Internal modulation of proteolysis in vascular extracellular matrix remodeling: role of ADAM metallopeptidase with thrombospondin type 1 motif 5 in the development of intracranial aneurysm rupture

ADAMTS-5 is decreased in the arterial wall of aneurysmal cerebral arteries

First, we investigated the presence of ADAMTS-5 in the aneurysmal arteries of human tissue and compared them with normal cerebral arteries by immunostaining. The CTA imaging data of the two patients who provided human IA samples in the study are shown in Figure 1B. Human IA sample 1 and sample 2 were obtained from IA patient 1 and patient 2, respectively. Extensive ADAMTS-5-positive immunostaining was revealed in the major parts of the media and intima/adventitia for normal cerebral arteries, with only slight ADAMTS-5-positive staining in the luminal part of the intima of IA arteries (Figure 1A). To further verify the decrease in ADAMTS-5 in IA arteries, IA samples obtained from mouse models were used. Immunostainings performed from IA mice samples also revealed the reduction of ADAMTS-5 within the arterial wall of IA arteries (Figure 1C, 1D).

Figure 1. Decreased presence of ADAMTS-5 within the aneurysmal wall. (A) Representative immunofluorescence performed on intracranial aneurysm samples harvested from human IA patients after microsurgical clipping, and the normal cerebral artery harvested from a human brain trauma patient, showing the presence of ADAMTS-5. ADAMTS-5 and Collagen IV are displayed in red and green, respectively. Scale bars: 20 μm. (B) CTA identification of aneurysms from the patients included in this study. Human IA sample 1 and sample 2 were obtained from IA patient 1 and patient 2, respectively. (C, D) Representative immunofluorescence performed on mouse samples displaying the healthy control artery and aneurysmal artery for ADAMTS-5. Scale bars: 20 μm. ATS5, ADAMTS-5; COLL IV, Collagen IV.

Administration of rADAMTS-5 protects against IA rupture

Given the obvious reduction in the cerebral arterial ADAMTS-5 expression observed in human IA and mouse IA, we sought to determine whether ADAMTS-5 played a protective role against IA development using the approach that giving a consecutive number of days of intraperitoneal injections of recombinant ADAMTS-5 protein (rADAMTS-5) in mice that received IA induction. Until the end of the 15-day experiment, no significant difference was found in the overall incidence of aneurysms between the control (CTRL) and rADAMTS-5 treatment group (55.56% versus 75%; n=18 versus n=16; Figure 2A). Nevertheless, rADAMTS-5 treatment significantly reduced the aneurysm rupture rate (Figure 2A; control versus rADAMTS-5: 80% versus 33.33%; P<0.05). The mouse group treated with rADAMTS-5 showed significantly better symptom-free survival than the control group (Figure 2B; Kaplan-Meier log-rank P<0.05). Figure 2D shows normal cerebral arteries, Figure 2C shows an unruptured aneurysm from a mouse without symptomatic signs throughout the experimental period, and Figure 2E shows a ruptured aneurysm with subarachnoid hemorrhage from a mouse that exhibited symptoms of aneurysmal rupture at day 7 after aneurysm induction. Additionally, the blood pressure was monitored before aneurysm induction surgery and every 5 days until day 15 of the study. No significant difference was found in the blood pressure between the control and treatment groups at any time point (Supplementary Figure 2 in the Supplementary Materials). The animal experimental procedure is shown in Supplementary Figure 1 (Supplementary Materials).

Figure 2. Effects of recombinant protein ADAMTS-5 on the development of aneurysmal rupture in male mice. (A) Incidence of aneurysm and rupture rate in sham mice, control (CTRL) IA mice, and recombinant protein ADAMTS-5-administered (rADAMTS-5) IA mice. (B) Symptom-free curve (Kaplan-Meier analysis curve) demonstrated a significant increase in survival in the rADAMTS-5 treatment cohort (log-rank P<0.05). (C) Unruptured aneurysm. (D) No aneurysm. (E) Ruptured aneurysm. *P<0.05, **P<0.01.

rADAMTS-5-administered IA mice exhibit reduced cerebral arterial matrix degradation

After determining of the protective effect of rADAMTS-5 administration in the IA mouse model, we examined the role of ADAMTS-5 in protecting against arterial ECM degradation. As expected, hematoxylin and eosin staining showed conspicuous thickening of the arterial wall with massive matrix degradation and remodeling in elastase-induced IA mice (Figure 3A). Mice administered rADAMTS-5 showed reduced adventitial atrophy and less lamellar fragmentation than control IA mice (Figure 3A). Next, elastin Verhoeff-van Gieson (EVG) staining was performed to characterize the elastin integrity of IA arteries. The staining demonstrated extensive elastin degradation within the aneurysmal wall of elastase-induced IA mice compared with intact elastin found in sham mice. However, the group of mice that received rADAMTS-5 exhibited a decreased tendency of elastin degradation (Figure 3A). Semiquantitative measurements of elastin degradation showed significantly reduced elastin degradation in rADAMTS-5-administered mice compared with control IA mice (Figure 3B). To further assess collagen distribution in IA, we also performed picrosirius red staining (PRS) for collagen within the aneurysmal tissue by polarization microscopy. Polarized light for picrosirius staining analysis confirmed low mural collagen existence localized within the adventitial layer of the aneurysm section acquired from elastase-induced control IA mice. The overall picrosirius red staining area was lower in the control IA arterial wall than in rADAMTS-5-administered mice (Figure 3C).

Figure 3. Recombinant protein ADAMTS-5 protected elastase-induced IA mice from vascular matrix degradation. (A) Representative hematoxylin and eosin (H&E) and elastin Verhoeff-Van Gieson (EVG)-stained section of cerebral arterial sections of sham mice, control (CTRL) IA mice, and recombinant protein ADAMTS-5-administered (rADAMTS-5) IA mice. Scale bar: 20 μm. (B) Quantification histogram showing elastin filament degradation. Arterial wall elastin filament degradation was evaluated based on the degree of degradation in elastin fibers (graded 0–4) as described in the Materials and Methods. **P<0.01. (C) Picrosirius red staining for collagen content within the arteries captured by polarization microscopy imaging from the sham mice, control IA mice, and recombinant protein ADAMTS-5-administered (rADAMTS-5) IA mice. Compared with the control group, the rADAMTS-5-administered group showed increased collagen birefringence under polarization. Scale bar: 20 μm.

Macrophage infiltration and gene expression in the cerebral arteries of rADAMTS-5-administered mice

Infiltrating macrophages in the IA wall and alterations in inflammatory mediators are associated with the formation and rupture of aneurysms. And macrophage infiltration is a common phenomenon that exists in aneurysm formation. Thus, the localization of macrophages in mouse IA arterial walls was identified by immunostaining. Sections of aneurysmal arteries of control IA mice showed a significantly higher staining area for macrophages (monocyte and macrophage antibody staining area) than those of mice receiving rADAMTS-5 during the 2 weeks of IA induction (Figure 4A). Thereafter, we assessed the role of ADAMTS-5 in the regulation of specific inflammatory mediators during IA formation and rupture. After aneurysm induction, rADAMTS-5-administered mice showed a significant difference in the expression of multiple inflammatory mediators (tumor necrosis factor-α [TNF-α], matrix metalloproteinase [MMP]-2, interferon-γ [IFN-γ], nuclear factor kappa B [NF-κB]-p65, and nuclear factor kappa B [NF-κB]-p50) when compared with control IA mice (Figure 4B). By contrast, no significant increase was found in the expression of genes encoding the mediator of monocyte chemoattractant protein-1 (MCP-1) (Figure 4B).

Figure 4. Effects of rADAMTS-5 on monocyte/macrophage infiltration and inflammatory mediators within the IA artery. (A) Immunostaining for macrophages (white arrow) with MOMA-2 (red) of arterial sections of the sham group, control group (CTRL), and rADAMTS-5-administered group. Scale bar, 40μm. (B) Effects of rADAMTS-5 treatment on the mRNA levels of inflammatory mediators. *P<0.05, **P<0.01.

rADAMTS-5 administration in IA mice reduces cerebral arterial apoptosis

Cell apoptosis of the arterial wall is a significant feature of aneurysmal disease and may contribute to arterial destruction and disease development [9, 10]. Therefore, we examined the role of ADAMTS-5 in arterial cell apoptosis. The in vivo TUNEL assay was performed to evaluate cell apoptosis in the aneurysmal wall. TUNEL-positive cells were abundant in the arterial wall of control IA mice but were significantly reduced in the arterial wall of rADAMTS-5-administered IA mice (Figure 5A, 5B). The finding suggests the potential involvement of ADAMTS-5 in apoptosis during IA development and rupture.

Figure 5. Decreased apoptosis in the IA arteries of rADAMTS-5-administered mice. (A) Representative images of TUNEL staining indicating less apoptosis (displayed in red) in arteries from rADAMTS-5-administered mice than in those from control (CTRL) mice. (B) Quantification of the TUNEL (+) cells in arteries from the control (CTRL) mice and rADAMTS-5-administered mice. **P<0.01.

ADAMTS-5-related proteolysis in the cerebral arteries of IA mice

Members of the ADAMTS family are the enzymes responsible for aggregated proteoglycan cleavage [11–13]. Versican and aggrecan can be cleaved by different proteases including MMPs and several members of the ADAMTS family [14, 15]. Immunofluorescence staining revealed the increased presence of versican in the aneurysmal wall of control IA mice compared with that in the healthy arterial wall of sham mice. Additionally, an increased tendency was observed in the cleavage products of versican (neoepitope of versican) in the aneurysmal wall of rADAMTS-5-administered IA mice compared with that in the arteries of control IA mice (Figure 6A), indicating the involvement of ADAMTS-5 in versican degradation during IA formation. Furthermore, aggrecan, another large aggregating proteoglycan, was also accumulated in the aneurysmal artery wall after IA induction in mice. We also found that the administration of rADAMTS-5 in IA mice reduced the aberrant accumulation of aggrecan. This reduction was accompanied by an opposite increase in the degradation products of the proteoglycan aggrecan (neoepitope of aggrecan) (Figure 6B).

Figure 6. Effects of rADAMTS-5 on proteolysis and detection of the main proteoglycans in IA arteries. Representative immunofluorescence staining images showing (A) versican (VCAN) degradation and (B) aggrecan (ACAN) degradation in IA arteries from sham mice and control (CTRL) mice and in arteries from rADAMTS-5-administered mice. Images in columns 3 and 5 represent higher magnification views of the areas contained within the white boxes in the left-hand columns. Versican and aggrecan are displayed in red, and the neoepitopes of versican and aggrecan are displayed in green. Scale bar: 25 μm.

Validation of the role of ADAMTS-5 in cerebral vessels in the zebrafish animal model

ADAMTS-5 inhibitor (Supplementary Figure 3 in Supplementary Materials) treatment was performed in zebrafish embryos. Compared with the fish embryos of the control group (exposed to 0.1% DMSO, v/v), which exhibit normal development, intracranial hemorrhage was observed in the group treated with ADAMTS-5 inhibitor (ATS5-in 0.5 μg/mL) from 36 hpf to 72 hpf (Figure 7A). O-Dianisidine staining at 72 hpf confirmed the presence of intracranial hemorrhage in those embryos (Figure 7B). We further determined the effect of ADAMTS-5 inhibitor using the Tg (flk: EGFP; gata1: DsRed) transgenic zebrafish line, in which vascular endothelial cells are labeled with EGFP (enhanced green fluorescent protein), and red blood cells are labeled with DsRed (Discosoma sp. red fluorescent protein). The results of confocal microscopy showed that intracranial hemorrhage occurred in the midbrain and hindbrain of the fish embryos (Figure 7C). According to the statistical data, the rate of intracranial hemorrhage was significantly higher in the high-dose ADAMTS-5 inhibitor treatment group than in the low-dose group and DMSO-treatment control group (Figure 7D). The results demonstrated that ADAMTS-5 inhibition may weaken the integrity of the brain blood vessels, leading to intracranial hemorrhage.

Figure 7. Inhibition of ADAMTS-5 causes intracranial hemorrhage in zebrafish. (A) Zebrafish embryos treated with DMSO (control, CTRL group), and intracranial hemorrhage in embryos treated with 0.5 μg/mL of ADAMTS-5 inhibitor (ATS5-in). The black arrow indicates the bleeding site. (B) O-Dianisidine staining confirming intracranial hemorrhage at 72 hpf. (C) Confocal microscopy showing cerebral blood vessel leakage of the Tg (flk: EGFP; gata1: DsRed) embryo. (D) Incidence rates of intracranial hemorrhage of the control group (CTRL) and ADAMTS-5 inhibitor treatment group (ATS5-in) at 0.25 μg/mL and 0.5 μg/mL. Scale bar: 50 μm. **P<0.01.

Animal study design

Experiments or animal surgery procedures were conducted in accordance with the guidelines approved by the Tianjin Medical University General Hospital, Institutional Animal Care and Use Committee.

Mouse model study

IAs were induced in mice as previously described [49, 50], all in a C57BL/6 background and 8~10 weeks old. Briefly, a single injection of elastase (35 mU in 2.5 μL; Sigma-Aldrich, St. Louis, MO, USA) into the cerebrospinal fluid at the right basal cistern was realized in anesthetized animals, followed by subcutaneous implantation of an osmotic pump (Durect Corporation, Cupertino, CA, USA) filled with angiotensin II (1000 ng/kg/min; Sigma-Aldrich) to induce systemic hypertension. Both the vehicle control group (CTRL group) and the rADAMTS-5 group received elastase injection and angiotensin II infusion, the sham group received sterile saline. Systolic blood pressure was assessed by repeated measurements using the tail-cuff method. To detect IA rupture, neurological examination was performed daily by 2 blinded observers, as previously described [50, 51]. Animals were analyzed twice a day for the detection of neurological symptoms during 15 days postinduction. Neurological symptoms were scored as follows: 0, normal function; 1, reduced eating or drinking activity demonstrated by a weight loss >2.0 g of body mass (≈10% weight loss) over 24 hours; 2, flexion of the torso and forelimbs on lifting the whole animal by the tail; 3, circling to one side with a normal posture at rest; 4, leaning to one side at rest; and 5, no spontaneous activity. Symptomatic mice (neurological symptom score, 1-5) were immediately euthanized (within 3 hours) to identify intracranial hemorrhage (ruptured aneurysms). Before symptoms occur, the mice were considered as symptom-free surviving animals, and all asymptomatic mice were euthanized 15 days after aneurysm induction. The brain samples were perfused with phosphate-buffered saline followed by gelatin containing blue dye to visualize cerebral arteries. Aneurysms are defined as a localized outward bulging of the vascular wall whose diameter is greater than the parent artery diameter [49, 52].

We started the administration of recombinant protein ADAMTS-5 (R&D SYSTEMS, Minneapolis, MN, USA) 1 day before aneurysm induction and continued the treatment for 2 weeks. Eighteen mice were administered rADAMTS-5 at a dosage of 7.5 μg/kg/day through intraperitoneal injection (200 μL injection volume), and sixteen mice in the vehicle control group received a phosphate-buffered saline injection.

Zebrafish husbandry and chemical treatments

Zebrafish (Danio rerio) husbandry and embryo maintenance were performed under standard laboratory conditions according to the Institutional Animal Care and Use Committee protocols. The AB wild-type strain and transgenic line Tg (flk: EGFP; gata1: DsRed; endothelial cells are marked by EGFP, and erythrocytes are indicated by DsRed) were used in this study. For chemical treatment, the embryos were incubated from 5 to 72 hpf in E3 egg water supplemented with ADAMTS-5 inhibitor (APExBIO, Houston, TX, USA) or DMSO (dimethyl sulfoxide, Sigma-Aldrich). The embryos were treated with 0.003% 1-phenyl-2-thiourea (PTU, Sigma-Aldrich) to avoid pigmentation. Thirty embryos were incubated in each group.

O-Dianisidine staining

Hemoglobin leakage was detected at 3 dpf by performing o-dianisidine staining as previously described [53]. Dechorionated embryos were collected and stained for 15 min in the dark with 0.6 mg/mL of o-dianisidine (Sigma-Aldrich), 0.01 M sodium acetate (pH 4.5), 0.65% H₂O₂ and 40% (v/v) ethanol at room temperature, followed by three washes with PBS. The stained embryos were immediately analyzed by microscopy (Olympus, Tokyo, Japan).

Immunostaining

Human samples

Human aneurysm samples were obtained during the surgeries by resecting the aneurysm sac distal to the clip closing the neck. Samples were obtained from two patients: a 69-year-old woman who had subarachnoid hemorrhage after a rupture of the posterior communicating artery IA (patient 1); a 40-year-old man who had subarachnoid hemorrhage after a rupture of the anterior communicating artery IA (patient 2). Normal cerebral vascular specimens were acquired from emergency surgery for brain trauma. The tissues were immediately stored for 2 hours in a fixative and then were cryoprotected before freezing in Tissue-Tek (Sakura, Tokyo, Japan). Experiments with the clinical samples were performed according to the principles expressed in the Declaration of Helsinki, and informed consent was obtained.

Mouse tissue samples

Each mouse was anesthetized and transcardially perfused with cold phosphate-buffered saline, followed by 20 mL of fixative solution (4% paraformaldehyde). The brains were cryoprotected before freezing in Tissue-Tek (Sakura).

Staining and confocal microscopy

For both human and mouse tissue samples, cryomicrotome-cut transversal sections (10 μm) were collected on polylysine slides and stored at −80° C before staining. Briefly, mouse and human tissue sections were blocked with 5% normal goat serum containing 0.1% Triton X-100 for 60 min (room temperature). Sections were incubated overnight with the following primary antibodies: collagen IV (Southern Biotech, Birmingham, AL, USA); ADAMTS-5, MOMA-2, versican-neo (the DPEAAE neoepitope of versican, neoepitope on versican generated by ADAMTS cleavage), and aggrecan (Abcam, Cambridge, MA, USA); aggrecan-neo (the NITEGE neoepitope of aggrecan) and versican (Thermo Fisher Scientific, Waltham, MA, USA). The sections were then incubated with the appropriate secondary antibody (Alexa Fluor 488, 594, or 647, and FITC or Cy3, Abcam). The sections were washed and coverslipped with antifade medium containing DAPI (Abcam), and images were digitally captured using an Olympus FluoView 1200 confocal microscope, followed by analysis using Image J (NIH, USA) software.

For zebrafish, living larvae were embedded in 1.2% low melting agarose, which was dissolved in E3 medium and Tricaine (Sigma-Aldrich). Tg (flk: EGFP; gata1: DsRed) embryos at 48 hpf with intracranial hemorrhage were captured by confocal microscopy (Carl Zeiss, Jena, Germany).

Gene expression in mouse cerebral arteries

Cerebral arteries (Circle of Willis, including the middle cerebral arteries and basilar artery) were isolated from the mouse brain when possible and dissolved in TRIzol (Invitrogen, Carlsbad, CA, USA). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify the mRNA levels. Quantitative values were obtained from the threshold cycle value (CT), and the data were analyzed by the 2^−∆∆CT method. We assessed the mRNA expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), matrix metalloproteinase-2 (MMP-2), and nuclear factor kappa B (NF-κB). The transcript amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified as an internal RNA control.

Histological assessments

Aneurysmal artery sections were subjected to hematoxylin and eosin staining (Solarbio, Beijing, China), Verhoeff-van Gieson elastin staining (Abcam), and picrosirius red staining (Solarbio) according to the manufacturer’s instructions. Two independent observers who were blinded to the animal group allocation examined 3-6 arterial sections from mice per group. The extent of elastin fiber integrity was scored on a scale of 0 to 4 (0, no elastin degradation; 1, mild degradation; 2, moderate; 3, moderate to severe; and 4, severe elastin degradation).

TUNEL assay

To study apoptosis in the aneurysmal artery, we performed TUNEL assays using an in situ cell death detection kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions. In brief, frozen sections of the cerebral artery were incubated with 1% of Triton X-100 in PBS for 5 min at room temperature (RT). After a wash in PBS, the sections were then incubated with 50 μL of TUNEL reaction mixture for 60 min at 37° C in a humidified atmosphere in the dark. Subsequently, the nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) for 10 min in the dark. After staining, the sections were observed using an Olympus FV1200 confocal microscope. For each treatment group, images from three randomly selected views were captured. For each picture, the number of positive cells and total nuclei were quantified, and the percentage of positive cells was calculated.

Statistical analysis

All of the results were expressed in the form of means ± SD. Fisher’s exact test was used to analyze the incidence of aneurysms (number of mice with any ruptured or unruptured aneurysms/total number of mice), the incidence of ruptured aneurysms (number of mice with ruptured aneurysms/total number of mice), and rupture rate (number of mice with ruptured aneurysms/number of mice with any aneurysms). Survival analyses were performed using the Kaplan–Meier method and the log-rank test. Mice that did not develop aneurysms were excluded in the survival analysis. The mRNA levels, intracranial hemorrhage rate, and blood pressure data were analyzed using Student’s t test or two-way ANOVA. GraphPad Prism 6 (GraphPad Software Inc.) was used for statistical analysis, and a P value <0.05 was considered statistically significant.