David McMullin

Proteomic Temporal Profile of Human Brain Endothelium After Oxidative Stress

Background and Purpose— Because brain endothelial cells exist at the neurovascular interface, they may serve as cellular reporters of brain dysfunction by releasing biomarkers into the circulation. Methods— We used proteomic techniques to screen conditioned media from human brain endothelial cultures subjected to oxidative stress induced by nitric oxide over 24 hours. Plasma samples from human stroke patients were analyzed by enzyme-linked immunosorbent assay. Results— In healthy endothelial cells, interaction mapping demonstrated cross-talk involving secreted factors, membrane receptors, and matrix components. In oxidatively challenged endothelial cells, networks of interacting proteins failed to emerge. Instead, inflammatory markers increased, secreted factors oscillated over time, and endothelial injury repair was manifested as changes in factors related to matrix integrity. Elevated inflammatory markers included heat shock protein, chemokine ligand-1, serum amyloid-A1, annexin-A5, and thrombospondin-1. Neurotrophic factors (prosaposin, nucleobindin-1, and tachykinin precursors) peaked at 12 hours, then rapidly decreased by 24 hours. Basement membrane components (fibronectin, desomoglein, profiling-1) were decreased. Cytoskeletal markers (actin, vimentin, nidogen, and filamin B) increased over time. From this initial analysis, the high-ranking candidate thrombospondin-1 was further explored in human plasma. Acute ischemic stroke patients had significantly higher thrombospondin-1 levels within 8 hours of symptom onset compared to controls with similar clinical risk factors (659±81 vs 1132±98 ng/mL; P<0.05; n=20). Conclusions— Screening of simplified cell culture systems may aid the discovery of novel biomarkers in clinical neurovascular injury. Further collaborative efforts are warranted to discover and validate more candidates of interest.

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.

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.

Pharmaco-proteomics opportunities for individualizing neurovascular treatment.

Abstract Neurovascular disease often involves multi-organ system injury. For example, patent foramen ovale (PFO) related ischemic strokes involve not just the brain, but also the heart, the lung, and the peripheral vascular circulation. For higher-risk but high-reward systemic therapy (e.g., thrombolytics, therapeutic hypothermia (TH), PFO closure) to be implemented safely, very careful patient selection and close monitoring of disease progression and therapeutic efficacy are imperative. For example, more than a decade after the approval of therapeutic hypothermic and intravenous thrombolysis treatments, they both remain extremely under-utilized, in part due to lack of clinical tools for patient selection or to follow therapeutic efficacy. Therefore, in understanding the complexity of the global effects of clinical neurovascular diseases and their therapies, a systemic approach may offer a unique perspective and provide tools with clinical utility. Clinical proteomic approaches may be promising to monitor systemic changes in complex multi-organ diseases – especially where the disease process can be ‘sampled’ in clinically accessible fluids such as blood, urine, and CSF. Here, we describe a ‘pharmaco-proteomic’ approach to three major challenges in translational neurovascular research directly at bedside – in order to better stratify risk, widen therapeutic windows, and explore novel targets to be validated at the bench – (i) thrombolytic treatment for ischemic stroke, (ii) therapeutic hypothermia for post-cardiac arrest syndrome, and (iii) treatment for PFO related paradoxical embolic stroke. In the future, this clinical proteomics approach may help to improve patient selection, ensure more precise clinical phenotyping for clinical trials, and individualize patient treatment.

Patent foramen ovale (PFO), stroke and pregnancy

Patent foramen ovale (PFO)-related stroke is increasingly recognized as an important etiology of ischemic embolic stroke—accounting for up to 50% of strokes previously considered ‘cryptogenic’ or with an unknown mechanism. As a ‘back door to the brain,’ PFO can allow venous clots to enter arterial circulation via interatrial right-to-left shunting, potentially resulting in ischemic stroke. We observe that clinically, PFO-related stroke affects women of childbearing age, and that pregnancy—owing to major changes in hemocoagulative, hormonal, and cardiovascular parameters—can enhance stroke risks. However, no systematic study has been performed and little is known regarding complications, pregnancy outcomes and treatment for PFO-related stroke during pregnancy. To identify and characterize the complications and clinical outcomes related to PFOs during pregnancy, we performed a literature review and analysis from all reported cases of pregnancy with PFO-related complications in the medical literature from 1970 to 2015. We find that during pregnancy and post-partum, PFO is associated with complications affecting multiple organs, including the brain, heart and lung. The three principal complications reported are stroke, pulmonary emboli and myocardial infarction. In contrast to other pregnancy-related stroke etiologies, which peak during later pregnancy and postpartum, PFO-related stroke peaks during early pregnancy (first and second trimester—60%), and most patients had good neurological outcome (77%). In patients with PFO with recurrent stroke during pregnancy, additional key factors include high-risk PFO morphology (atrial septal aneurysm), larger right-to-left shunt, multiple gestation and concurrent hypercoagulability. Compared to strokes of other etiologies during pregnancy, most PFO stroke patients experienced uneventful delivery (93%) of healthy babies with a good clinical outcome. We conclude with recommended clinical treatment strategies for pregnant patients with PFO suggested by the data from these cases, and the clinical experience of our Cardio-Neurology Clinic.

15: PFO CLOSURE REDUCES PLASMA LEVELS OF SEROTONIN IN A LONG TERM FOLLOWUP OF STROKE PATIENTS

Purpose of Study PFO allows venous clots and vasoactive factors to bypass pulmonary filtration and remain in circulation. We previously identified an immediate reduction of procoagulant serotonin (5-HT) in left atrial blood post PFO closure. To understand the long-term effect of PFO closure, we report the change of 5-HT in peripheral venous blood in 1-year followup. Methods Used 97 PFO-related stroke patients were recruited on IRB approval. Venous blood was collected at baseline (BL) and 1 year follow-up (FU) of treatments (PFO closure and medical therapy). Plasma 5-HT was quantified by mass spectrometry. Patients with serotonin modifying medications (ie. SSRIs) or conditions (anxiety/depression) were excluded. Summary of Results 5-HT level in peripheral venous blood was significantly reduced by 27.27% (BL: 7.57±8.04 µM; FU: 5.51±5.72 µM; p=0.0034) in 61 patients receiving PFO closure (figure 1A). In the 37 PFO patients treated with medicine alone, no changes were observed (BL: 5.79±7.15 µM; FU: 6.25±6.68 µM; p=0.4050) (figure 1B). 5-HT reduction was independently associated with PFO closure after adjusting for age, gender, medical history and medication status in a multivariate regression (figure 1C). Conclusions We found that PFO closure independently reduced 5-HT level in peripheral venous blood. These results support the hypothesis that PFO related right-to-left interatrial shunt may foster higher level of procoagulant and vascoactive substances in circulation. While PFO closure decrease prothrombotic markers immediately post closure, this effect is sustained in long term followup up to 1 year. Further studies on the clinical outcome of these PFO patients with respect to their prothrombotic circulatory profiles are ongoing. Abstract 15 Figure 1

MP13: IMPORTANT ROLE OF PROTHROMBIN TIME (PT) AND PARTIAL THROMBOPLASTIN TIME (PTT) IN PREDICTING TPA-RELATED HEMORRHAGIC TRANSFORMATION

Purpose of Study IV tPA is not routinely followed by blood work due to its reputed short half life. While there has been much focus on tPAs extra-vascular effects on the neurovascular unit in the context of hemorrhagic transformation (HT), little is known about its intravascular efficacy, where it has its intended effect. Emerging data suggest that tPA may be most effective in microvasculature and IV therapy may be a good adjunct to intra-arterial therapy. We previously found that the effect of tPA can last more than 72 hr after stroke onset. Now, we report that even routine blood labs can potentially predict HT. Methods Used 72 storke patients with IV tPA were recruited on IRB approval. Clinical coagulation profile (PT, PTT, fibrinogen and D-dimer) were performed at 12, 24, 72 hr post tPA. Patients on medications (e.g. anticoagulants) or with conditions (e.g. liver dysfunction, infection) that may affect these labs were excluded. Summary of Results Compared to those without HT, HT patients had significantly higher PT and PTT (Fig A,B) as early as 12 hr post IV tPA and throughout the first 3 days of treatment. ROC analysis suggested PT/PTT at 12 and 24 hr has potential to predict tPA-induced HT (Fig C,D. PT: AUC=0.848, p=0.001; PTT: AUC=0.877; p=0.003). Conclusions Higher PT/PTT level within 72 hr of IV tPA is early marker of tPA-induced HT. Whether these routine labs have value in symptomatic hemorrhage will require further study in a larger cohort. But this proof-of-concept study suggests that tPA efficacy can potentially be followed in real time. The development of a reliable blood test would be of clinical utility to gauge thrombolytic efficacy in real time to guide and triage adjunct treatments. Abstract MP13 Figure 1

MP6: HOMOCYSTEINE LEVEL IN PFO RELATED STROKE PATIENTS WITH RESPECT TO MEDICAL THERAPY VS PFO CLOSURE

Purpose of Study Homocysteine is an independent risk factor of ischemic stroke by promoting vascular endothelial dysfunction and thrombotic process through oxidative stress. We previously found that PFO closure may reduce total homocysteine level (tHcy) in plasma. Here, we compare the effect of PFO closure and medical treatment in reducing mild homocysteinemia in PFO-related stroke patients. Methods Used 28 PFO-related stroke patients with mildly elevated tHcy (>12 µmol/l) were prospectively recruited in accordance with IRB. 14 received PFO closure and 14 were treated by medical therapy (antiplatelet/anticoagulant) alone. None of the patients were on folate or vitamin B supplementation. Plasma was collected at baseline and 1 year follow-up after treatment. tHcy level was determined by selected reaction monitoring using mass spectrometry. Summary of Results Compared to medical therapy, PFO closure resulted in a lower tHcy level during follow-up (PFO closure: 11.13±3.94 µmol/L, medical therapy: 15.48±3.55 µmol/L, p=0.006), with no difference at baseline (PFO closure: 17.77±4.39 µmol/L, medical therapy: 16.47±7.50 µmol/L, p=0.575). Mild hyperhomocysteinemia patients post PFO closure had a significant reduction of tHcy by 37.34% (p=0.0005), with 71.43% of the patients (10 of 14) having tHcy levels back to normal (<12 µmol/l), while most of medically treated patients (13 of 14) stayed abnormal (p=0.4820) (χ2-test, adjusted p=0.002). Conclusions We found that compared with routine medical therapy, PFO closure reduced tHcy level in patients with mild hyperhomocysteinemia. Since PFO stroke patients tend to be younger, the life-time risk of even mildly elevated tHcy may be important for future thrombotic risk. Understanding the mechanism of PFO-related tHcy changes is important in optimizing medical treatment (e.g, folate replacement); studies are ongoing. Abstract MP6 Figure 1