Heart-brain signaling in patent foramen ovale-related stroke: differential plasma proteomic expression patterns revealed with a 2-pass liquid chromatography-tandem mass spectrometry discovery workflow

Abstract

Patent foramen ovale (PFO) is highly prevalent and associated with more than 150,000 strokes per year. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear. Therefore, mapping the changes in systemic circulation of PFO-related stroke is crucial in understanding the pathophysiology to individualize the best clinical treatment for each patient. We initiated a study using a novel quantitative, 2-pass discovery workflow using high-resolution liquid chromatography-mass spectrometry/mass spectrometry coupled with label-free analysis to track protein expression in PFO patients before and after endovascular closure of the PFO. Using this approach, we were able to demonstrate quantitative differences in protein expression between both PFO-related and non-PFO-related ischemic stroke groups as well as before and after PFO closure. As an initial step in understanding the molecular landscape of PFO-related physiology, our methods have yielded biologically relevant information on the synergistic and functional redundancy of various cell-signaling molecules with respect to PFO circulatory physiology. The resulting protein expression patterns were related to canonical pathways including prothrombin activation, atherosclerosis signaling, acute-phase response, LXR/RXR activation, and coagulation system. In particular, after PFO closure, numerous proteins demonstrated reduced expression in stroke-related canonical pathways such as acute inflammatory response and coagulation signaling. These findings demonstrate the feasibility and robustness of using a proteomic approach for biomarker discovery to help gauge therapeutic efficacy in stroke.

Figure 1

Anatomy of normal versus PFO. Normally, the septum separates the left and right chambers of the heart. PFO is a congenital condition whereby the opening between the left and right atrial chambers fails to close after birth. 1a. Normal septum; 1b. PFO

Figure 2

Diagram of the Two Pass workflow. Left panel: full scan MS spectrum. Center panel: Screen capture of SIEVE analysis illustrating differential expression of a frame. Right panel: Fragmentation spectrum of a targeted mass from inclusion list generated from SIEVE differential analysis.

Figure 3

Number of identified proteins using the selected frame filter for Pass 2. Filter syntax: Ratio of PFOstroke/Normal > 1.5* Ratio of NonPFOstroke/Normal AND Ratio of NonPFOstroke/Normal > Ratio of Normal/Normal

Figure 4

Differentially expressed proteins (Pvalue <0.01) in PFO stroke samples vs Non PFO. stroke samples. Protein search stringency parameters: FDR <1%, peptides>1.

Figure 5

Protein expression patterns before, during and after PFO endovascular closure. As described in the methods, longitudinal blood samples were taken from a cohort of 4 patients upon admission, from left and right chambers immediately before and after closure and at 3 month follow up. A. Proteins with increased expression levels (ratio >1.2, Pvalue <0.01) in 3 month follow up post-closure venous plasma (relative to pre-closure venous plasma) samples. Corresponding expression levels of PSLA, PSRA, PRLA and PRRA plasma for each patient are also shown. B. Proteins with decreased expression levels (ratio <0.5, Pvalue<0.01) in 3 month follow up post-closure venous plasma (relative to pre-closure venous plasma) samples. Matched expression levels of PSLA, PSRA, PRLA and PRRA plasma for each patient are also shown.

Figure 6

Protein expression pattern before, during and after PFO endovascular closure. * Pvalue < 0.05 ** Pvalue < 0.001

Figure 7

IPA canonical pathway analysis of PFO closure dataset. P-value <0.0001