Fibroblast

SMC

EC

Elastin

SUMMARY

Qualitative Metabolic Flux Study Reveals the Metabolism Change in Pulmonary Arterial Smooth Muscle Cells From Patient and Donor Control

RESULTSBACKGROUNDPulmonary arterial hypertension (PAH) is life-threatening with progressive resistance to pulmonary blood flow that culminated in heart failure. Except for lung transplant, there is still no effective therapies for PAH. Hyperproliferative pulmonary arterial smooth muscle cells (PASMC) that occlude the lumen of pulmonary arterial could be therapeutic targets for PAH. We used cultured cells from patients with hereditary or idiopathic (H/I) PAH and donor control to identify the abnormal cell metabolism in the pathogenesis of PAH.

Flux analysis [U-13C6] glucose was applied as the labeled tracer. For the measurement of the turnover rate of the carbohydrate metabolism process. The key metabolites in both the glycolysis and TCA cycle was qualified at different time point to characterize the turnover rate of the pathways.

Qualitative flux analysis is most powerful when used to follow the fate of a stable labeled isotope tracer through the context of biological pathways. To visualize the Profinder results in a pathways context, a network was created in Omix Premium from the human BioCyc database and Profinder results were imported into Omix Premium. Figure 4 shows the resul ts o f label incorporation of metabolites in glycolysis and TCA cycle in donor control PASMC, PAH PASMC and PAH PASMC with siALDH.

ACKNOWLEDGEMENT

Dan Li1, Songjie Chen2, Yuqin Dai3, Michael Snyder2, Marlene Rabinovitch1

1Vera Moulton Wall Center for Pulmonary Vascular Diseases, Stanford Cardiovascular Institute, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, 2Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, 3Agilent Technologies, 5301 Stevens Creek Blvd, Santa Clara, CA 95051

Symptom: • Shortness of breath • Reduced exercise tolerance • Dizziness • Chest pain • Syncope

Pathology • Narrowing and stiffness of pulmonary arteries • High pressure in pulmonary arteries • Enlarged right ventricle

Medial Hypertrophy of Muscular Arteries • Swelling of ECs • Fragmented elastin • Proliferation of fibroblasts and SMC

Ctrl PAH

HE staining of lung tissue from donor control and PAH patient.

Figure 1. Agilent MassHunter VistaFlux Solution

[U-13C6] glucose was applied as a tracer to study the dynamic energy flux changes. Agilent MassHunter VistaFlux solution (Figure 1) was used for qualitative targeted flux analysis. As shown in Figure 1, Agilent MassHunter Pathways to PCDL and PCDL Manager was used to create a target metabolite list. The Pathways to PCDL software is designed to facilitate the creation of a custom Agilent Personal Compound Database and Library (PCDL) from metabolites present in pathway content contained within popular databases such as BioCyc, KEGG, and WikiPathways. Multiple pathways can be selected for investigating more complex biological networks. The resulting PCDL can be viewed and edited in Agilent MassHunter PCDL Manager. Metabolites that are not relevant to the experiment can be deleted, and additional compounds can be appended to the PCDL. For targeted isotopologue analysis, retention times are added for all metabolites of interest. This custom PCDL is then used for targeted mining of isotopologues in LC/MS data using Agilent MassHunter Profinder software. The Profinder batch results corrected for 13C natural abundance, were exported to Omix Premium as a Profinder archive file (.PFA), which includes sample group information, compound identifiers, and isotopologue abundances. Omix Premium offers graphical options specifically designed for the visualization and interpretation of qualitative flux data including the ability to change properties of the metabolite node, and to add associated charts and graphics.

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Figure 2: Agilent MassHunter Profinder results summary for a 13C label time course flux study

Figure 3: Agilent MassHunter Profinder Isotopologue display of lactic acid (A) donor control, (B) PAH, and (C) PAH with siALDH

1. PAH PASMC has higher glycolytic function compared to donor control reflected by the turnover rate of lactate.

2. siALDH in PAH PASMC decreases the glycolytic function in PAH PASMC.

3. PAH PASMC has lower TCA cycle turnover rate compared to donor control.

4. PAH PASMC has faster turnover rate of acetyl-CoA compared to donor control.

5. siALDH decreases the turnover rate of acetyl-CoA in PAH PASMC.

CONCLUSION

1. The hyperproliferative phenotype of PASMC in PAH might be resulted from the higher energy production from glycolysis.

2. siALDH decreases the hyperproliferative phenotype of PASMC in PAH, which might be due to the lower energy production from glycolysis and TCA cycle.

3. The faster incorporation rate of acetyl-CoA at 0.5 hr and lower rate at 1 hr suggested that the PAH PASMS may convert the acetyl-CoA to acetylate histones.

4. siALDH decreases turnover rate of acetyl-CoA, which resulted in a decrease of the histone acetylation.

5. ALDH may become a potential promising target for PAH therapy.

6. This study also revealed a novel mechanism that may help understanding the biological process in some aggressive cancer subtypes marked by ALDH.

ALDH is found to be increased in PAH PASMC. Depleting ALDH decreases the proliferation of PAH PASMC and relive pulmonary hypertension in mice model.

WORKFLOW

Figure 4: Summary of the [U-13C6] glucose labelling of the TCA and glycolysis metabolites.

The isotopologue display in Profinder provides useful insight into the incorporation of the stable label, as well as reproducibility of replicates with a sample group. This can be viewed as raw values or % corrected values, with and without an additional correction for natural isotope abundance. Figure 3 shows the isotopologue results for lactic acid. Results are displayed as natural abundance-corrected values grouped by time point. Initial inspection of the data shows that over time a decrease in lactic acid M+0 is observed with a corresponding increase in lactic acid M+3, revealing that all three carbons of lactic acid are being derived from the [U-13C6] glucose tracer, and that after 8 hours the majority of the lactic acid has become labeled. We analyzed the dynamic metabolomic changes in PASMC of donor control, PAH and PAH with siALDH.

METHODOLOGYLung Tissue (pulmonary arterial smooth muscle cells): We received lung tissue from PAH patients and Controls through the Pulmonary Hypertension Breakthrough Initiative (PHBI). All patients and controls have consented to have their explanted lungs donated to investigators for research purposes. PASMC are obtained from unused donor lungs and from patients with PAH. All material will be de-identified. Cell culture: Control or PAH PASMC were cultured in SM culture medium and 5% FBS. The passage 3-8 of the Control or PAH PASMC were used for this study. siALDH was carried out by siRNA.

Flux study: PASMC were washed with glucose-free medium and subsequently incubated with 1 g/mL of [U-13C6] glucose. Cells were quenched at different time points of 0.1, 0.5, 1, 2, and 8 hrs by a rapid wash with ice-cold PBS, lysed, snap frozen, and extracted using 90%MeOH.

LC/MS method: An IP-RP Q-TOF LC/MS method was used for separation and detection of metabolites from selected cell extracts. Flux data were acquired on a high resolution accurate mass Agilent 6545 Q-TOF LC/MS, and Agilent VistaFlux software was used for the data analysis.

RESULTSTargeted Isotopologue Extraction MassHunter Profinder is a fast batch-processing software for mass spectrometric data that provides quality review and visualization of metabolite isotopologue incorporation. After batch isotopologue extraction in Profinder, results were reviewed and manually curated as necessary. For each metabolite, Profinder displays a compound detail table, the isotopologue information, the compound extracted ion chromatogram, and the compound mass spectrum for each LC/MS file in the batch (Figure 2).

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For Research Use Only. Not for use in diagnostic procedures.