20-HETE synthesis inhibition promotes cerebral protection after intracerebral hemorrhage without inhibiting angiogenesis

Abstract

20-HETE, an arachidonic acid metabolite synthesized by cytochrome P450 4A, plays an important role in acute brain damage from ischemic stroke or subarachnoid hemorrhage. We tested the hypothesis that 20-HETE inhibition has a protective effect after intracerebral hemorrhage (ICH) and then investigated its effect on angiogenesis. We exposed hippocampal slice cultures to hemoglobin and induced ICH in mouse brains by intrastriatal collagenase injection to investigate the protective effect of 20-HETE synthesis inhibitor N-hydroxy-N′-(4-n-butyl-2-methylphenyl)-formamidine (HET0016). Hemoglobin-induced neuronal death was assessed by propidium iodide after 18 h in vitro. Lesion volume, neurologic deficits, cell death, reactive oxygen species (ROS), neuroinflammation, and angiogenesis were evaluated at different time points after ICH. In cultured mouse hippocampal slices, HET0016 attenuated hemoglobin-induced neuronal death and decreased levels of proinflammatory cytokines and ROS. In vivo, HET0016 reduced brain lesion volume and neurologic deficits, and decreased neuronal death, ROS production, gelatinolytic activity, and the inflammatory response at three days after ICH. However, HET0016 did not inhibit angiogenesis, as levels of CD31, VEGF, and VEGFR2 were unchanged on day 28. We conclude that 20-HETE is involved in ICH-induced brain damage. Inhibition of 20-HETE synthesis may provide a viable means to mitigate ICH injury without inhibition of angiogenesis.

Introduction

20-Hydroxyeicosatetraenoic (20-HETE) acid is a major bioactive metabolite of arachidonic acid that is synthesized by cytochrome P450 (CYP) 4A enzymes. It acts as a vasoconstrictor in the microvasculature.¹ Elevated levels of 20-HETE in cerebrospinal fluid and plasma of patients with ischemic stroke are associated with poor outcomes.^2,3 Additionally, N-Hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine (HET0016), a highly selective inhibitor of the 20-HETE synthesizing enzyme,⁴ was shown to protect against ischemic stroke independent of vascular effects.^5,6 Intracerebral hemorrhage (ICH) is one of the most lethal stroke types. However, little is known about the relationship between 20-HETE and ICH.⁷ We hypothesized that 20-HETE participates in ICH injury and that inhibiting its synthesis would promote histologic and functional recovery.

In addition to being a vasoconstrictor of cerebral microvasculature, 20-HETE regulates proliferation and migration of endothelial progenitor cells through the angiogenic growth factor cellular pathway.⁸ Inhibition of 20-HETE synthesis reduces vascular epithelial growth factor (VEGF)- and fibroblast growth factor-induced angiogenesis and tumor growth by preventing vascularization.^9,10 Angiogenesis in the perihematomal region is a critical adaptation for brain repair after ICH.¹¹ Nevertheless, little is known about how inhibiting 20-HETE synthesis affects angiogenesis in the ICH brain. Thus, the goal of this study was to determine whether inhibition of 20-HETE synthesis by HET0016 protects ICH brain and whether it suppresses angiogenesis.

Material and methods

Animals and study design

All experimental procedures followed the ARRIVE (http://www.nc3rs.org.uk/arrive), STAIR, and RIGOR guidelines¹² and were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University School of Medicine; 8- to 10-week-old (23–25 g) and 16- to 18-month-old (28–34 g) C57BL/6 male mice were obtained from Charles River Laboratories (Frederick, MD). A total of 435 mice were used. Animals were randomly assigned to different study groups by using the randomizer form at http://www.randomizer.org.¹³ Sample size calculations based on our pilot studies indicated that eight mice/group would provide at least 80% power for detecting a 20% decrease in lesion volume at α = 0.05 (two-sided). Investigators blinded to the treatment groups evaluated outcomes in all mice and performed analysis. Animals that died during surgery or shortly after ICH were excluded from analysis.

ICH model

We used the collagenase-induced ICH model to assess the role of 20-HETE inhibition in cerebral protection and angiogenesis after ICH. Mice were anesthetized by 3% isoflurane inhalation and maintained by 1% isoflurane during surgery. ICH was induced by injecting 0.5 µL of 0.075 U collagenase VII-S (Sigma-Aldrich) into the left striatum (0.8 mm anterior and 2.0 mm lateral of the bregma, and 3.0 mm in depth).^14,15 Sham-operated mice underwent the same protocol but without collagenase injection. Rectal temperature was monitored and maintained at 37.0 ± 0.5℃ by the DC Temperature Controller (FHC Inc., Bowdoin, ME) throughout the experimental and early recovery periods.

HET0016 treatment

Mice were randomly assigned to receive HET0016 (10 mg/kg, Cayman Chemical) or vehicle (hydroxypropyl-β-cyclodextrin, Sigma-Aldrich) intraperitoneally at 2 h after ICH. The delivery route, dosing, and treatment regimens were based on previous work in mice and our pilot studies.¹⁶ For the angiogenesis study, mice received HET0016 injections at 2 h after ICH and then every 12 h for three days.

Organotypic hippocampal slice cultures

Organotypic hippocampal slice cultures (OHSCs) were cultured as described previously.^17,18 Brains were rapidly removed from seven-day-old C57BL/6 mice and cut coronally into 350-µm-thick slices with a Mcllwain tissue chopper. Hippocampal slices were immediately placed on a hydrophilic PTFE cell culture insert (30-mm Millicell-CM, Millipore). Culture medium consisted of 50% Minimal Essential Medium, 25% horse serum, and 25% Hanks’ Balanced Salt Solution supplemented with 6.5 mg/mL D-glucose, 2 mmol L-glutamine, 100 U/mL penicillin G potassium, and 100 µg/mL streptomycin sulfate. Culture medium was changed every two days. At 12–14 days in vitro, cultured slices were incubated for 24 h in serum-free medium and then exposed to 10 µmol/L hemoglobin (Sigma, H7379, human hemoglobin) for 18 h with HET0016, 20-HETE agonist (20-5,14-HEDGE), or 20-HETE antagonist (20-6,15-HEDGE). Slices were incubated with propidium iodide (PI) or dihydroethidium for 30 min before images were observed with an inverted fluorescence microscope (TE2000-E, Nikon, Japan). The PI fluorescence intensity before hemoglobin treatment was recorded as P0. Pmax was induced by 100 µmol N-methyl-D-aspartate (NMDA). Cell death was calculated by the formula: (Px – P0)/(Pmax – P0) × 100%. The 20−5,14-HEDGE and 20-6,15-HEDGE were gifts from Professor John R. Falck (University of Texas Southwestern Medical Center).

Quantification of lesion volume, hematoma size, and edema

For quantification of lesion volume, we stained a group of cryosections with Luxol fast blue (for myelin, Sigma Aldrich) and Cresyl violet (for surviving neurons, Sigma-Aldrich).¹⁹ Mice were perfused with phosphate-buffered saline followed by 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde overnight and then transferred to 30% sucrose. Brains were cut into 25-µm-thick coronal sections at 15 rostral-caudal levels spaced 200 µm apart and then stained and digitized with a 10 × objective. The lesion is evident by a lack of staining. Areas in individual sections were measured by Image J software (NIH 1.47t). The total lesion volume was calculated as the sum of the total lesion area multiplied by the distance between the sections (200 µm). The total lesion volume was also corrected for brain swelling: corrected lesion volume =volume of nonhemorrhagic hemisphere – (volume of hemorrhagic hemisphere – lesion volume).¹⁵ For quantification of hematoma size at 24 h post-ICH, brains were cut into 1-mm sections and fixed by 4% paraformaldehyde. Hematoma size was measured by Image J software and calculated as the sum of the total hemorrhagic area multiplied by 1 mm.¹⁹

At three days after ICH induction, brain water content was measured with the wet-dry weight method. The brains were removed and divided into ipsilateral and contralateral striatum and cerebellum (as an internal control). Samples were immediately weighed on an analytical balance to obtain the wet weight and then dried at 100℃ for 24 h to obtain the dry weight. The brain water content was calculated as [(wet weight – dry weight)/ wet weight] × 100%.¹⁹

Neurologic deficit

Neurologic deficits of mice were evaluated by a 24-point scoring test, the corner turn test, and the wire hanging test at 24 h and 72 h post-ICH, as previously reported.^18,19 In the neurologic deficit scoring system,²⁰ we scored mice on six parameters, including body symmetry, gait, climbing, circling behavior, front limb symmetry, and compulsory circling. Each test was graded from 0 to 4, establishing a maximum deficit score of 24. Animals that had a neurologic deficit score higher than 20 at 24 h were excluded from analysis.

For the corner turn test,²¹ the mouse was allowed to proceed into a 30℃ corner. The mouse could freely turn left or right to exit the corner. The choice of direction during 10 repeats was recorded, and the percentage of left turns was calculated.

For the wire hanging test,²² a metallic wire (1 mm × 55 cm) was stretched horizontally between two posts, 50 cm above the ground. We covered the hind limbs of the mice with adhesive tape to prevent them from using all four paws and placed a pillow beneath them to prevent injury from falls. The mice were placed on the wire, and latency to fall was recorded. Mice that had a latency time less than 10 s before and after ICH were excluded from analysis.

Immunofluorescence

We stained 25-µm-thick brain coronal sections by standard immunohistochemistry procedures as previously described.¹⁴ The primary antibodies for immunohistochemistry were anti-CYP4A (1:200, Abcam ab3573), anti-NeuN (1:200, Millipore, MAB377), anti-glial fibrillary acidic protein (GFAP; 1:500, Sigma, G3893), anti-Iba1 (1:500, Wako, 019-19741), anti-CD11b (1:500, AbD Serotec, MCA275FT), anti-CD68 (1:200, AbD Serotec, MCA1957), anti-myeloperoxidase (1:200, DAKO, A0398), and anti-CD31 (1:200, Abcam, ab56299). Nuclei were labeled with DAPI (1:1000, Life Technologies, R37606). Omission of the primary antibody was used as a control. Images were observed under a Nikon Eclipse 90i fluorescence microscope and analyzed with Image J software (NIH, 1.47t). Images were captured from five optical fields (20 × 10 magnification) in each of three sections per animal. The average fluorescence intensity was expressed as percentage increase in the perihematomal area normalized to the contralateral side of the same section.

LC/MS/MS analysis of 20-HETE

Ipsilateral hemispheres were cut into 4-mm-thick slices at 6, 24, and 72 h and seven days after ICH. Tissue samples were stored at –80℃ until LC/MS/MS analysis of 20-HETE.²³ Briefly, mouse brains were homogenized in oxygenated Krebs buffer on ice and then incubated with 1 mM NADPH (Calbiochem) for 1 h at 37℃. The reaction was stopped by acidification to pH 4.0 with 10% acetic acid. In brief, samples were loaded onto preconditioned solid-phase extraction Phenomenex Strata C18-E columns (Phenomenex) with d₆-20-HETE as an internal standard (0.5 ng; Cayman Chemical). Columns were then washed with 10% methanol and eluted with 2 mL of 100% methanol. Samples were concentrated under nitrogen and stored at −80℃ until LC/MS/MS analysis. 20-HETE production was quantified with a Shimadzu UFMS Triple Quadrupole Mass Spectrometer LCMS-8050 combined with a Nexera UHPLC using negative ionization MRM mode. The quantification limit of this ultrasensitive method is 1 pg of 20-HETE.

Western blot, gelatin gel zymography, and ELISA

Total protein from the ipsilateral striatum was extracted by T-PER reagent (Pierce) with protease inhibitor cocktail (Roche Molecular Biochemicals). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were probed with primary antibodies against phospho-Src (Tyr416, 1:1000, Cell Signaling, 2101), total-Src (1:1000, Cell Signaling, 2108), carbonyl proteins (OxyBlot protein oxidation detection kit, S7150, Millipore), 3-nitrotryosine proteins (clone 1A6, 1:1000, Millipore, 05-233), VEGF (1:1000, R&D Systems, AF-493-NA), VEGFR2 (1:500, BD Pharmingen, 555307), VEGFR3 (1:500, BD Pharmingen, 552857), and β-actin (1:5000, Santa Cruz Biotechnology, sc-47778) at 4℃ overnight. The membranes were incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibody (1:3000, Santa Cruz Biotechnology) for 1 h. Protein signals were visualized in chemiluminescence solution and exposed under an ImageQuant ECL Imager (GE Healthcare). Images were analyzed by Image J. Optical density values were normalized to the corresponding loading control intensity and expressed as fold change.

Levels of pro-matrix metalloproteinase (MMP)-9 and pro-MMP-2 were measured by gelatin gel zymography.¹⁴ Protein from the ipsilateral striatum was extracted by protein extraction reagent (T-PER, Pierce) and quantified by BCA protein assay. Equal amounts of protein were purified with gelatin-sepharose 4B (GE Healthcare). Then, proteins were separated on a 10% Tris-glycine gel with 0.1% gelatin and incubated with developing buffer for 36 h at 37℃. The gel was stained with 0.5% Coomassie blue and then exposed to UV light. Gelatinase standard was mixed with mouse pro-MMP-9 and pro-MMP-2 (R&D Systems, 909-MM and 924-MP). Pro-MMP-9/2 activity was evaluated by optical density and normalized to that of the sham group.

Concentrations of IL-1β, IL-6, and TNF-α in OHSC medium were measured by ELISA (R&D System).¹⁹

Reverse transcription real-time PCR

Total RNA was isolated from ipsilateral striatum by using the miRNeasy Mini Kit (Qiagen). Equal amounts of mRNA were reverse transcribed with a cDNA synthesis kit (Life Technologies). Real-time PCR was carried out with the TaqMan master mix and the following primers: angiogenin1, Mm00833184_s1; angiopoietin-1, Mm00456503_m1; angiopoietin-2, Mm00545822_m1; MMP19, Mm00491296_m1; and angiogenin inhibitor1, Mm00836405 (Life Technologies). The relative gene expression was expressed as fold of change (ΔΔCt) and normalized to that of the sham group.²⁴

Statistical analysis

Data are presented as means ± SD. Data for mRNA expression are presented as mean ± SEM. Student’s t test was used for comparisons of two groups. Differences among multiple groups were analyzed by one-way or two-way ANOVA with Bonferroni post hoc test. We evaluated survival data using a log-rank (Mantel Cox) test. All analysis was carried out with SigmaPlot 12.5 software. A probability value of p < 0.05 was considered statistically significant.

Results

HET0016 decreases levels of 20-HETE after collagenase-induced ICH

To clarify the cell type that expresses CYP4A after ICH, we first performed double immunolabeling with cell-type-specific markers. In the intact brain, CYP4A was present exclusively in NeuN⁺ cells (Figure 1(a)). After ICH, GFAP⁺ cells, but not CD11b⁺ cells, expressed CYP4A in the perihematomal region (Figure 1(b) and (c)). Next, we evaluated the level of 20-HETE in ipsilateral hemisphere by LC/MS/MS. The concentration of 20-HETE was increased by 79.6 ± 26.2% at 6 h, 71.0 ± 10.6% at 24 h, and 102.8 ± 27.4% at 72 h after ICH. HET0016 treatment (10 mg/kg) reduced 20-HETE expression at 24 h after ICH (Figure 1(d)).

Figure 1.

Induction of CYP4A expression after intracerebral hemorrhage (ICH). (a) NeuN⁺/CYP4A⁺ cells were distributed in contralateral and ipsilateral striatum. Green, NeuN; red, CYP4A; n = 6. Scale bars: 50 µm, inset: 10 µm. (b) At 72 h after hemorrhage, GFAP⁺/CYP4A⁺ cells were observed in the perilesional region. Three GFAP⁺/CYP4A⁺ cells are indicated by arrows. Green, GFAP; red, CYP4A; n = 6. Scale bars: 50 µm, inset: 10 µm. (c) CD11b⁺ cells around the perilesional area did not colocalize with CYP4A at 72 h after ICH. Green, CD11b; red, CYP4A; n = 6. Scale bars: 50 µm, inset: 10 µm. (d) The concentration of free 20-HETE in ipsilateral hemisphere after ICH was measured by LC/MS/MS. Statistical significances were analyzed by one-way ANOVA with Bonferroni post hoc test. n = 5–8/group. *p < 0.05 vs. sham group, ^##p < 0.01 vs. 24-h ICH. Data are presented as means ± SD.

HET0016 improves neuronal survival and reduces inflammatory cytokines and reactive oxygen species in OHSCs

We have reported recently that neurons in OHSCs are selectively vulnerable to hemoglobin toxicity.¹⁸ To investigate whether HET0016 protects against hemoglobin-induced hippocampal injury in vitro, we first measured neuronal survival via PI staining in dose-response experiments. HET0016 (10 µmol/L) reduced CA1 cell death by 16.7% in slices exposed to 10 µmol/L hemoglobin for 18 h. 20-HETE antagonist 20-6,15-HEDGE at 10 µmol/L decreased PI uptake by 23.4%. In contrast, 20-HETE agonist 20-5, 14-HEDGE increased neuronal death by 28.5% after hemoglobin exposure (Figure 2(a) and (b)). We further analyzed the concentrations of proinflammatory cytokines in OHSC medium by ELISA. HET0016 reduced TNF-α and IL-1β levels by 36.3 ± 12.1% and 32.9 ± 6.6%, respectively, whereas IL-6 level was unchanged (Figure 2(c)). Next, we measured reactive oxygen species (ROS) accumulation in situ by assessing the oxidation of dihydroethidium to ethidium, which correlates with superoxide radical formation.²⁵ ROS content was lower in HET0016-treated slices than in vehicle-treated slices after 18 h of hemoglobin exposure (Figure 2(d)).

Figure 2.

HET0016 treatment improves neuronal survival and reduces inflammatory cytokines and reactive oxygen species in organotypic hippocampal slice cultures. (a) Hippocampal injury was induced by 10 µmol/L hemoglobin (hemo) exposure. From left to right: HET0016-induced reduction in propidium iodide (PI) uptake was dose-dependent (n = 23–39 slices/group), 20-HETE agonist (20-5,14-HEDGE; 10 µmol/L) increased PI uptake (n = 20–43 slices/group), and 20-HETE antagonist (20-6,15-HEDGE; 10 µmol/L) decreased PI uptake (n = 18–34 slices/group). **p < 0.01, ***p < 0.001 vs. sham group; ^#p < 0.05, ^###p < 0.001 vs. hemoglobin + vehicle group; one-way ANOVA with Bonferroni post hoc test. (b) Representative images and quantification of PI fluorescence in hippocampal slices exposed to saline (sham) or 10 µmol/L hemoglobin with vehicle, 10 µmol/L HET0016, 10 µmol/L 20-HETE agonist, or 10 µmol/L 20-HETE antagonist. Scale bar: 500 µm. n = 18–40 slices/group; *p < 0.05 vs. hemoglobin + vehicle group; one-way ANOVA with Bonferroni post hoc test. (c) Culture medium was collected after slices were exposed to 10 µmol/L hemoglobin for 18 h. The concentrations of TNF-α, IL-1β, and IL-6 were measured by ELISA. n = 8; *p < 0.05 vs. hemoglobin + vehicle group; one-way ANOVA with Bonferroni post hoc test. (d) Oxidation of dihydroethidium (HEt) to ethidium correlates with superoxide radical formation. HET0016 reduced the HEt fluorescent intensity in slice cultures incubated with 10 µmol/L hemoglobin for 18 h. Red, HEt fluorescent indicator. Scale bar = 200 µm. n = 5–9; ***p < 0.001 vs. sham group; ^###p < 0.001 vs. hemoglobin + vehicle group; one-way ANOVA with Bonferroni post hoc test. All data are presented as means ± SD.

HET0016 mitigates ICH-induced brain injury, brain edema, and neurobehavioral deficits

HET0016 did not alter the mortality of mice after ICH (Figure 3(a)). However, Luxol fast blue/Cresyl violet staining at 72 h after ICH showed that lesion volume was 20.7% smaller in mice treated with HET0016 (10 mg/kg) than in those treated with vehicle (Figure 3(b)). The lesion volume corrected for brain swelling showed similar results (Supplementary Figure 1(a)). HET0016 also significantly decreased brain water content in the ipsilateral striatum (Figure 3(c)). A lower dose of HET0016 (1 mg/kg) was not protective (Supplementary Figure 1(b)). Aged mice treated with HET0016 (10 mg/kg) also exhibited a reduction in lesion volume (Supplementary Figure 1(c)).

Figure 3.

HET0016 treatment reduces lesion volume and improves neurobehavioral outcomes after intracerebral hemorrhage (ICH). (a) Survival curves showed no significant difference between the vehicle-treated and the HET0016-treated groups. Log-rank (Mantel-Cox) test, p = 0.598. (b) Luxol fast blue and Cresyl violet-stained brain sections at 72 h after ICH. Lesions are circled in white; scale bar: 1 mm. Intraperitoneal injection of HET0016 (10 mg/kg) reduced lesion volume. n = 8; **p < 0.01 vs. ICH + vehicle; t-test at each time point. (c) HET0016 reduced striatal edema at 72 h after collagenase injection. n = 5; **p < 0.01 vs. ICH + vehicle; t-test. ips-stri: ipsilateral striatum; con-stri: contralateral striatum; cerebel: cerebellum. (d) HET0016 decreased neurologic deficit score at 24 and 72 h after ICH. n = 9; *p < 0.05; Mann–Whitney U test at each time point. Results are shown as box-and-whisker plots. The middle horizontal line within the box represents the median, the boxes extend from the 25th to the 75th percentile, and the whiskers represent 95% confidence intervals. (e) HET0016 improved corner turn test performance of mice (n = 10) at 24 and 72 h after ICH and increased latency to fall in the wire-hanging test (n = 15) at 72 h. ***p < 0.001 vs. sham; ^#p < 0.05 vs. ICH + vehicle; one-way ANOVA with Bonferroni post hoc test at each time point. Values are presented as mean ± SD.

Next we assessed neurologic function of the mice at 24 and 72 h after ICH. Mice treated with HET0016 had lower neurologic deficit scores than did the vehicle-treated group at both time points (Figure 3(d)). HET0016 also improved performance in the corner turn test at 24 and 72 h after ICH and increased falling latency in the wire hanging test at 72 h (Figure 3(e)).

To address whether HET0016 affects the hematoma formation, we evaluated the hematoma size. HET0016 did not change the hematoma size at 24 h post-ICH (Supplementary Figure 1(d)).

HET0016 inhibits neuronal death and inflammatory response in vivo after ICH

To investigate whether HET0016 preserves cell viability after ICH in vivo, we assessed neuronal injury in the perihematomal region at 72 h after ICH. Mice post-treated with HET0016 had fewer FJB⁺ and TUNEL⁺ cells than did vehicle-treated mice (Figure 4(a)). Moreover, HET0016 reduced CD68⁺ microglial numbers and Iba1 expression in the perihematomal area (Figure 4(b)). We further evaluated the response of astrocyte and neutrophil infiltration in the perilesional area. HET0016-treated mice exhibited less GFAP expression and fewer myeloperoxidase-positive cells at 72 h after ICH than did vehicle-treated mice (Figure 4(b) and (c)). These data indicate that HET0016 attenuated the inflammatory reaction after ICH.

Figure 4.

HET0016 treatment reduces neuronal death and inflammatory response in vivo after intracerebral hemorrhage (ICH). (a) HET0016 reduced the number of FJB⁺ and TUNEL⁺ cells at 72 h after ICH. n = 8/group, scale bar = 50 µm. (b) HET0016 decreased the intensity of Iba1-positive and GFAP-positive cells and the number of CD68-positive cells at 72 h after ICH. n = 8/group, scale bars = 50 µm. (c) HET0016 reduced the number of myeloperoxidase (MPO)-positive cells. n = 8/group, scale bar = 25 µm. *p < 0.05, **p < 0.01 vs. ICH + vehicle group; t-test. Data are presented as means ± SD.

HET0016 reduces MMP gelatinolytic activity, ROS, reactive nitrogen species, and phosphorylated Src kinase expression in vivo after ICH

MMP-9 activity is associated with blood–brain barrier disruption and inflammatory response after ICH.²⁶ We found that HET0016 reduced pro-MMP-9 and pro-MMP-2 activity at 24 h after ICH (Figure 5(a)). It is also known that Src kinase is involved in MMP-9 regulation and blood toxicity. Our data showed that HET0016 decreased phospho-Src at 72 h after ICH (Figure 5(b)). Although intact hemoglobin can bind NO and reduce reactive nitrogen species (RNS)-mediated injury, autoxidation and metabolism of hemoglobin can stimulate generation of ROS and RNS,^27,28 which cause protein damage and disrupt cellular function.²⁹ We measured ROS and RNS by assaying for oxidized carbonyl proteins and 3-nitrotyrosine proteins. HET0016 decreased protein carbonylation and nitrosylation at 72 h post-ICH (Figure 5(c) and (d)).

Figure 5.

HET0016 treatment reduces matrix metalloproteinase (MMP) gelatinolytic activity, reactive oxygen species, reactive nitrogen species, and phosphorylated Src kinase expression in vivo after intracerebral hemorrhage (ICH). (a) HET0016 reduced the gelatinase activity of pro-MMP-9 and pro-MMP-2 at 24 h after ICH. n = 8; *p < 0.05, ***p < 0.001 vs. sham; ^#p < 0.05, ^##p < 0.01 vs. ICH + vehicle; one-way ANOVA with Bonferroni post hoc test. (b) 20-HETE inhibition reduced the expression of phosphorylated Src kinase at 24 h after ICH. n = 8/group; *p < 0.05, ***p < 0.001 vs. sham; ^#p < 0.05 vs. sham + vehicle; one-way ANOVA with Bonferroni post hoc test. (c–d) HET0016 decreased levels of carbonyl protein (c) and 3-nitrotryosine protein (d) at 72 h after ICH. n = 8/group; *p < 0.05, ***p < 0.001 vs. sham; ^#p < 0.05, ^###p < 0.001 vs. ICH + vehicle; one-way ANOVA with Bonferroni post hoc test. All data are presented as means ± SD. O.D: optical density.

HET0016 does not inhibit angiogenesis in vivo at day 28 after ICH

VEGF induces endothelial progenitor cell proliferation and migration. To evaluate whether HET0016 inhibits angiogenesis after ICH, we assessed the expression of VEGF and VEGF receptors. We injected mice intraperitoneally with HET0016 (10 mg/kg) once at 2 h after ICH or every 12 h for three days. Expression levels of VEGF and VEGFR2 were decreased at 7 and 14 days after ICH compared with those of the sham group. VEGFR2 level recovered by 21 days, but the reduction in VEGF expression continued at least until day 28 (Figure 6(a)). Neither single nor multiple HET0016 injections significantly altered VEGF or VEGFR2 expression from that in the vehicle-treated mice. VEGFR3, which promotes sprout fusion in angiogenesis and limits VEGFR2 activity,^30,31 was decreased at day 7 in all ICH groups but did not differ from that in the sham group at any other time point (Figure 6(a)). The density and diameter of CD31-positive blood vessels in the perihematomal area were decreased at 28 days after ICH but were unaffected by HET0016 (Figure 6(b)). To identify angiogenesis after ICH, we injected EdU (200 mg/kg body weight) 2 h before the mice were sacrificed. Immunostaining revealed that EdU⁺/lectin⁺ cells appeared at seven days post-ICH (Figure 6(c)).

Figure 6.

HET0016 treatment does not inhibit angiogenesis in vivo at 28 days after intracerebral hemorrhage (ICH). (a) HET0016 did not reduce the expression of VEGF, VEGFR2, or VEGFR3 at 28 days after ICH. No significant difference was present between mice that received a single intraperitoneal injection of HET0016 at 2 h post-ICH (single IP) and those that received injections at 2 h and then every 12 h for three days (multiple IP). n = 8; *p < 0.05, **p < 0.01, ***p < 0.001 vs. sham; one-way ANOVA with Bonferroni post hoc test. (b) HET0016 did not decrease CD31 expression or average blood vessel diameter in the perilesional region at 28 days after ICH. Green, CD31. Scale bar = 50 µm. n = 6/group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. sham; one-way ANOVA with Bonferroni post hoc test. (c) EdU⁺ cells were observed in lectin-positive blood vessels at 14 days after ICH. Scale bar = 15 µm. All data are presented as means ± SD.

To determine whether other pro-angiogenic factors are inhibited by HET0016, we analyzed the mRNA expression of angiogenin1, angiopoietin-1, angiopoietin-2, MMP19, and angiogenin inhibitor1. Overall, HET0016 decreased mRNA levels of angiogenin1, angiopoietin-2, and MMP19 at 7 and 14 days. The group that received multiple HET0016 injections exhibited less angiopoietin-2 than the single-injection group at 14 days post-ICH (Supplementary Figure 2(a) and (b)). However, consistent with the results of VEGF and VEGFR2, we observed no differences between the HET0016-treated and vehicle-treated groups at 21 and 28 days post-ICH (Supplementary Figure 2(c) and (d)).

Discussion

This study showed that inhibition of 20-HETE synthesis with HET0016 significantly reduces lesion volume and brain edema, improves neurologic deficits, decreases the inflammatory response, and reduces ROS production in the early stage after ICH, but does not suppress angiogenesis in the later stage after ICH.

CYP4A is expressed in neurons and can be upregulated by oxidative stress.^32,33 Cultured astrocytes also express CYP4A and synthesize 20-HETE when exposed to arachidonic acid.³⁴ We found that CYP4A was expressed in neurons and induced in astrocytes at 72 h after ICH and that 20-HETE concentration increased within the first three days after ICH. These data indicate that both neuronal and astrocyte CYP4A contribute to 20-HETE synthesis after ICH. The role of astrocyte-produced CYP4A and 20-HETE needs further study.

Inflammation contributes to ICH-induced secondary injury.^26,35 Inflammatory responses include activation and polarization of microglia and astrocytes.³⁶ Therefore, we investigated whether HET0016 reduces inflammation after ICH. We found that inhibition of 20-HETE synthesis in OHSCs exposed to hemoglobin for 18 h decreased IL-1β and TNF-α levels in the culture medium. However, it should be noted that the glucose concentration used in the OHSC culture medium is relatively high (36 mM). Although slice culture medium often has a high glucose concentration, presumably to account for consumption over a few days, this high glucose might exaggerate an inflammatory response that may be amenable to 20-HETE antagonism. Actually, this in vitro slice culture model is relevant to ICH in the setting of uncontrolled diabetes. HET0016 also reduced the number of activated CD68⁺/Iba1⁺ microglia and myeloperoxidase-positive neutrophils and decreased GFAP immunoreactivity at 72 h after ICH in vivo. It should be noted that these results represent only one in vivo model. Although some believe that collagenase enhances the inflammatory response compared with that in the blood injection ICH model, we chose it for its other advantages. Unlike the blood model, the collagenase model has features of vascular rupture, hematoma expansion, and increased intracranial pressure that resemble those of clinical ICH.^35,37 Taken together, our results indicate that HET0016 attenuates proinflammatory responses after ICH in vivo and in vitro. Because astrocytes express CYP4A after ICH, reductions in astrocyte activation might decrease 20-HETE synthesis and release. The mechanisms involved in 20-HETE-induced inflammation need further study.

20-HETE causes an increase of superoxide generation.³⁸ Inhibition of 20-HETE synthesis decreased oxidative stress in an OHSC model of oxygen-glucose deprivation and protected neurons after ischemia-reperfusion injury.³⁹ We showed that HET0016 reduced ROS production and that both HET0016 and 20-HETE antagonist 20-6,15-HEDGE reduced cell death in OHSCs exposed to hemoglobin. HET0016 also reduced the levels of protein carbonylation and nitrosylation and reduced neuronal death early after ICH in vivo. Thus, the HET0016-induced reduction in neuronal death might result from decreases in oxidative stress. Additional work is needed to determine whether HET0016 provides neuroprotection in the late stage after ICH and whether it has the same protection in the blood injection ICH model.

Src kinase signaling is involved in ischemic⁴⁰ and hemorrhagic stroke injury.^14,26 We showed previously that MMP-9 was a downstream target of Src kinase signaling after ICH.¹⁵ In the current study, pro-MMP-9 and pro-MMP-2 activities and Src kinase expression were all reduced by HET0016, suggesting that Src kinase-MMP signaling may be involved in 20-HETE-mediated brain injury after ICH.

Angiogenesis after stroke underpins functional recovery by supplying nutrients and creating a suitable microenvironment for regeneration.⁴¹ Apart from its role in vasoconstriction, 20-HETE works as an angiogenesis mediator. As a mediator upstream of growth factors, 20-HETE is involved in endothelium proliferation and vascular remodeling.⁴² The underlying mechanisms include activation of hypoxia-inducible factor (HIF)-1α, NADPH, ROS, MMP-9, VEGF, and VEGFR2.^1,43 To address the concern that 20-HETE inhibition might delay angiogenesis after ICH, we evaluated the expression of angiogenic factors. The protein levels of VEGF and VEGFR2 decreased markedly at 7 and 14 days after ICH. However, protein levels of VEGF and VEGFR2 and angiogenesis in the perihematomal region were no different from those in the sham group on days 21 and 28. Similar results were reported in a post-hindlimb-ischemia angiogenesis model.²³ Although VEGF expression had not returned to normal by 28 days, strategies that can increase VEGF expression/activity may promote angiogenesis and ICH recovery.

Astrocytes and endothelial cells express VEGFR3 during development and after brain insults.^44,45 VEGFR3 regulates VEGFR2 expression and promotes sprout fusion when VEGFR2 is blocked.^30,31 Our results showed that expression of VEGFR3 was reduced at 7 days after ICH but recovered by 14 days. Based on these findings, VEGF/VEGFR2 might be the primary pathway that regulates angiogenesis after ICH. Moreover, we evaluated mRNA levels of several proangiogenic factors. HET0016 temporarily reduced the mRNA levels of angiogenin-1, angiopoietin-2, and MMP19 at 7 and 14 days post-ICH. However, these differences disappeared at 21 and 28 days. Thus, HET0016 did not inhibit angiogenesis at the late stage after ICH.

Inhibition of vascular 20-HETE formation lowers arterial pressure and blocks vasoconstriction.¹ Moreover, hypertension is a major risk factor for patients with ICH. Systolic blood pressure increases within 3 h of ICH onset to levels beyond premorbid levels.⁴⁶ Thus, HET0016 might inhibit inflammation and also regulate blood pressure. However, previous studies indicated that treatment with HET0016 did not affect body temperature, blood gases, arterial pressure, or cerebral blood flow in ischemic stroke, suggesting that its neuroprotective effect is not due to alterations in physiologic parameters.^16,47 Moreover, HET0016 did not alter hematoma size at 24 h post-ICH.

Epoxyeicosatrienoic acids (EETs) are also metabolites of arachidonic acid formed by P450 enzymes and can modulate vasodilation and angiogenesis.^48,49 The imbalances of 20-HETE and EET levels and the effects of EETs on brain injury and angiogenesis after ICH need to be addressed.

In summary, HET0016 exhibits cerebroprotective activity without inhibiting angiogenesis after ICH. Thus, 20-HETE synthesis inhibition might be a therapeutic strategy for treating patients with ICH.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by NIH Grants: R01NS078026, R01AT007317, R56NS096549, R01NS102583, and R21NS101614 (JW); HL034300 (M.L. Schwartzman); AHA Grants: 17GRNT33660766 (JW); 14POST20140003 and 16SDG30980031 (XH); Stimulating and Advancing ACCM Research (StAAR) grants (XH and JW) from the Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University; and the Robert A. Welch Foundation (I-0011) to JRF.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

Xiaoning Han, Xiaochu Zhao, and Jian Wang designed the experiments and wrote the manuscript. Austin M. Guo provided 20-HETE LC/MS/MS analysis. John R. Falck provided 20-HETE antagonist and agonist. Zengjin Yang, Qian Li, Xi Lan, John R Falck, and Raymond C. Koehler contributed in critical revision of the manuscript. Xiaoning Han, Xiaochun Zhao, Xi Lan, Qian Li, Yufeng Gao, Xi Liu, Jieru Wan, Xuemei Chen, and Weidong Zang collected and analyzed the data.

Supplementary material

Supplementary material for this paper can be found at the journal website: http://journals.sagepub.com/home/jcb