The aim of the study was to discover the metabolomic changes in plasma that occur during human Ischemia-Reperfusion (I/R) injury and to evaluate the diagnostic utility of plasma metabolomic ...biomarkers for determination of myocardial injury. Deciphering the details of plasma metabolome in ST-segment elevation myocardial infarction (STEMI) patients before and after primary percutaneous coronary interventions (PPCI) would allow for better understanding of the mechanisms involved during acute myocardial ischemia and reperfusion in humans. We performed a detailed non-targeted metabolomic analysis of plasma from 27 STEMI patients who had undergone PPCI in the first 48 hrs employing a LC-MS approach. Plasma metabolome at ischemic condition was compared to multiple time points after PPCI which allowed us to focus on changes in the reperfusion phase. Classification of the differential metabolites based on chemical taxonomy identified a major role for lipids and lipid-derived molecules. Biochemical pathway analysis identified valine, leucine and isoleucine biosynthesis, vitamin B6 metabolism and glutathione metabolism as the most significant metabolic pathways representing early response to I/R injury. We also identified phenyl alanine, tyrosine, linoleic acid and glycerophospholipid metabolism as the most significant pathways representing late response to I/R injury. A panel of three metabolites pentadecanoic acid, linoleoyl carnitine and 1-linoleoylglycerophosphocholine was discovered to have diagnostic value in determining the extent of I/R injury based on cardiac biomarkers. Using a non-targeted LC-MS approach, we have successfully generated the most comprehensive data to date on significant changes in the plasma metabolome in STEMI patients who had undergone PPCI in the first 48 hrs showing that lipid metabolites represent the largest cohort of molecules undergoing significant change.
Atherosclerosis is usually the underlying cause of heart attacks, strokes and peripheral vascular diseases – collectively known as cardiovascular diseases. Oxidation of low density lipoprotein (LDL) ...and its lipid content has an important role in the formation of lipid-laden atherosclerotic plaques. Not much is known about the impact of oxidative stress on bioactive oxylipin molecules present in LDL. The aim of this study is to understand the changes in oxylipin molecules present in LDL characterized by varying degrees of LDL oxidation.
LDL was isolated from the pooled plasma of healthy normolipidemic volunteers and was subjected to in vitro copper-catalyzed oxidation for varying time intervals (0 h, 6 h, 12 h, 24 h and 30 h). At each time interval, oxylipins were isolated through solid phase extraction and quantified using a targeted LC/-MS/MS approach employing stable isotope dilution method.
Our results demonstrate that different forms of oxidized LDL (OxLDL) are characterized by specific oxylipin distribution and concentration. Compared to non-oxidized LDL, there is a significant increase in oxylipin generation (p ≤ 0.05) in OxLDL subjected to 12 h and 24 h of oxidation. Though linoleate derived oxylipins are the most abundant in OxLDL extracts, the concentration of particular oxylipin species differed with different degrees of oxidation. Specifically, two pro-inflammatory linoleate-derived triols, namely 9,10,13-triHOME and 9,12,13-triHOME, exhibited a concentration increase of ~25 fold in 12h-OxLDL compared to non-oxidized LDL. Moreover, Partial least squares Discriminant Analysis (PLS-DA) identified 10 oxylipins, primarily prostaglandins, which could serve as additional biomarkers for oxidative stress or cardiovascular risk assessment.
Our data suggests that oxidative stress induces profound changes in the oxylipin content of LDL and the pattern of change is based on the extent of oxidation.
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•Oxylipin profile of LDL changes dramatically during copper oxidation.•Oxidation of LDL generates new oxylipins, with both pro- and anti-inflammatory properties.•Oxylipin profile of LDL can determine the level of oxidation of the LDL particle.
Oxidized phospholipids (OxPLs) promote inflammation as well as low density lipoprotein (LDL) uptake in a variety of physiological and pathological states. Given the anti-inflammatory role of the ...cytokine IL-10, we investigated its modulatory effect on the production of oxidized phosphatidylcholines (OxPCs) as well as lipid metabolic responses in global myocardial ischemia/reperfusion (I/R) injury. Increased OxPCs levels, by 1-Palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (POVPC), promoted oxidative stress (OS) and cell death. OxPCs-mediated-OS, resulted in oxidized low-density lipoprotein receptor 1 (LOX-1) activation and upregulated the expression of toll-like receptor 2 (TLR2). IL-10-induced increase in proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulated LOX-1 as well as TLR2 inflammatory responses. Under stress conditions, phosphorylation of sterol regulatory element binding protein 1c (SREBP 1c) was prevented by IL-10. The latter also prevented the generation of OxPCs and reduced their ratio (OxPCs/PCs) during injury. LOX-1 activation also promoted SREBP1c-mediated TGF-βRII expression which was inhibited by IL-10. Both fragmented and non-fragmented OxPCs were elevated during I/R and this effect was attenuated by IL-10. The largest impact (two-threefold change at log
) was on PAzPC, (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine)-a fragmented OxPC. Thus it appears that among different OxPCs, IL-10 significantly reduces a single molecule (PAzPC)-mediated lipid metabolic responses in cardiomyocytes thereby mitigating inflammation and cell death.
Repetitive Transcranial Magnetic Stimulation rTMS is increasingly being used to treat Major Depressive Disorder MDD. Given that not all patients respond to rTMS, it would be clinically useful to have ...reliable biomarkers that predict treatment response. Oxidized phosphatidylcholine OxPC and some oxylipins are important plasma biomarkers of oxidative stress and inflammation. Not only is depression associated with oxidative stress, but rTMS has been shown to have anti-oxidative effects.
To investigate whether plasma oxolipidomics profiles could predict treatment response in patients with treatment resistant MDD.
Fourty-eight patients undergoing rTMS treatment for MDD were recruited along with nine healthy control subjects. Plasma OxPCs and oxylipins were extracted and analyzed through high performance liquid chromatography coupled with mass spectrometry. Patients with a Hamilton Depression Rating Scale score Ham-D ≤7 post-treatment were defined as having entered remission.
Fifty-seven OxPC and 32 oxylipin species were identified in our subjects. MDD patients who entered remission following rTMS had significantly higher pre-rTMS levels of total and fragmented OxPCs compared to non-remitters and controls one-way ANOVA, p<0.05. However, no significant changes in OxPC levels were found as a result of rTMS, regardless of treatment response p>0.05. No differences in plasma oxylipins were found between remitters and non-remitters at baseline.
Certain categories of OxPCs may be useful predictive biomarkers for response to rTMS treatment in MDD. Given that elevated oxidized lipids may indicate higher levels of oxidative stress and inflammation in the brain, patients with this phenotype of depression may be more receptive to rTMS treatment.
As an emerging platform technology, metabolomics offers new insights into the pathomechanisms associated with complex disease conditions, including cardiovascular diseases. It also facilitates ...assessing the risk of developing the disease before its clinical manifestation. For this reason, metabolomics is of growing interest for understanding the pathogenesis of acute coronary syndromes (ACS), finding new biomarkers of ACS, and its associated risk management. Metabolomics-based studies in ACS have already demonstrated immense potential for biomarker discovery and mechanistic insights by identifying metabolomic signatures (e.g., branched-chain amino acids, acylcarnitines, lysophosphatidylcholines) associated with disease progression. Herein, we discuss the various metabolomics approaches and the challenges involved in metabolic profiling, focusing on ACS. Special attention has been paid to the clinical studies of metabolomics and lipidomics in ACS, with an emphasis on ischemia/reperfusion injury.
Smurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor ...of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer.
Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation.
CNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines.
Altogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf2-CNKSR2 interaction may serve as a common strategy to control proliferation of human breast cancer cells by modulating CNKSR2 protein stability.
Chikungunya virus (CHIKV), a positive-stranded RNA virus, can cause neurological complications by infecting the major parenchymal cells of the brain such as neurons and astrocytes. A proteomic ...analysis of CHIKV-infected human astrocytic cell line U-87 MG revealed tight functional associations among the modulated proteins. The predominant cellular pathways involved were of transcription–translation machinery, cytoskeletol reorganization, apoptosis, ubiquitination, and metabolism. In the proteome, we could also identify a few proteins that are reported to be involved in host–virus interactions. One such protein, Nucleophosmin (NPM1)/B23, a nucleolar protein, showed enhanced cytoplasmic aggregation in CHIKV-infected cells. NPM1 aggregation was predominantly localized in areas wherein CHIKV antigen could be detected. Furthermore, we observed that inhibition of this aggregation using a specific NPM1 oligomerization inhibitor, NSC348884, caused a significant dose-dependent enhancement in virus replication. There was a marked increase in the amount of intracellular viral RNA, and ∼105-fold increase in progeny virions in infected cells. Our proteomic analysis provides a comprehensive spectrum of host proteins modulated in response to CHIKV infection in astrocytic cells. Our results also show that NPM1/B23, a multifunctional chaperone, plays a critical role in restricting CHIKV replication and is a possible target for antiviral strategies.
ST-segment elevation myocardial infarction (STEMI) occurs as a result of acute occlusion of the coronary artery. Despite successful reperfusion using primary percutaneous coronary intervention ...(PPCI), a large percentage of myocardial cells die after reperfusion, which is recognized as ischemia/reperfusion injury (I/R). There are rapid changes in plasma lipidome during myocardial reperfusion injury. However, the impact of coronary artery reperfusion on plasma oxylipins is unknown. This study aimed to investigate alterations in the oxylipin profiles of STEMI patients during ischemia and at various reperfusion time points following PPCI. Blood samples were collected from patients presenting with STEMI prior to PPCI (Isch, n = 45) and subsequently 2 h following successful reperfusion by PPCI (R-2 h, n = 42), after 24 h (R-24 h, n = 44), after 48 h (R-48 h, n = 43), and then 30 days post PPCI (R-30 d, n = 29). As controls, blood samples were collected from age- and sex-matched patients with non-obstructive coronary artery disease after diagnostic coronary angiography. High-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) using deuterated standards was used to identify and quantify oxylipins. In patients presenting with STEMI prior to reperfusion (Isch group), the levels of docosahexaenoic acid (DHA)-derived oxylipins were significantly higher when compared with controls. Their levels were also significantly correlated with the peak levels of creatine kinase (CK) and troponin T(TnT) before reperfusion (CK: r = 0.33,
= 0.046, TnT: r = 0.50,
= 1.00 × 10
). The total concentrations of oxylipins directly produced by 5-lipoxygenase (5-LOX) were also significantly elevated in the Isch group compared with controls. The ratio of epoxides (generated through epoxygenase) to diols (generated by soluble epoxide hydrolysis (sEH)) was significantly lower in the Isch group compared with the controls. Following reperfusion, there was an overall reduction in plasma oxylipins in STEMI patients starting at 24 h post PPCI until 30 days. Univariate receiver operating characteristic (ROC) curve analysis also showed that an elevated ratio of epoxides to diols during ischemia is a predictor of smaller infarct size in patients with STEMI. This study revealed a large alteration in plasma oxylipins in patients presenting with STEMI when compared with controls. Total oxylipin levels rapidly reduced post reperfusion with stable levels reached 24 h post reperfusion and maintained for up to 30 days post infarct. Given the shifts in plasma oxylipins following coronary artery reperfusion, further research is needed to delineate their clinical impact in STEMI patients.
The primary aim of the study is to investigate the temporal changes in plasma lipidome before and after reperfusion in patients with ST-segment elevation myocardial infarction (STEMI) and their ...association with myocardial injury. We found that 56% of the identified lipid species were significantly altered (corrected p< 0.05) in the first 24 h following reperfusion in patients with STEMI. Three lipid species, namely, acylcarnitine 18:2, TG 51:0, and LPC 17:1 were associated with a change in troponin concentration (delta troponin) and in-hospital cardiovascular events. Of these, acylcarnitine 18:2, and LPC 17:1 and their respective whole class levels, were significantly higher (p < 0.05) in the STEMI population than the age/sex-matched control subjects. Overall, our analyses showed a large shift in plasma lipidome in patients that undergo myocardial reperfusion. The differences found for acylcarnitines and LPC species and their association with both cardiac markers and cardiac outcomes need further validation.
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•Human plasma lipidome rapidly shifts during myocardial reperfusion injury•Novel plasma lipids are associated with cardiovascular events•Acylcarnitines and lysoPCs correlate with the extent of myocardial injury•Acute MI results in elevated plasma AC 18:2 and LPC 17:1 compared to controls
Cardiovascular medicine; Lipidomics
ST-segment Elevation Myocardial Infarction (STEMI) occurs as a result of acute occlusion of the coronary artery. Despite successful reperfusion using percutaneous coronary intervention (PCI), a large ...percentage of myocardial cells die after reperfusion which is recognized as ischemia/reperfusion injury (I/R). Oxidized phosphatidylcholines (OxPCs) are a group of oxidized lipids generated through non-enzymatic oxidation and have pro-inflammatory properties. This study aimed to examine the roles of OxPCs in a clinical setting of myocardial I/R.
Blood samples were collected from STEMI patients at presentation prior to primary PCI (PPCI) (Isch) and at 4 time-points post-PPCI, including 2 h (R-2 h), 24 h (R-24 h), 48 h (R-48 h), and 30 days (R-30 d) post-PPCI. As controls, blood samples were collected from patients with non-obstructive coronary artery disease after diagnostic coronary angiography. Aspiration thrombectomy was also performed in selected STEMI patients. High-performance lipid chromatography-electrospray mass spectrometry (LC-MS/MS) was used for OxPCs analysis.
Twenty-two distinct OxPC species were identified and quantified in plasma samples in patients presenting with STEMI. These compounds were categorized as fragmented and non-fragmented species. Total levels of OxPCs did not significantly differ between Isch and control groups. However, total levels of fragmented OxPCs increased significantly in the ischemic period compared with controls (Isch: 4.79 ± 0.94, Control: 1.69 ± 0.19 ng/μl of plasma,
< 0.05). Concentrations of non-fragmented OxPCs had significant reductions during ischemia compared to the control group (Isch: 4.84 ± 0.30, Control: 6.6 ± 0.51 ng/μl,
< 0.05). Levels of total OxPCs in patients with STEMI were not significantly different during reperfusion periods. However, fragmented OxPCs levels were elevated at 48 h post-reperfusion and decreased at 30 days following MI, when compared to R-2 h and R-24 h time points (Isch: 4.79 ± 0.94, R-2 h: 5.33 ± 1.17, R-24 h: 5.20 ± 1.1, R-48 h: 4.18 ± 1.07, R-30 d: 1.87 ± 0.31 ng/μl,
< 0.05). Plasma levels of two fragmented OxPCs, namely, POVPC and PONPC were significantly correlated with peak creatine kinase (CK) levels (
< 0.05). As with plasma levels, the dominant OxPC species in coronary aspirated thrombus were fragmented OxPCs, which constituted 77% of total OxPC concentrations.
Biologically active fragmented OxPC were elevated in patients presenting with STEMI when compared to controls. PONPC concentrations were subsequently increased after PPCI resulting in reperfusion. Moreover, levels of POVPC and PONPC were also associated with peak CK levels. Since these molecules are potent stimulators for cardiomyocyte cell death, therapeutics attenuating their activities can result in a novel therapeutic pathway for myocardial salvage for patients undergoing reperfusion therapy.
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