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Because brain endothelial cells exist at the neurovascular interface, they may serve as cellular reporters of brain dysfunction by releasing biomarkers into the circulation.
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.
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).
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.
The concept of the neurovascular unit suggests that human brain endothelial cells (HBEC), a major component of the blood–brain barrier, serve as critical regulators of neuronal integrity and have important roles in cell matrix signaling.1–3 Endothelial dysfunction contributes to neurological disease and, conversely, neuronal injury is reflected as perturbations in endothelial function.4 Perturbations in the blood–brain barrier and endothelial homeostasis are important in central nervous system diseases such as cerebral ischemia and neurodegeneration. Because HBEC can survey the entire brain, they may serve as cellular integrators and sensors of brain disease, releasing measurable biomarkers into the circulation.
Biomarkers may be important tools for clinical decision-making in stroke. However, finding new biomarkers at the bedside is difficult because of the complexity and heterogeneity of clinical stroke samples.5 In this study, we explored the feasibility of stroke biomarker screening using a simpler in vitro HBEC culture system to find candidates for further validation. HBEC were exposed to the nitric oxide donor, sodium nitroprusside (SNP), to induce oxidative stress, and conditioned media were screened using discovery proteomic methods. We hypothesized that analysis of newly identified proteins secreted by HBEC may help to identify important factors in brain endothelial response to oxidative stress and aid in the discovery of biomarkers for ischemic stroke.
HBEC (CSC-Cell Systems) were plated on 6-well plates using serum-free media to avoid abundant proteins in feeding media that may interfere with mass spectrometry (MS). At 80% confluent, medium was replaced with RPMI and treated with 50 μmol/L of the nitric oxide donor SNP, a validated trigger of neurovascular nitrosative/oxidative stress.6 This SNP concentration does not cause cell death.6 Cell culture media were collected at 0, 6, 12, and 24 hours after SNP treatment and compared with media from untreated cultures (Figure 1). Independent cell culture experiments were performed in triplicate.
Proteomic profiling of culture media was performed on samples before and after SNP from each time point using gel-based separation followed by tandem MS (MS/MS; ThermoLCQ; Figure 1). Raw MS/MS data were searched using SEQUEST. Independent triplicates were analyzed in random order to decrease “batch effect.” One-dimensional SDS gel separation of each sample was performed. To preserve molecular weight information before MS/MS analysis, each gel was sliced at molecular weight ranges of 300 to 220, 220 to 160, 160 to 100, 100 to 80, 80 to 60, 60 to 50, 50 to 30, 30 to 20, 20 to 15, and 15 to 0 kDa (referred to as slice 1 to 10, respectively). Fractions <50 kDa were considered likely to contain low-molecular-weight growth and secreted factors, as well as degraded fragments of larger substrates reflective of increased protease activity.7,8
Standard methods were used to analyze our proteomic data.9 Proteins were reported with respect to time and molecular weight and were categorized into 3 groups: (1) found in both SNP and control samples in at least 1 time point; (2) after SNP treatment only; (3) in controls only. Semiquantitative protein amounts found in both SNP-treated samples and controls were obtained from areas under peaks, and ratios (SNP-treated/control) representing protein fold changes were calculated. A ratio of 1 indicates no change, a ratio >1.0 is considered an increased trend, and a ratio <1.0 is considered a decreased trend after SNP. Principle component analysis was performed using Matlab 6.5.1.
Acute ischemic stroke patients were recruited within 8 hours of symptom onset following our Institutional Review Board-approved protocol. Ischemic infarction is defined as an appropriate clinical syndrome, with MRI or CT findings consistent with ischemic stroke and without evidence of structural disease. Patient samples were age-, comorbidity-, and stroke severity-matched, and samples were measured by enzyme-linked immunosorbent assay (human thrombospondin-1 [TSP-1], chemokine ligand-1, and nidogen-1).
All proteins identified in HBEC conditioned media have been deposited into the EMBL/PRIDE database (accession number 8647) and are also listed in the Supplementary Table (available online at http://stroke.ahajournals.org). Following standard proteomic practice, proteins found in only 1 sample (n=116) were excluded from analysis to avoid false-positive results. Remaining multi-sample hits (n=277) were found in both control and SNP-treated samples (n=224), SNP-treated samples only (n=37), and controls only (n=16) (Supplementary Table). Table 1 lists selected proteins discussed.
For all proteins found, principle component analysis demonstrates intra-sample reproducibility by showing that the greatest variance in data comes from difference in treatment state, ie, triplicates from SNP-treated vs controls co-cluster in 3-D principle component analysis space at 6 hours (Figure 2A).
In selected protein classes likely to be important in endothelial signaling (eg, secreted factors, membrane receptors, matrix components), bioinformatic mapping of protein–protein interactions10,11 demonstrated potential “cross-talk” in control undamaged endothelial cells (Figure 2B). However, similar networks of interacting proteins failed to emerge in SNP-treated endothelial cells.
For individual proteins, observed changes in expression appear to be time-dependent and treatment-specific, as indicated by a global heat map analysis (Figure 2C). Because SNP treatment did not alter markers of cell lysis (lactate dehydrogenase, 0.6705 vs 0.6665 in control vs SNP-treated), the protein differences are most likely attributable to endothelial response to oxidative stress, rather than to nonspecific cell death.
In oxidatively stressed cells, the number of secreted proteins peaked at 12 hours after SNP exposure. Because protein size information was preserved from 1-D in-gel separation, we were able to analyze protein number with respect to their approximate molecular weight (Figure 2D). More proteins came from the lower-molecular-weight fragments, potentially containing secreted factors or degraded substrates, in gel slices 7 to 10 (molecular weight <50 kDa).
In a subset of proteins found in at least 1 time point in both control and SNP-treated samples, we performed semiquantitative analysis of the temporal fold ratio change. Calculated SNP/control ratios suggest proteins are either upregulated (ratio >1) or downregulated (ratio <1) over time after oxidative stress. These proteins appeared to segregate into 3 dynamic categories with distinct temporal profiles. Table 1 lists selected proteins from Figure 3 discussed, with respect to protein size and fold ratio changes over time.
The first category (Figure 3A) comprised “rising” proteins with SNP/control ratios that increased over time (ie, peaked later in SNP-treated or degraded later in controls, n=62 proteins). This category included proteins representing a variety of stress/inflammatory markers (eg, TSP-1, heat shock protein, chemokine ligand-1, serum amyloid-A1, annexin-A5) and protease inhibitors (eg, tissue inhibitor of matrix metalloproteinase-2). Cytoskeletal markers of endothelium, such as actin, vimentin, nidogen, and filamin B, also increased over time.
The second category (Figure 3B) comprised “falling” proteins with SNP/control ratios that reach a maximum and then decrease over time (ie, early peak in SNP-treated or early degradation in control samples, n=16 proteins). This category mostly included proteins representing cellular machinery and endothelial extracellular matrix integrity (eg, basement membrane component, fibronectin-1, tau tubulin kinase, desmoglein-1, profilin-1).
The third category (Figure 3C) comprised “oscillating” proteins that went up and down over time (n=121 proteins). These mostly consisted of signaling proteins, membrane receptors, and secreted factors, eg, neutrophic factors and signaling factors (prosaposin, nucleobindin-1, and precursor tachykinin-1, which converts to various neuropeptides such as substance p and neurokinin), annexin-A2, calsyntenin-1, calsyntenin-3, protease inhibitors (tissue inhibitor of matrix metalloproteinase-1), and inflammatory signals (transforming growth factor β2, IL-6, and IL-8).
Initial analysis revealed a large number of endothelial proteins, some of which showed degradation into smaller fragments over time, indicative of protease activity.7,8 We hypothesized that in our study, temporal relationships between full-size and smaller fragments of the same secreted factors may indicate active turnover of proteins involved in endothelial response to oxidative stress. In terms of statistical significance, one of the highest-ranking candidates was TSP-1. TSP-1 was identified both in high-molecular-weight (160 –220 kDa) and low-molecular-weight (0 –15 kDa) gel slices (Supplementary Table). TSP-1 may functionally exist as a trimer at 450 kDa in vivo.12 In our cell culture system, we detect both the 150-kDa full-size monomer and smaller fragments of proteolytically processed TSP-1. Full-size TSP-1 is upregulated after 12 hours of oxidative stress, and smaller degraded TSP-1 fragments (<15 kDa) appeared in SNP-treated samples at 12 and 24 hours (Table 2). Interestingly, enolase, a protease known to degrade TSP-1, increased at 12 hours as well, consistent with the timing of the appearance of the degraded product of TSP-1 (Table 2). Overall, this may suggest that TSP-1 is actively produced and degraded in response to oxidative stress.
To further explore these in vitro findings, we measured TSP-1 by enzyme-linked immunosorbent assay in acute ischemic stroke patients with exact time onset of ischemia. All stroke patients had middle cerebral artery territory infarct, without hemorrhagic transformation on CT at 48 to 72 hours, and were not administered tissue plasminogen activator. Their clinical characteristics are comparable to controls without stroke (Table 3). Patients with acute ischemic stroke had significantly higher TSP-1 levels within 8 hours of symptom onset compared to controls with similar clinical risk factors (1132±89 ng/mL in stroke vs 659±81 ng/mL in controls; P<0.05; Figure 3D).
To test the dynamic range of our approach, we measured 2 other candidates from the rising category, chemokine ligand-1 and nidogen-1. Both markers showed a trend toward higher values in stroke patients within 8 hours of symptom onset (Figure 3D).
In this proof-of-concept study, we demonstrate the feasibility of temporal proteomic profiling of cell culture media for HBEC response to oxidative stress, followed by exploratory bedside plasma testing. This novel approach may provide a quick and reproducible screening tool for candidate markers and/or targets of stroke therapy, in which it otherwise would be difficult to find low-abundance biomarkers using discovery proteomic technology. Our data showed that there were clear differences between healthy HBEC vs oxidatively stressed HBEC. Healthy cells developed networks of cross-talk involving secreted factors, membrane receptors, and matrix components,13 whereas stressed HBEC released potential biomarkers, demonstrating different patterns over time.
In general, the endothelial response to oxidative stress was highly dynamic and cellular machinery and inflammatory markers peaked gradually, secreted factors tended to oscillate over time, and endothelial injury and repair were manifested in dynamic changes in factors related to matrix integrity. Reactive/inflammatory markers specific to ischemia increased over time, as represented by TSP-1, chemokine ligand-1, heat shock protein-1, serum amyloid-A1, annexin-5, and serum amyloid-A1. Neurotrophic factors and neuropeptides such as prosaposin, nucleobindin-1, annexin-2, calsyntenin-3, calsyntenin-1, protease inhibitors (eg, tissue inhibitor of matrix metalloproteinase-1), and tachykinin precursors (tachykinin-1, which converts to various neuropeoptides such as substance p and neurokinin) tended to peak mid-treatment (12 hours) and then rapidly decreased by 24 hours (Table 1). In general, these responses are consistent with the idea that cellular stress leads to an overall downregulation of beneficial trophic mediators and an increase in potentially deleterious inflammatory signals.14 Loss of vascular trophic coupling may be an important part of stroke pathophysiology.6,15
In addition to trophic and inflammatory markers, our data also suggested that oxidative stress may trigger deterioration and active remodeling of endothelial extracellular matrix. Over time, basement membrane components decreased (fibronectin-1, desmoglein-1, profiling-1), whereas other cytoskeleton components (nidogen-1, actin, vimentin, and filamin B) appeared to increase over time after oxidative stress (Table 1). In the context of brain endothelium, these responses are consistent with blood–brain barrier alterations. Our findings are supported by other proteomic studies demonstrating that cytoskeletal proteins contribute to the dynamic blood–brain barrier responses when bovine brain endothelial cells are co-cultured with astrocytes.16 In comparison to the study by Haqqani et al in which both rat brain endothelial cellular and secreted proteins were studied by 2-D gel (n=38 proteins) vs isotope-based (n=138 proteins) proteomics, our study only measured protein secreted by human brain endothelial cells and found a higher number (n=277) of lower-abundance proteins, correlating to human plasma at a low picogram level, a concentration previously difficult to achieve by direct human plasma screening.17,18 Taken together, our findings suggest that brain endothelium should be a rich source of brain injury-specific biomarkers comprising trophic, inflammatory, and barrier properties that then become accessible in the systemic circulation. There is an overlap between proteins found in our endothelial cell culture and the published human plasma proteome.19
In this initial study, a large number of potential markers were profiled. One of the high-ranking candidates was TSP-1, a pleiotropic antiangiogenic factor involved in coagulation and atherosclerosis.12 In our brain endothelial cells, TSP-1 was produced and then actively degraded after 12 hours of oxidative stress. Our data are consistent with those of previous studies of mouse models of cerebral ischemia in which TSP-1 increases within 1 hour after occlusion.20
Because, in stroke, focal ischemia leading to oxidative injury begins with a specific clinical event with an onset that can be timed, we explored our finding further by measuring TSP-1 levels in a small cohort of acute ischemic stroke patients whose exact time of stroke onset was known. Consistent with our in vitro findings, TSP-1 levels were higher in acute stroke, within 8 hours of initial symptom onset, compared to age-matched controls with similar clinical risk factors. Two other rising markers (chemokine ligand-1 and nidogen-1) also appeared to be higher in stroke patients, although these results did not reach statistical significance. Importantly, our cell culture markers showed a good dynamic range of pg to ng/mL concentration in human plasma, as confirmed by our enzyme-linked immunosorbent assay measurements, highlighting the potential for detecting low-abundance candidates otherwise not feasible in direct human plasma proteomic screening.18 However, it must be acknowledged that there are many caveats. We did not assess potential markers from the oscillating category because it will be difficult to match the timing of our cell cultures to variable stroke onset and sample collection times in patients. The same is true for proteins from the falling category. For example, although fibronectin-1 is listed in this category, its temporal profile peaks at 6 hours and then falls over the next 24 hours (Table 1). So, depending on the time of sample collection, fibronectin-1 levels may be high or low in patients. However, our cell-based finding of an early fibronectin-1 peak is at least consistent with a previous study showing elevated plasma fibronectin levels within the first 6 to 8 hours after stroke onset.21 Similarly, elevations in markers such as IL-6 and tissue inhibitor of matrix metalloproteinase-2 also have been reported in stroke patients previously.22,23 Ultimately, our initial attempt at validation is obviously limited by our sample size. However, our findings are consistent with the idea that specific markers derived from our cell culture model were measurable in actual human stroke samples.
Proteomic profiling of mouse and rat endothelium after ischemia has been actively investigated.17,24 However, to our knowledge, temporal proteomic profiles of human brain endothelial responses have not been reported. Differences between human and rodent cells are not fully understood. For example, our findings of increased vimentin and decreased fibronectin at 24 hours are similar to those of the Haqqani et al17 study that looked at rat endothelial cells. However, our highest-ranking and clinically validated marker TSP-1 was not detected in the rat cells at all.17
The dynamic changes of secreted factors may have important roles in cell signaling after ischemia. Furthermore, analysis of fragment sizes may help identify neurovascular proteases. Although our data are driven by oxidative stress because this is the central trigger after stroke,25 this relatively simple methodology can be generalizable to other insults. Because it is challenging to characterize unknown low-abundance secreted factors by discovery proteomics directly at the bedside, an initial screening in a “cleaner” cell culture system may yield candidates that later can be tested clinically. Ultimately, larger collaborative efforts are required to validate more candidates of interest, as advocated by major proteomic and research organizations such as the Human Proteome Organization and the National Institutes of Health.
The present study provides proof of concept. A full in vivo validation of all potential candidate biomarkers is outside the scope of our initial study. This type of label-free gel-based intact protein discovery proteomics, even when optimized in a serum-free cell-culture system, still may not be able to discover exceedingly low levels of secreted factors or give the most accurate quantification. Isotope-labeled techniques offer more accurate quantitation26 than the semiquantitative label-free ratio we report. Because endothelial cells do not work in isolation, contribution by neurons, astrocytes, and hematologic agents may alter their secretory function. In addition, oxygen glucose deprivation also might be a better “stroke mimic.” In our model in which SNP is used, nitrosylated proteins would be of interest for future study. And dose-response is important because U-shape curves also may be present, depending on the protein involved. Further studies using RNA, quantitative protein microarrays for target validation, co-cultures with glial cells, validation in other model systems with dose-response curves, and larger clinical cohorts with timed samples to validate other candidates are required to confirm our findings.
In conclusion, our proof-of-concept study suggests that high-throughput systemic bench-to-bedside screening may be used to explore therapeutic targets and clinically relevant biomarkers in neurovascular injury. Further analysis and validation of this approach are warranted.
Sources of Funding This work is supported in part by grants from the National Institutes for Neurological Disorders and Stroke (R21-NS052498, K23-NS051588, R01-NS48422, R37-NS37074, P01-NS55104, R01-NS067139).
The online-only Data Supplement is available at http://stroke.ahajournals.org/cgi/content/full/STROKEAHA.110.585703/DC1.