Vascular Cell Senescence Contributes to Blood-Brain Barrier Breakdown

Abstract

Background and purpose: Age-related changes in the cerebrovasculature, including blood-brain barrier (BBB) disruption, are emerging as potential risks for diverse neurological conditions. Because the accumulation of senescent cells in tissues is increasingly recognized as a critical step leading to age-related organ dysfunction, we evaluated whether senescent vascular cells are associated with compromised BBB integrity.

Methods: Effects of vascular cell senescence on tight junction and barrier integrity were studied using an in vitro BBB model, composed of endothelial cells, pericytes, and astrocytes. In addition, tight junction coverage in microvessels and BBB integrity in BubR1 hypomorphic (BubR1(H/H)) mice, which display senescence cell-dependent phenotypes, were examined.

Results: When an in vitro BBB model was constructed with senescent endothelial cells and pericytes, tight junction structure and barrier integrity (evaluated by transendothelial electric resistance and tracer efflux assay using sodium fluorescein and Evans blue-albumin were significantly impaired. Endothelial cells and pericytes from BubR1(H/H) mice had increased senescent-associated β-galactosidase activity and p16(INK4a) expression, demonstrating an exacerbation of senescence. The coverage by tight junction proteins in the cortical microvessels were reduced in BubR1(H/H) mice, consistent with a compromised BBB integrity from permeability assays. Importantly, the coverage of microvessels by end-feet of aquaporin 4-immunoreactive astrocytes was not altered in the cortex of the BubR1(H/H) mice.

Conclusions: Our results indicate that accumulation of senescent vascular cells is associated with compromised BBB integrity, providing insights into the mechanism of BBB disruption related to biological aging.

Keywords: aging; brain; endothelial cells; pericytes; permeability; tight junctions.

Figure 1. Characterization of cerebrovascular endothelial cells and pericytes from young and middle-aged mice

A, Senescence-associated β-galactosidase (SA-β-gal) activities were detected in primary ECs and PCs from young (5 to 8-week-old) and middle-aged (40 to 50-week-old) C57BL/6 WT mice. B, Number of SA-β-gal-positive cells was quantified comparing ECs and PCs from young and middle-aged mice. C, The mRNA levels of p16^INK4a and p21 in the cells were quantified by RTPCR. D, Number of cells was counted at 5 and 10 days after plating of the cells. Bar represents 200 μm. Data are plotted as mean ± S.E.M. (n = 3). *, p < 0.05, **, p < 0.01, n.s., not significant. Student’s t test was employed for the statistical analysis.

Figure 2. Cellular senescence attenuates barrier integrity and endothelial TJ in an in vitro BBB model

A, Left panels: Schematic representation of the triple co-culture for in vitro BBB model. ECs were cultured on semipermeable filter inserts and PCs were plated on the bottom side of the filters, while astrocytes were cultured into the well of culture plate. Right panels: Representative ECs, PCs and astrocytes cultures stained for CD31 (endothelial cell marker; red), PDGFR-β (pericyte maker; green) and GFAP (astrocyte marker; green), respectively. Nuclei were counterstained with DAPI (blue). B, In vitro BBB models composed of ECs/PCs from middle-aged mice (senescent BBB model) or young mice (young BBB model) were prepared. Barrier integrity of standard and senescent BBB models was evaluated by TEER measurement (leftl), permeability coefficient for NaF (middle) and EB-albumin (right) at 5 days after plating for co-culture, respectively. C, ECs in standard and senescent BBB models were subjected to staining for ZO-1, occludin or Claudin-5 (green) at day 5 of culture. White arrows indicate the tight junctions with frayed appearance. Dotted white arrows indicate the abnormal cells with the smaller cytoplasm highly immune-reactive for TJ proteins. D, Percentages of ECs with frayed borders as demonstrated by staining for TJ proteins were calculated in standard and senescent BBB models. Data are plotted as mean ± S.E.M. (n = 3-4). *p < 0.05. Student’s t test was employed for the statistical analysis.

Figure 3. Increased brain vascular cell senescence in BubR1^H/H mice

A, SA-β-gal activities were detected in ECs and PCs from BubR1^+/+ mice and BubR1^H/H mice (5 to 8-week-old). B, Number of SA-β-gal-positive cells was quantified comparing ECs and PCs from BubR1^+/+ mice and BubR1^H/H mice. C, The mRNA levels of p16^INK4a and p21 in the cells were quantified by RT-PCR. Bar represents 200 μm. Data are plotted as mean ± S.E.M. (n = 4). *, p < 0.05, **, p < 0.01, n.s., not significant. Student’s t test was employed for the statistical analysis.

Figure 4. Impaired BBB integrity and reduced TJ protein coverage in BubR1^H/H mice

A, Left panels (upper): Representative confocal images and fluorescence histograms showing perivascular leakage of EB dye in the cortex of 5-month-old BubR1^+/+ littermates and BubR1^H/H mice. EB dye (2%; 4 mL/kg of body weight) was injected intraperitoneally, and EB leakage in the brains was analyzed after 6 hours of injection. Left panels (lower): Fluorescence intensities across the section (white lines) including MVs stained with anti-collagen type IV antibody (red) and EB dye (green) were quantified to confirm the EB leakage around MVs. Right panel: Quantifications of extravasated EB dye in the brains of 1- and 5-month-old BubR1^+/+ littermates and BubR1^H/H mice. B, C, D, Frozen sections of cortex and hippocampus from 5-month-old BubR1^+/+ littermates and BubR1^H/H mice were co-stained for TJ proteins (green; ZO-1, occludin or Claudin-5) and an endothelial marker Isolectin-B4 (red). TJ coverage was quantified against EC layers as determined by Isolectin-B4 immunoreactivity in different brain regions. Bar represents 100 μm. Data are plotted as mean ± S.E.M. (n = 4-5). *, p < 0.05, **, p < 0.01, n.s., not significant. Student’s t test was employed for the statistical analysis.

Figure 5. Accelerated activation of astrocytes and microglia in BubR1^H/H mice

A, GFAP and Iba1 were immunostained in cortices of BubR1^+/+ littermates and BubR1^H/H mice at 5 months of age. Bar represents 1 mm. B, Slides were subjected to digital analysis using the Aperio ImageScope software and the expression levels of GFAP and Iba1 were quantified. C, Expression levels of GFAP, synaptophysin (Syp) and PSD-95 were analyzed by Western blotting in the brains of littermate controls and BubR1^H/H mice at 1 and 5 months of age. Data are plotted as mean ± S.E.M. (n = 4-5). *, p < 0.05. n.s., not significant. Student’s t test was employed for the statistical analysis.

Figure 6. Increased association of MVs with reactive astrocytes in BubR1^H/H mice without affecting MV coverage by aquaporin 4-positive astrocytic end-feet

A, Representative confocal images of cortices from 1- and 5-month-old BubR1^+/+ littermates and BubR1^H/H mice co-stained with anti-GFAP (green) and anti-collagen type IV antibody (red). B, Quantifications of MVs associated with reactive astrocytes in the cortices. Reactive astrocytes were identified by strong GFAP immunoreactivities and hypertrophic morphologies with thick proximal processes. The percentage of MVs associated with reactive astrocytes were calculated by dividing the numbers of collagen type IV-positive MVs that overlap with reactive astrocytes by those of total collagen type IV-positive MVs. C, Representative confocal images of cortices from 1- and 5-month-old BubR1^+/+ littermates and BubR1^H/H mice co-stained with anti-AQP4 (green) and Isolectin B4 (red). D, Quantifications of MVs covered by AQP4-positive astrocytic end-feet in the cortices. Bar represents 100 μm. Data are plotted as mean ± S.E.M. (n = 4-5). **, p < 0.01. n.s., not significant. Student’s t test was employed for the statistical analysis.