that is likely to originate from an ancient common ancestor of Middle-Eastern Arab Muslims and Jews who lived approximately 5,000 years ago.