Proteomic Temporal Profile of Human Brain Endothelium After Oxidative Stress

Proteomic Temporal Profile of Human Brain Endothelium After Oxidative Stress MingMing Ning, MD, MMsc; David A. Sarracino, PhD; Alvin T. Kho, PhD; Shuzhen Guo, PhD; Sun-Ryung Lee, PhD; Bryan Krastins, MS; Ferdinando S. Buonanno, MD; Juan A. Vizcaíno, PhD; Sandra Orchard; David McMullin, PhD; Xiaoying Wang, MD, PhD; Eng H. Lo, PhD 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. (Stroke. 2011;42:37-43.) Key Words: biomarker 䡲 cerebral ischemia 䡲 human brain endothelial cells 䡲 oxidative stress 䡲 proteomics

T

he 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 decisionmaking in stroke. However, finding new biomarkers at the bedside is difficult because of the complexity and heteroge-

neity 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.

Materials and Methods Cell Culture HBEC (CSC-Cell Systems) were plated on 6-well plates using serum-free media to avoid abundant proteins in feeding media that

Received March 30, 2010; accepted May 11, 2010. From the Clinical Proteomics Research Center (M.M.N., A.T.K., F.S.B., D.M., E.H.L.), Neuroprotection Research Laboratory (M.M.N., S.G., S.R.L., X.W., E.H.L.), Stroke/Neurocritical Care (M.M.N., F.S.B.), Massachusetts General Hospital, Harvard Medical School, Boston, Mass; Children’s Hospital Boston (A.T.K.), CHIP, Boston, Mass; Harvard Partners Center for Genetics and Genomics (D.A.S., B.K.), Boston, Mass; EMBL (J.A.V., S.O.), European Bioinformatics Institute, Cambridge, UK. The online-only Data Supplement is available at http://stroke.ahajournals.org/cgi/content/full/STROKEAHA.110.585703/DC1. Correspondence to Eng H. Lo, MGH East 149-2401, Charlestown, MA 02129. E-mail [email protected]; and to MingMing Ning, MGH WACC-739C, 15 Parkman Street, Boston, MA 02114. E-mail [email protected] © 2010 American Heart Association, Inc. Stroke is available at http://stroke.ahajournals.org

DOI: 10.1161/STROKEAHA.110.585703

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Figure 1. Schematic of experimental design. The workflow consisted of screening of cell cultures of human brain endothelial cells conditioned media before and after stimuli. Mass spectrometry was performed on predigested bands preserving molecular weight information. All identified markers were then quantitatively analyzed, followed by clinical sampling to validate highest-ranking candidates.

may interfere with mass spectrometry (MS). At 80% confluent, medium was replaced with RPMI and treated with 50 ␮mol/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.

dard 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.

Mass Spectrometry

Global Changes Specific to Treatment and Timing

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.” Onedimensional 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-molecularweight growth and secreted factors, as well as degraded fragments of larger substrates reflective of increased protease activity.7,8

Proteomic Data Analysis 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.

Human Stroke Patients 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).

Results Total Protein Database 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 stan-

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).

Semiquantitative Analysis of Proteins Found in Both Controls and SNP-Treated Samples 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

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Ning et al Table 1.

Brain Endothelium Proteome and Oxidative Stress

Selected Proteins Discussed in Results Section With Respect to Temporal Fold Ratio Change and Protein Size* A) Rising SNP/Control Ratio (62 Proteins)

GI Number 4503747

39

Ratio SNP/Control

Symbol

Name

Cellular Component and Function

Slice Number

0 hr

6 hr

12 hr

24 hr

FLNB

Filamin B, beta (actin binding protein 278)

Plasma membrane, integral to membrane

2

…

…

…

1.4

4505395

NID1

Nidogen (enactin)

Basement membrane, extracellular matrix

2

…

…

1.3

1.6

4507485

TSP-1/THBS1

Thrombospondin 1

Extracellular

2

…

…

0.6

1.5

4501885

ACTB

Actin, beta

Actin filament, cytoskeleton

8

…

…

2.0

2.6

4502107

ANXA5

Annexin-A5

Unknown

8

…

…

0.6

SNP

4507511

TIMP2

Tissue inhibitor of metalloproteinase-2

Basement membrane, extracellular matrix

8

…

…

0.8

1.5

4504153

CXCL1

Chemokine (C-X-C motif) ligand-1

Extracellular space, soluble fraction,

10

…

…

0.3

0.4

4504523

HSPE1

Heat shock 10-kDa protein-1 (chaperonin 10)

Unknown

10

…

C

1.1

3.5

4507895

VIM

Vimentin

Cytoskeleton, intermediate filament

10

…

…

1.4

1.5

4506777

SAA-1

Serum amyloid A1

Extracellular

10

…

…

…

2.0

B) Falling SNP/Control Ratio (16 Proteins)

Ratio SNP/Control

GI Number

Symbol

Name

Cellular Component

Slice Number

0 hr

6 hr

12 hr

24 hr

16933542

FN1

Fibronectin 1

Extracellular matrix, extracellular

1

…

1.4

0.8

0.6

28466991

TTBK2

Tau tubulin kinase-2

Cytoskeleton, intermediate filament

8

1

1.0

…

…

4503401

DSG1

Desmoglein-1

Plasma membrane, cell junction

10

1

SNP

2.5

C

4826898

PFN1

Profiling-1

Cytoskeleton, cytoplasm, actin

10

…

4.7

2.8

2.1

C) Oscillating SNP/Control Ratio (121 Proteins)

Ratio SNP/Control

GI Number

Symbol

Name

Cellular Component

Slice Number

0 hr

6 hr

12 hr

24 hr

7662268

CSTN3

Calsyntenin-3

Cell, integral to membrane, membrane

3

…

…

1.6

…

7662374

CSTN1

Calsyntenin-1

Cell, membrane

3

…

…

SNP

0.2

20070228

NUCB1

Nucleobindin-1

Extracellular space, cytoplasm,extracellular

6

…

…

1.3

0.5

4757756

ANXA2

Annexin-A2

Plasma membrane, soluble fraction,

7

…

…

0.8

0.4

4507509

TIMP1

Tissue inhibitor of metalloproteinase-1

Extracellular matrix, extracellular

8

1

0.8

1.1

0.3

10834984

IL-6

Interleukin-6 (interferon, beta-2)

Extracellular space, extracellular

8

…

SNP

C

1.8

4503571

ENO1

Enolase-1 (alpha)

Phosphopyruvate hydratase complex

10

…

C

1.8

1.4

4507463

TGF-␤2

Transforming growth factor beta-2

Extracellular space, extracellular

10

…

…

1.4

C

7770075

TAC1

Tachykinin precursor-1 (substance K, substance P, neurokinin-1, neurokinin-2, neuromedin L, neurokinin alpha, neuropeptide K)

Extracellular space, extracellular

10

…

…

1.1

…

10834978

IL-8

Interleukin-8

Extracellular space, soluble fraction

10

…

…

0.7

C

11386147

PSAP

Prosaposin

Extracellular space, membrane

10

…

…

2.0

C

C indicates proteins found in control only; SNP, proteins found in sodium nitroprusside-treated only; . . ., proteins not detected in either condition at this time point. Proteins found in both controls and SNP-treated samples have calculated ratio of SNP/control (ratio ⬎1, increased trend after SNP; ratio ⬍1, decreased trend after SNP). All proteins are arranged in order of molecular weight (gel slice 1–10 in order of 300 –220, 220 –160, 160 –100, 100 – 80, 80 – 60, 60 –50, 50 –30, 30 –20, 20 –15, and 5– 0 kDa). *The full list of all proteins is available in the Supplementary Data Table at http://stroke.ahajournals.org/cgi/content/full/STROKEAHA.110.585703/DC1.

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).

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Figure 2. Global profiles of identified proteins. A, Principle component analysis (PCA) shows co-clustering of control vs sodium nitroprusside (SNP)-treated samples at 6 hours, demonstrating intrasample reproducibility. B, Specific protein–protein interaction analysis of important functional categories, such as secreted factors, membrane receptors, and matrix-related components, revealed increased potential signaling networks over time in control healthy endothelial cells at 24 hours. Empty boxes represent factors only found at other time points not shown here. C, Heat map of all proteins demonstrates stimulus- and timespecific changes. D, Protein number vs time with respect to molecular weight. Protein number peaked at 12 hours after SNP, and most were found in the lowermolecular-weight (⬍50 kD) region containing important secreted factors and protease substrates.

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 ␤2, IL-6, and IL-8).

Analysis of TSP-1 as a Representative Brain Endothelial Response 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

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Brain Endothelium Proteome and Oxidative Stress

41

Figure 3. Semiquantitative analysis of protein expression over time, plotted as ratio of sodium nitroprusside (SNP)/control (ie, fold change induced by SNP stress). X-axis indicates time. Y-axis indicates fold change ratio. All proteins graphed here are listed in Supplementary Table. A, Rising proteins (n⫽62) comprise a variety of inflammatory markers and protease inhibitors. B, Falling proteins (n⫽16) mostly comprise cellular machinery and extracellular matrix integrity. C, Oscillating proteins (n⫽121) include signaling mediators, membrane receptors, and secreted factors. D, Enzyme-linked immunosorbent assay of human plasma comparing controls with stroke patients described in Table 3. Thrombospondin-1 levels were significantly elevated in stroke samples (P⬍0.05). Elevations in nidogen-1 and chemokine ligand-1 were not statistically significant.

candidates was TSP-1. TSP-1 was identified both in high-molecular-weight (160 –220 kDa) and lowmolecular-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 SNPtreated 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. Table 2. Quantitative Changes in TSP-1 and Enolase Over Time Baseline

6 hr

12 hr

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). Table 3. Clinical Characteristic of Acute Ischemic Stroke Patients

24 hr

TSP-1 (full-length monomer, MW⫽150 kDa) Controls

ND

ND

1782⫹974

2708⫹2215

SNP

ND

ND

1136⫹1313 4082⫹95

TSP-1 (degraded fragment, MW ⬍15 kDa) Controls

ND

ND

ND

ND

SNP

ND

ND

91⫹56*

79⫹9*

Enolase

Risk Factors

Stroke (n⫽10)

Control (n⫽10)

P

Age (mean)

51.6

51.8

NS

Gender (% male)

50

40

NS

Hypertension

3

3

NS

Heart disease

0

0

NS

Atrial fibrillation

1

1

NS

Hyperlipidemia

2

3

NS

Smoking

0

0

NS

Diabetes

0

0

NS

Controls

ND

108⫹187

317⫹33

313⫹306

Obesity

1

1

NS

SNP

ND

ND

559⫹88*

665⫹357

Sedentary lifestyle

2

2

NS

Alcohol (mean)

0

0

NS

NIHSS (onset)

7.4

0

NA

Modified Rankin scale score (3 mo)

1.2

0

NA

⬍8 hr (2–8)

NA

NA

MW indicates molecular weight; ND, not detected within the sensitivity limits of our system; SNP, sodium nitroprusside; TSP-1, thrombospondin-1. Values expressed as arbitrary units (mean⫹SD) derived from quantitation of peak area under curve. SNP-treated samples had significant increase in TSP-1 degraded fragment and enolase at 12 hours. *P⬍0.05 comparing controls vs SNP-treated cells.

Initial onset of stroke to time of blood sampling

NA indicates not applicable; NS, not significant.

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Discussion 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 amyloidA1. Neurotrophic factors and neuropeptides such as prosaposin, nucleobindin-1, annexin-2, calsyntenin-3, calsyntenin-1, protease inhibitors (eg, tissue inhibitor of matrix metalloproteinase1), 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 lowerabundance 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 lowabundance 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

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Ning et al 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 lowabundance 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 serumfree 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.

Brain Endothelium Proteome and Oxidative Stress

7. 8. 9. 10.

11.

12. 13. 14.

15.

16.

17.

18.

Conclusion In conclusion, our proof-of-concept study suggests that highthroughput 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, K23NS051588, R01-NS48422, R37-NS37074, P01-NS55104, R01-NS067139).

Disclosures

19.

20.

21.

22.

None.

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Proteomic Temporal Profile of Human Brain Endothelium After Oxidative Stress MingMing Ning, David A. Sarracino, Alvin T. Kho, Shuzhen Guo, Sun-Ryung Lee, Bryan Krastins, Ferdinando S. Buonanno, Juan A. Vizcaíno, Sandra Orchard, David McMullin, Xiaoying Wang and Eng H. Lo Stroke. 2011;42:37-43; originally published online December 16, 2010; doi: 10.1161/STROKEAHA.110.585703 Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2010 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628

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Supplementary Table. (1). All proteins found in BOTH Control and SNP treated (Ratio >1 – increased trend post-SNP; Ratio