High wall shear stress-dependent podosome formation in a novel murine model of intracranial aneurysm

Abstract

High wall shear stress (HWSS) contributes to intracranial aneurysm (IA) development. However, the underlying molecular mechanisms remain unclear, in part due to the lack of robust animal models that develop IAs in a HWSS-dependent manner. The current study established a new experimental IA model in mice that was utilized to determine HWSS-triggered downstream mechanisms. By a strategic combination of HWSS and low dose elastase, IAs were induced with a high penetrance in hypertensive mice. In contrast, no IAs were observed in control groups where HWSS was absent, suggesting that our new IA model is HWSS-dependent. IA outcomes were assessed by neuroscores that correlate with IA rupture events. Pathological analyses confirmed these experimental IAs resemble those found in humans. Interestingly, HWSS alone promotes the turnover of collagen IV, a major basement membrane component underneath the endothelium, and the formation of endothelial podosomes, subcellular organelles that are known to degrade extracellular matrix proteins. These induced podosomes are functional as they degrade collagen-based substrates locally in the endothelium. These data suggest that this new murine model develops IAs in a HWSS-dependent manner and highlights the contribution of endothelial cells to the early phase of IA. With this model, podosome formation and function was identified as a novel endothelial phenotype triggered by HWSS, which provides new insight into IA pathogenesis.

Keywords: cerebrovascular integrity; endothelial dysfunction; high wall shear stress; intracranial aneurysm; murine model; podosomes.

FIGURE 1

HWSS is a major trigger for IA formation in mice. (A) A schematic view of the left common carotid artery (LCCA) ligation model. CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery; MCA, middle cerebral artery; OA, olfactory artery; ACA, anterior cerebral artery. (B) An experimental protocol for IA induction and assessment in mice. All mice underwent LCCA ligation or sham operation followed by a stereotaxic injection of elastase (12 mU) into the right cistern magna. After infusion of angiotensin-II (750 ng/kg/min), daily neurological scores and body weight were recorded and IA-related assays were performed after all mice were euthanized due to severe neurological deficits or at the end of two weeks. (C) LCCA ligation (IA group) vs. sham operation (control group) were performed in mice followed by elastase injection and angiotensin infusion indicated in (B). After IA induction with sham operation (n = 8) or LCCA ligation (n = 10), cerebral arteries in the circle of Willis (CoW) were visualized by vascular perfusion of bromophenol blue/gelatin mixture. A representative image of cerebral blood vessels from control group was shown in (C1, C2). Representative images were shown of one IA at the left posterior communicating artery (C3, C4) or of multiple IAs (C5–C7) at different locations from LCCA-ligated group. (D) The rate of IA incidence in mice with sham operation or LCCA ligation. Four mice were excluded as their brains were too soft to be help up for postmortem analyses after sudden death. (E) The comparison between unruptured and ruptured IA formation rate in LCCA-ligated mice.

FIGURE 2

Experimental IA outcomes. (A–C) Mouse survival curve, neurological deficit scores, and body weight were recorded as indicated in both sham-operated (n = 8, black line or curve, control group) and LCCA-ligated mice (n = 10, red line or curve, IA group) after the completion of IA induction surgeries (*p < 0.05).

FIGURE 3

Histological analyses of the experimental IAs. (A) Representative light microscopic images of frozen sections with H&E (nuclei: dark blue; cytoplasm: pink) in control normal arteries (A1, A2) and IA samples (A3, A4). (B) Internal elastic lamina was highlighted by Verhoeff Van-Gieson (VVG) staining (dark blue to black, B1–B4) in normal arteries and IA samples, respectively. (C) Masson-trichrome staining shows the distribution of smooth muscle cells (red in C1–C4). Blacked boxed regions were selected for the images in higher magnifications. n = 8 for each group. Scale bars: 50μm.

FIGURE 4

Immunofluorescence analyses of vascular cells in experimental IAs. (A–C) Frozen sections from control arteries or IA samples were immunostained for CD45, F4/80, CD31, and αSMA, respectively. CD45: a pan-inflammatory cell marker (red in A1–A4); F4/80, a marker for macrophages (red in B1–B4); CD31, an endothelial cell marker (red in C1–C4), and αSMA, a smooth muscle marker to highlight vascular structure (green in all panels). Nuclei were stained with 4′ ,6-diamidino-2-phenylindole (DAPI, blue in all panels). White boxed regions were selected for the images in higher magnifications. n = 5 for each group. Scale bars: 50μm.

FIGURE 5

HWSS up-regulates metalloproteinases expressions and collagen IV degradation. (A) Immunostaining of the CoW frozen sections from sham-operated or LCCA-ligated mice (3 days after surgery) against endothelial cell marker CD31 (red) and pan-inflammation marker CD45 (red) as well as smooth muscle cell marker αSMA (green). Nuclei were highlighted by DAPI staining (blue). n = 6 for each group. (B, C) Total cell lysates (Triton soluble vs. insoluble) were extracted from the CoW in mice (3 days after surgery) under sham operation or LCCA ligation. Western blot was performed to reveal the expression of collagen I and collagen IV, respectively. The level of each protein was normalized to GAPDH loading control and further quantified in (C). (D) Based on RNA-seq profiling, expression levels of metalloproteinases [Fragments Per Kilobase Million (FPKM) >2.0] were shown while all other metalloproteinases with FPKM lower than 2.0 were excluded. (E) Quantitative RT-PCR (RT-qPCR) revealed the expression levels of MMP1, MMP2, and MMP14 in the CoW that was exposed to sham operation or LCCA ligation. The expression level of each gene was normalized to GAPDH. n = 3 independent experiments for (C, E), respectively. n.s., not significant; *p < 0.05, **p < 0.01. Scale bar: 50μm.

FIGURE 6

HWSS induces endothelial podosome formation and function. (A) Representative images of podosomes were shown by en face immunostaining against cortactin (green) in the dissected CoW from mice after sham operation or LCCA ligation. Endothelial lining was labeled by anti-VE-cadherin antibody (red). (B) The rate of podosome formation was calculated by the number of cells having podosomes over the total number of cells counted. At least nine different fields under 20X objective were selected for calculations. n = 5 for sham operation or n = 6 for LCCA ligation group. (C) Double en face immunostaining against two podosome markers including cortactin (green) and p-SRC-Y416 (red) in the dissected CoW from mice after LCCA ligation for 3 days. (D) The percentage of co-localization incidence was measured by the number of yellow-positive puncta over total number of green-positive puncta. More than 120 different cells were examined. n = 5 for mice after sham operation or LCCA ligation, respectively. (E) The podosome activity was visualized by the released green fluorescence due to the substrate degradation based on in situ zymography. See green dots (white arrows) in the endothelium. (F) Total fluorescence intensity was quantified by Image J and normalized to that from sham control. More than 12 different frozen sections were examined in either sham-operated (n = 6) or LCCA-ligated mice (n = 5). White boxed regions were selected for images under higher magnification. Student’s t-test used for statistical analysis. ***p < 0.001, **p < 0.01.