Endothelial-Ercc1 DNA repair deficiency provokes blood-brain barrier dysfunction

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Jüttner, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4358616/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2025 Read the published version in Cell Death & Disease → Version 1 posted You are reading this latest preprint version Abstract Aging of the brain vasculature plays a key role in the development of neurovascular and neurodegenerative diseases, thereby contributing to cognitive impairment. Among other factors, DNA damage strongly promotes cellular aging, however, the role of genomic instability in brain endothelial cells (EC) and its potential effect on brain homeostasis is still largely unclear. We here investigated how endothelial aging impacts blood-brain barrier (BBB) function by using excision repair cross complementation group 1 (ERCC1)-deficient human brain ECs and an EC-specific Ercc1 knock out (EC-KO) mouse model. In vitro, ERCC1-deficient brain ECs displayed increased senescence-associated secretory phenotype (SASP) expression, reduced BBB integrity and higher sprouting capacities due to an underlying dysregulation of the Dll4-Notch pathway. In line, EC-KO mice showed more P21 + cells, augmented expression of angiogenic markers and a concomitant increase in the number of brain ECs and pericytes. Moreover, EC-KO mice displayed BBB leakage and enhanced cell adhesion molecule expression accompanied by peripheral immune cell infiltration into the brain. These findings were confined to the white matter, suggesting a regional susceptibility. Collectively, our results underline the role of endothelial aging as a driver of impaired BBB function, endothelial sprouting and increased immune cell migration into the brain, thereby contributing to impaired brain homeostasis as observed during the aging process. Biological sciences/Neuroscience/Neural ageing Biological sciences/Physiology/Ageing Endothelial aging BBB ERCC1 senescence white matter angiogenesis immune cell migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Brain endothelial cells (ECs) line the interior wall of cerebral microvasculature and establish the blood-brain barrier (BBB), which maintains the delicate homeostasis of the central nervous system (CNS). Brain endothelial tight junctions (i.e. Claudin-5) and adherens junction proteins (i.e. VE-cadherin) ensure the BBB-specific paracellular resistance, which prevents uncontrolled entry of blood components and infiltration of peripheral immune cells into the brain ( 1 ). Additionally, BBB-specific transporters, such as the efflux transporter P-glycoprotein (P-gp) and major facilitator superfamily domain-containing protein 2a (Mfsd2a), regulate the metabolite exchange between CNS and periphery ( 2 ), ensuring optimal brain performance. During aging, brain EC fitness and function are severely affected ( 3 ), leading to abnormal vascular responsiveness to cerebral blood flow and disruption of the BBB ( 4 – 7 ). Aging-related alterations in BBB function include reduced integrity, altered transport mechanisms ( 8 ) and abnormal angiogenesis ( 9 ). Physiological angiogenesis describes the multistep process of new vessel formation from the existing vasculature and is crucial to respond to the tissues oxygen needs ( 10 ). In elderly, impaired angiogenesis and pathological vascular remodelling is suggested to contribute to microvascular rarefaction and potentially reduced tissue perfusion ( 11 – 17 ). Although dysfunction of brain ECs is recognized as a significant factor in the onset and progression of age-related neurodegenerative diseases such as stroke and different forms of dementia, including Alzheimer’s disease and vascular dementia, the underlying mechanisms remain elusive ( 18 – 22 ). With age, cellular repair mechanisms are known to gradually deteriorate, leading to the accumulation of DNA damage and the advancement of cellular aging ( 23 ). DNA damage response can induce aging via several mechanisms, including metabolic changes, transcriptional stress and senescence ( 24 , 25 ). Cells can progress into senescence at the end-stage of their replicative capacity, marked by irreversible cell cycle arrest ( 26 ). Senescent cells are metabolically active and acquire a cell-specific senescence-associated secretory phenotype (SASP) characterized by cytokine (i.e. Interleukins IL-6, IL-1β), chemokine (i.e. CXCL1, CXCL10), vasoactive mediator and growth factor (i.e. VEGF, TGF-β) production. Mouse models employing the deletion of the Excision repair cross complementation group 1 ( Ercc1 ), a DNA repair endonuclease, have been successfully used to study human aging and senescence ( 24 , 27 ). Ercc1 knock out mice ( Ercc1 Δ/− KO) closely mimic human vascular aging by displaying increased vascular stiffness, extracellular matrix remodelling and reduced vasodilator function ( 28 , 29 ), thereby supporting the link between DNA damage and age-related vascular impairments. Nevertheless, it remains unclear how specifically endothelial aging affects BBB function. In this study, we investigated the role of EC aging in BBB dysfunction and inflammation in vitro and in vivo . We report that ERCC1 deficiency in human brain ECs results in SASP expression, reduced BBB function and enhanced endothelial sprouting via a dysregulation of the Dll4-Notch axis. In line, EC-KO mice demonstrate increased angiogenic marker expression as well as higher numbers of endothelial cells and pericytes, specifically in the white matter (WM). EC-KO mice also display BBB leakage, glial reactivity at the vasculature and leukocyte infiltration in WM areas. Together, we suggest that senescent brain ECs accumulate during aging, thereby promoting BBB impairment and excessive sprouting, which in turn might contribute to the pathogenesis of neurodegenerative diseases. Materials and Methods Animals All animal procedures were performed at the Erasmus Laboratory Animal Science Center following the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and approved by the National Animal Care Committee and the administration within Erasmus University Medical Center Rotterdam (protocol number 118-13-03). Endothelial-specific Ercc1 KO animals were bred as described previously ( 28 ). In brief, the Cre-loxP system was used to generate a conditional mouse model to knock out endonuclease Ercc1 in ECs (B6.Cg-Tg(Tek-cre)12Flv/J, The Jackson Laboratory, Bar Harbor, USA). In the resulting litters, Tie2 cre+/-: Ercc1 fl/- mice have an Ercc1 KO in ECs, where Cre-recombinase is active (from here on referred to as EC-KO mice). Tie2 cre+/-: Ercc1 fl/+ animals were used as wild type (WT) controls. Mice (male and female) were kept in individually ventilated cages, in a 12-h light/dark cycle with food and water ad libitum . Animals were euthanized around the age of 22 weeks by cardiac perfusion with ice-cold phosphate-buffered saline (PBS), after which brains were collected. Each brain was divided sagittally and one hemisphere snap-frozen, the other post-fixed in 1.6% paraformaldehyde (PFA) for 24 hours followed by incubation in 30% sucrose for 24 hours. Sagittal brain slices (10 µm) were cut (CryoStar NX70, Thermo Fisher Scientific, Waltham, USA) and stored at -80 ⁰C upon use. For details on the animals see Table 1. Immunohistochemistry Cryosections were defrosted, permeabilized and blocked with 5% normal goat serum and 0.1% Triton-X100 in PBS (Sigma-Aldrich, Saint Louis, MO, USA), PBS only or Tris-buffered saline (TBS). Primary antibodies were incubated overnight at 4°C. Tissue slides were incubated for 1 hour at room temperature with secondary antibodies coupled to Alexa Fluor 488, 555 or 647 fluorophores (Molecular Probes, Eugene, OR, USA). The tissue slides were then counterstained with Hoechst (Molecular Probes, Eugene, OR, USA), embedded in Mowiol (in-house) mounting medium, and stored in the dark at 4°C until microscopic evaluation. Antibody details are listed in Table 2. Immunocytochemistry (ICC) was performed similarly to IHC on human cerebral microvascular endothelial cells (hCMEC/D3) transduced with lentiviral constructs (see Lentiviral short hairpin RNA knock down for ERCC1 ). Shortly, cells were seeded in 8-well µ-slides (#80826, Ibidi, München, Germany) and fixed with 1.6% PFA, ice-cold methanol or acetone for 10 min at room temperature and permeabilized for 5 minutes using 0.05% Triton-X100 in PBS. Blocking, primary and secondary antibody incubation occurred as described above. Nuclei were visualized using Hoechst and wells were filled with Mowiol before imaging. Antibody details are listed in Table 2. Microscopy and image acquisition Images were acquired with wide field imaging using the Olympus VS200 (Olympus, Tokyo, Japan) slide scanner or confocal imaging using the Leica SP8 confocal microscope (Leica, Wetzlar, Germany) with a 60x or a 63x oil immersion objective, respectively. Regions of interest (ROI) were acquired as z-stacks of 4 or 8 µm and step size of 266 nm or 130 nm. For wide field images, deconvolution was performed using Huygens Professional 21.10 software (Scientific Volume Imaging B.V., Hilversum, The Netherlands). NIS elements (version 5.30.03, Nikon Europe B.V., Amsterdam, The Netherlands), FIJI and QuPath-0.2.3/-0.4.4 were used for automated and manual analysis. Three ROIs per brain region per animal were imaged in the white matter (WM; corpus callosum, dorsal fornix and anterior commissure), the hippocampus (HC, supra and infra-pyramidal molecular layer) and the cortex (CRTX). For the vascular analysis, arterioles were defined as αSMA + vessels with smooth muscle cells (SMCs) (αSMA + , often PDGFRβ + cells) and capillaries as αSMA − , PDGFRβ + vessels with pericytes (PCs) (PDGFRβ + cells) ( 30 ). Immune cell counts were performed blinded and manually by two researchers using QuPath-0.2.3/-0.4.4. Migrated immune cells were defined by their proximity to the vasculature as perivascular (still in contact with the abluminal side of the vessel marker) or parenchymal (minimal 10 µm distanced from the vessel). Human brain endothelial cell culture The immortalized hCMEC/D3 cell line was a kind gift provided by Prof.dr. IA Romero (Open University, Milton Keynes, UK) and Prof.dr. PO Coureaud (Université Paris Descartes, France) ( 31 ). Cells were cultured from passages 29 to 39 in endothelial basal medium-2 (EBM-2) supplemented with 2.5% (v/v), heat-inactivated fetal bovine serum, growth supplement kit (#CC-3156, #CC-4147; Lonza, Basel, Switzerland), and 1% (v/v) penicillin-streptomycin (#15140-122; Gibco, Thermo Fisher Scientific, Waltham, USA). hCMEC/D3 cells were grown on bovine skin collagen I-coated culture flasks (#C5533; Sigma-Aldrich) until confluent unless stated otherwise. For culture, cells were maintained at 37°C and 5% CO 2 and routinely screened for the presence of mycoplasma. Lentiviral short hairpin RNA knock down of ERCC1 Short hairpin RNAs were used to knock down ERCC1 (shERCC1) expression in hCMEC/D3 as previously described ( 32 – 34 ). Sub-confluent HEK 293T cells were co-transfected with the specific expression plasmids and packaging plasmids (pMDLg/pRRE, pRSV-Rev and pMD2G) using calcium phosphate as transfection reagent. Infectious lentiviral particle-containing supernatant was collected after 48 hours, concentrated using Amicon Ultra15 filters (UFC910024; Merck, Darmstadt, Germany) and stored at − 80°C upon further use. hCMEC/D3 cells were transduced at passage 30 by adding the concentrated supernatant 4–6 hours after seeding and stable cell lines were selected 24 hours later using puromycin treatment (2 ng/ml, P7255; Sigma Aldrich). The knock down efficiency was assessed using quantitative real-time PCR (qRT-PCR) and Western blot. Constructs (TRCN0000049920) with 84% knock down efficiency were used for subsequent experiments. shERCC1 encodes for 5’- CAAGAGAAGATCTGGCCTTAT-3’. hCMEC/D3 cells transduced with lentivirus expressing non-targeting shRNA (NTC; SHC002, Sigma-Aldrich) were used as control cells. Transduced shERCC1 and NTC cells were used from passage (P) 1–7. The assessed gene expression in shERCC1 cells compared to NTC showed the same effect between P1 and P7, with varying effect sizes over the different passages. Induced pluripotent stem cell-derived brain pericytes Human induced pluripotent stem cells (hiPSC) were differentiated to neural crest (NC)-derived brain pericytes (hiBPC) using previously published protocols ( 33 , 35 , 36 ). Briefly, episomal hiPSC line (#A13700, Gibco, Thermo Fisher Scientific, Leusden, The Netherlands) was cultured in mTeSR Plus medium (STEMCELL Technologies, Vancouver, Canada) and grown on vitronectin-coated plates (Invitrogen, Thermo Fisher Scientific). HiPSCs were passaged as single cells, seeded onto Matrigel-coated plates (2 x 105 cells/cm 2 ) and cultured for 5 days in NC induction medium, consisting of DMEM/F12 GlutaMAX™ (Gibco, Thermo Fisher Scientific), 1× B27 (Gibco, Thermo Fisher Scientific), 0.5% bovine serum albumin and 3 µM CHIR 99021 (Tocris, Bristol, United Kingdom). The resulting NC cells were seeded onto 0.1% gelatin-coated plates (2.5 x 104 cells/cm 2 ) and cultured for additional 5 days in pericyte medium (ScienCell, Carlsbad, CA, USA). Induced brain pericytes (iBPCs) were characterized by immunocytochemistry (ICC) and RT-qPCR ( 33 ). iBPC were used between passages 2–4 in spheroid-based sprouting experiments . Electric Cell-substrate Impedance Sensing (ECIS) The transendothelial electrical resistance (TEER) of shERCC1 and NTC cells was assessed using the ECIS™ Model 1600R (Applied BioPhysics, Troy, NY) as previously reported ( 37 , 38 ). In short, cells were seeded at a density of 100.000 cells into 8W10 + ECIS arrays (#72040, Ibidi). Impedance was measured at multiple frequencies over a time course of 120 hours. To quantify the maximum resistance [ohm], the data at 4000 Hz was normalized to the resistance at time before medium replacement. Scratch-wound and spheroid-based sprouting assay For the scratch-wound assay, NTC and shERCC1 cells were grown to confluence and the scratch was induced diagonally with a plastic pipette tip. Cell migration was imaged for 22 hrs at 10x magnification, bright field, at 37°C with the Nikon Ti2 live cell imaging system (Nikon, Tokyo, Japan). Spheroid-based sprouting assays were performed as previously reported ( 33 ). In brief, NTC and shERCC1 cells and iBPCs were re-suspended in a ratio of 20:1 in EGM-2 medium supplemented with 0.25% methylcellulose (4.000 cP, Sigma-Aldrich, Saint Louis, MO, USA). Cell suspension was seeded in a 24-well plate and flipped upside down. After 24h, the spheroids were collected and re-suspended in 1,5 mg/ml collagen type-I rat tail mixture (Enzo science, Farmingdale, NY, USA) and re-plated in a 24-well plate upside down until complete polymerization. 30 minutes after polymerization, EGM-2 medium was administered and wells were incubated at 37°C and 20% O 2 , 5% CO 2 for 5 days. Images were taken using the Nikon LIPSI Ti2 confocal spinning disk imaging system (Nikon, Tokyo, Japan), 10x objective, and adjusted for brightness/contrast in ImageJ. Sprouting number and length were analysed using the ImageJ plugin NeuronJ ( 39 ) . RNA isolation and real-time quantitative polymerase chain reaction (qRT-PCR) Total RNA was extracted from mouse whole brain homogenates (WBH) using the RNeasy Lipid Tissue Mini Kit (#174804, Qiagen) and from hCMEC/D3 using TRIzol (#15596-018, Thermo Fisher Scientific). RNA quantity was assessed by Nanophotometer (Implen, Westlake Village, USA). The High-Capacity cDNA Reverse Transcription Kit (#4368813, Thermo Fisher Scientific) was used to synthesize cDNA and transcripts of interest were detected with SYBR Green (#4309155, Thermo Fisher Scientific) using the QuantStudio™ 3 Real-Time PCR System (#A28567, Thermo Fisher Scientific). Expression was normalized to housekeeping genes β-actin (WBH) and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ; hCMEC/D3) using the 2 − ΔΔ CT relative quantification method. Primer sequences are summarized in Supplemental Table 1. Nuclear fractionation and Western Blot hCMEC/D3 cells were washed with cold PBS and lysed on ice with cell lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing protease and phosphatase inhibitors (Roche, Almere, The Netherlands, and Cell Signaling Technology, Boston, MA, USA, respectively). Nuclear fractions were isolated using the NE-PER extraction kit (Thermo Fisher Scientific), following the manufacturer’s instructions. All samples were diluted in Laemmli buffer (2x) (BioRad Hercules, CA, USA) (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue) and heated to 95°C for 3–5 min. Lysates were separated on SDS-PAGE followed by transfer to nitrocellulose for immune blot analysis. Blots were blocked with blocking buffer (Licor, Lincoln, USA) for 1 hour at room temperature. Subsequently, membranes were incubated in blocking buffer containing 0.1% Tween-20 with primary antibodies (Table 2) overnight at 4°C and detected and quantified by incubation with IRDye secondary antibodies (1 hour, room temperature) (LI-COR) and imaged by Azure Sapphire Biomolecular Imager (Azure Biosystems, Inc, Sierra CT, Dublin, CA, USA). Original Western blots are depicted in SI.1b. Statistical analysis All analyses were performed blinded and data are plotted as box plots with median ± quartiles and whiskers extend to minimum and maximum values. Statistical tests were performed using GraphPad Prism v9 (GraphPad Software, La Jolla, USA). We used Shapiro-Wilk test for data normality. For comparing two experimental groups, two-tailed Student’s t-test was used and non-parametric data was analysed by Mann-Whitney test. Statistical significance was set at p < 0.05 and nominal p-values are reported throughout the manuscript. For multiple Student’s t-test Benjamini–Hochberg correction was performed (q = 10%) and the q-values can be found in Supplementary Tables 2 and 3. Of note, all significantly different genes survived FDR correction. Test details are indicated in the corresponding Figure legend. For the creation of the gene expression heat map, we used the web-based tool MetaboAnalyst ( http://www.metaboanalyst.ca , accessed: 10/07/2023). Results ERCC1 deficiency induces BBB impairment in brain ECs To investigate the effect of aging on brain EC function in vitro , we generated an accelerated aging model by reducing the expression of Ercc1 (shERCC1) in a human brain EC cell line (hCMEC/D3). shERCC1 cells expressed less ERCC1 mRNA (83%, p = 0.010, Fig. 1 a) and showed less ERCC1 protein (Fig. 1 b, SI.1b) compared to the non-targeting control cells (NTC). With increasing passage (P) number, shERCC1 cells adopted an enlarged cell size (encircled, Fig. 1 c), which is characteristic of senescent cells ( 40 ). As a measure of DNA damage, we evaluated the phosphorylation of histone H2AX (yH2AX) ( 41 ). shERCC1 cells showed yH2AX foci (yellow arrowhead) and some pan-nuclei H2AX phosphorylation (green arrowhead) (Fig. 1 d). Among the tested senescence and SASP markers, we found a significant increase in IL-6 (p = 0.004), IL-1B (p = 0.016) and intercellular adhesion molecule 1 ( ICAM1 ) (p = 0.047) mRNA expression in shERCC1 cells compared to NTC, while no significant differences were observed in TNFA , CDKN1A and CDKN2A expression (Fig. 1 e). Next, we investigated the expression of BBB transporters and junction components upon silencing Ercc1. shERCC1 cells showed an increased expression of the transporters PGP (p = 0.003) and MFSD2A (p = 0.016) compared to NTC cells (Fig. 1 f). Junctional markers like claudin5 (Clnd5) and VE-cadherin (VE-Cad, CDH5 ) were decreased in shERCC1 cells, both in their RNA expression ( CLDN5; p = 0.0156, CDH5; p = 0.0010) and protein level (Fig. 1 g,h). Zona occludens-1 (ZO-1, TJP1 ) did not differ between conditions (Fig. 1 g, SI. 1a). In line with decreased CLDN5 and VE-Cad levels, shERCC1 cells displayed a significantly reduced barrier resistance compared to NTC cells (p = 0.017) (Fig. 1 i). Together, these results indicate that ERCC1 knock down induces DNA damage accumulation and BBB dysfunction in brain ECs. ERCC1 deficiency enhances migration and sprouting of brain ECs in vitro The reduction in BBB markers such as Cldn5 can underlie a (transient) loss of EC identity, which has been associated, among others, with angiogenesis and vascular remodeling ( 42 ). Thus, we evaluated the delta like canonical notch ligand 4 (Dll4)-Notch1 axis, which is fundamental in the regulation of EC sprouting angiogenesis ( 43 ). NOTCH1 (p = 0.0009) and DLL4 (p = 0.0313) mRNA expression were decreased in shERCC1 cells compared to NTCs (Fig. 2 a), and DLL4 density was reduced in shERCC1 cells (Fig. 2 b). Next, we assessed the mRNA expression of VEGFA and kinase insert domain receptor ( KDR , gene encoding vascular endothelial growth factor receptor 2), which are pivotal in the regulation of the Dll4-Notch1 pathway. We observed a significant increase of VEGFA (p = 0.016) and a decreasing trend for KDR (p = 0.059) in shERCC1 cells compared to NTCs (Fig. 2 a). Lastly, we assessed the mRNA expression of SNAI2 , a transcription factor which has been shown to directly regulate DLL4 expression in ECs ( 44 ). We report a significant increase in SNAI2 mRNA expression (p = 0.016) in shERCC1 cells compared to NTC cells (Fig. 2 a). To functionally assess the dysregulated Notch pathway in shERCC1 cells, we performed a scratch-wound assay and a sprouting assay. shERCC1 cells closed the scratch significantly faster than the NTC cells (Fig. 2 c). Further, shERCC1 cells showed a significant increase in the number of sprouts (p = 0.007) and cumulative sprout length (p = 0.007) compared to NTC cells (Fig. 2 d,e). The minimum length of the sprouts was significantly decreased in shERCC1 cells compared to NTC cells (p = 0.003) (Fig. 2 e). No change was observed in the mean or maximum sprout length (SI.2a). Together these data indicate a dysregulated Dll4-Notch1 axis in shERCC1 cells, which may explain their impaired angiogenic capacity. EC-specific Ercc1 KO mice show senescence and increased BBB transporters in white matter tissue To study the impact of EC-specific accelerated cellular aging on brain homeostasis in vivo , we utilized EC - KO mice ( 28 ). First, we examined the senescence profile of EC-KO mice compared to WT mice by using multiplex qPCR on whole brain homogenates (WBH) (Fig. 3 a). Ercc1 mRNA expression was reduced in EC-KO compared to WT brains (p = 0.0001). The mRNA expression of the senescence markers Cdkn1a (encoding P21) (p = 0.002), Tnfa (p = 0.002) and Icam1 (p = 0.004) were increased in EC-KO brains, and Il-6 (p = 0.06) showed a similar trend, while C dkn2a (encoding P16) and Il-1b did not differ between genotypes (Fig. 3 a). Immunohistochemical analysis also showed enhanced levels of P21 in EC-KO mice (p = 0.021) compared to WT mice which co-localized with Lectin, an endothelial cell marker (p = 0.021; Fig. 3 b,c). To evaluate the properties of the BBB in the EC-KO mice, we next investigated the mRNA expression of BBB-associated markers in WBH. We found a significant increase in Cldn5 and Cdh5 mRNA expression in EC-KO mice compared to WT (p = 0.009 and p = 0.0009, respectively), while Lama1, Tjp1, Mdr1a (encoding P-GP), and Mfsd2a were unchanged (Fig. 3 a). We then examined P-GP and MFSD2A levels in the different brain regions of EC-KO and WT using immunohistochemistry (Fig. 3 d). We found an increase of P-GP (p = 0.046) and MFSD2A (p = 0.003) immunoreactivity (fluorescent mean intensity (MI)) in the white matter (WM) and a trend towards increased P-GP expression in the hippocampus (HC) (p = 0.102) of EC-KO mice compared to WT (Fig. 3 e). Concomitantly, we found higher transporter coverage (reactivity area/Lectin area) of the vasculature (P-GP (p = 0.039); MFSD2A (p = 0.013)) in the WM of EC-KO mice. No differences were found in the cortex (CRTX) or HC. Focusing from now on the WM, we analyzed CLDN5 levels via immunohistochemistry. No differences were observed when comparing the number of CLDN5 + vessels and the MI of CLDN5 within the vasculature between EC-KO and WT (Fig. 3 f,g). In summary, endothelial specific Ercc1 -mediated aging increases P21 + cells and BBB transporter levels specifically in the WM. EC-KO mice increase angiogenic marker expression and show BBB leakage Following our findings in shERCC1 cells (Fig. 2 ), we studied angiogenesis-related markers in the WBH of EC-KO and WT mice. We observed an increase of Kdr (p = 0.026), platelet-derived growth factor receptor beta ( Pdgfrb) (p = 0.030), angiopoietin2 ( Angpt2 ) (p = 0.001) and a positive trend for Cd31 (p = 0.075) in EC-KO mice compared to WT (Fig. 4 a). Vegfa, Dll4, Notch1 and Snai2 mRNA did not change between the experimental groups, but we observed an increase in SNAI2 on protein level in the WM vasculature of EC-KO mice compared to WT (Fig. 4 b). Since we found an increase in angiogenic markers in EC-KO mice, we next investigated potential changes in the vascular architecture in EC-KO and WT mouse brains. We used a triple immunostaining with PDGFRβ, alpha-smooth muscle actin (αSMA) and LAMININ, to discriminate capillaries from arterioles (Fig. 4 c, SI. 4a) ( 45 ). The capillary ECs density was increased in the WM of EC-KO compared to WT mice (p = 0.043), but not in CRTX or HC (Fig. 4 d). In contrast, the absolute cell count and percentage of total ECs or arterial ECs did not differ between genotypes in all regions (Fig. 4 d, SI. 4b). Furthermore, the capillary density in the WM of EC-KO mice showed an increasing trend (p = 0.088), while overall vascular density and arterial density did not change between EC-KO in WT in all three brain regions (Fig. 4 d, SI. 4c). Finally, the number of PCs was significantly higher in the WM (p = 0.027) of EC-KO mice as well as in the HC (p = 0.028), while the PC coverage of the endothelium was unaffected (Fig. 4 e). No differences were detected in the number and coverage of smooth muscle cells (SMCs), nor in the mean expression levels of PDGFRβ and αSMA between EC-KO and WT (SI. 4d,e). Lastly, we assessed if the vascular changes resulted in BBB leakage. We found an increased IgG reactivity in the WM of EC-KO animals compared to WT (p = 0.040) (Fig. 4 f), while IgG reactivity did not differ between genotypes in the grey matter (average CRTX and HC) (SI. 4f). Taken together, these data indicate that EC-KO mice display microvascular changes in the WM which results in local BBB leakage. EC-KO mice display an inflamed BBB and immune cell infiltration in the white matter Based on the observed IgG leakage, specifically in the WM of EC-KO mice, we next focused on the possible presence of local inflammation. In the whole brain lysates, we found a significant increase of P2ry12 mRNA (p = 0.027), a homeostatic marker for microglia, and a similar trend for Gfap (p = 0.06), a marker for reactive astrocytes (Fig. 5 a). In WM tissue, we observed more IBA1 + cells in EC-KO mice compared to WT (Fig. 5 b). Furthermore, we found a decreased P2RY12 levels in the WM of EC-KO mice (p = 0.012) compared to WT (Fig. 5 c,d), which may indicate microglia activation. Of note, the GFAP-vessel co-localization was higher in the WM of EC-KO (P = 0.006) compared to WT. (Fig. 5 e, SI. 4a). Next, we analyzed BBB inflammation by the vascular expression of ICAM1. EC-KO mice displayed more ICAM1 + vessels (p = 0.001) and a trend towards higher vascular ICAM1 levels (p = 0.052) in the WM compared to WT (Fig. 5 f,g). Using CD45 to identify leukocytes and CD8 for cytotoxic CD8 + T cells specifically (Fig. 5 h), we found an increased density of immune cells in the WM of EC-KO mice (p = 0.018) compared to WT. Almost half of the cells were in the parenchymal tissue similarly in WT and EC-KO brains (Fig. 5 i). 27% of parenchymal cells were CD8 + T cells in the EC-KO mice compared to 9% of the perivascular cells (Fig. 5 i). In the WT brains, all parenchymal Cd45 + cells were positive for Cd8 (total count: 2 cells), while all perivascular cells were Cd8 − (SI. 4b). In summary, our findings show that endothelial aging coincides with BBB inflammation and increased peripheral immune cells migration into the brain, highlighting a key role of endothelial cells in CNS aging and subsequent inflammation, specifically in the WM. Discussion Preclinical and clinical studies indicate that aging is a critical factor inducing endothelial dysfunction ( 3 , 46 , 47 ). In line, brain endothelial dysfunction is frequently found during healthy brain aging as well as in neurological disorders such as stroke and Alzheimer’s disease ( 48 – 51 ). However, the role of brain endothelial aging and senescence in BBB impairment remains largely unknown. In this study, we evaluated the consequences of ERCC1 deficiency, a model for accelerated aging, in brain ECs in vitro and in vivo . We show that ERCC1-deficient brain ECs display reduced BBB integrity, increased transporter expression and more endothelial sprouting. We validated our in vitro findings in EC-KO mice, which display a higher expression of angiogenic genes and more capillary ECs and pericytes, specifically in the WM. Furthermore, the WM of the EC-KO animals demonstrates IgG leakage and increased glial cell reactivity near the vasculature, which coincided with immune cell infiltration in the brain parenchyma. Together, our work highlights the effect of endothelial cell aging on BBB dysfunction, angiogenesis and local inflammation. In this study, shERCC1 display classical hallmarks of DNA damage and cellular aging including H2AX phosphorylation and increased SASP component expression (i.e. IL-1B , IL-6 , VEGFA ). Conversely, expression of CDKN1A (P21) and CDKN2A (P16), known senescence markers ( 52 ), was unaffected in shERCC1 cells. SASP and P21/P16 elevation are not always concomitant ( 53 , 54 ), and the presence of the latter is not a pre-requisite for cell aging as exemplified by studies in post-mitotic cardiomyocytes ( 55 , 56 ). Furthermore, in our set-up, the high proliferative capacity of hCMEC/D3 cells combined with ERCC1 deficiency, may highlight cellular aging features directly associated to DNA damage-related cell stress, while overshadowing the P21/P16 expression present in the few senescent cells with halted cell cycle. Lastly, VEGF, highly expressed in the shERCC1 cells, is known to negatively regulate P21/P16 expression ( 57 ), which may explain the similarity with NTC cells. The cellular stress of shERCC1 cells is also accompanied by a reduced expression of Cldn5 and VE-cad, resulting in an impaired barrier integrity, as seen during aging in vivo and in vitro ( 58 , 59 ). Together, these results substantiate the role of DNA damage in inducing cell aging, and highlight the effect of brain EC aging on BBB function. In vivo , EC-KO mice displayed increased vascular P21 expression compared to WT animals. However, not all brain ECs were P21 + , which aligns with previous reports on this model suggesting partial efficiency of the Cre-lox system in deleting Ercc1 ( 28 , 60 , 61 ). P21 expression is increased in the brain endothelium of elderly compared to young individuals ( 60 , 62 ) and MRI studies positively associate age and enhanced BBB permeability in healthy elderly suggesting the possible effect of aging on BBB dysfunction ( 63 – 65 ). In our study, EC-KO mice displayed increased IgG leakage, possibly underlying reduced BBB resistance resulting from EC aging, which is in line with our in vitro findings on BBB dysfunction. Furthermore, we observed an increase in Cldn5 and Cdh5 expression in the whole brain homogenate of EC-KO animals compared to WT, but no changes in Cldn5 expression in the WM vessels. Interestingly, we show an increased number of brain EC in WM capillaries, which could partially explain the increase in junction mRNA expression. In sum, our findings present an in vivo model for accelerated vascular aging, which recapitulates some of the features, including reduced BBB integrity, observed during healthy cerebrovascular aging in humans. In EC-KO mice, the increased number of ECs in WM brain capillaries was concomitant with an enhanced expression of angiogenic markers ( Angpt2 , Kdr ), suggesting increased EC sprouting. Furthermore, we observed a general increase in vascular SNAI2 expression in the WM on EC-KO animals. High Snai2 expression has been previously shown to directly impair the Dll4-Notch1-axis, resulting in angiogenesis characterized by dysfunctional vessels ( 44 ). Similarly, shERCC1 cells displayed increased SNAI2 and reduced DLL4 and NOTCH1 expression together with increased sprout number. These findings may indicate a reactivation of the angiogenic program in the WM of EC-KO mice sustained by Snai2. In line with our findings, previous studies found increased angiogenic markers (i.e. Angpt2) in brain ECs isolated from the corpus callosum of aged mice compared to younger animals ( 66 ). Interestingly, in our study we found the major changes in the WM of EC-KO mice, while the CRTX and HC seemed to be less affected, suggesting a regional susceptibility to EC aging. In humans, the WM has been previously shown to be more susceptible to age-related pathologies including vascular dementia ( 67 , 68 ). A possible explanation may lie in the inherent lower capillary density of the WM, which makes this area more sensitive to hypoxic insults, a known trigger for angiogenesis via different pathways including Snai2 upregulation ( 66 , 67 , 69 – 73 ). Eventually, studies in aged mice and elderly show vessel rarefaction and reduced vessel length, which may be the result of dysfunctional angiogenesis ( 11 , 74 – 78 ). Together our data suggest that DNA-damage in brain ECs may sustain dysfunctional angiogenesis via dysregulated Dll4-Notch1 signalling, and that the WM is more susceptible for this process. However, more research is warranted to fully comprehend the mechanisms underlying brain vasculature maintenance and remodelling during aging. With age, a decrease in BBB transporter expression is observed ( 8 , 79 ). However, both our endothelial aging models show increased transporter (P-gp and Mfsd2a) expression, specifically in the WM of EC-KO mice. P-gp expression can be primarily regulated by inflammation and oxidative stress, as evidenced by increased P-gp levels in stroke and seizure studies ( 80 – 82 ). Further, other senescent mouse models showed higher P-gp brain vasculature expression, postulating a protective role for senescent cells in toxin efflux from the aging brain ( 83 , 84 ). Similarly, the increase in Mfsd2a might also be protective. Mfsd2a limits vesicle-mediated transcytosis, which is crucial to maintain BBB integrity, as shown by barrier leakage in Mfsd2a KO mice ( 85 , 86 ). Under homeostatic conditions, increased Mfsd2a expression induces characteristics of cellular aging, while Mfsd2a overexpression alleviates tissue damage after acute brain injury ( 23 , 87 , 88 ). These evidences highlight the multifaceted role of Mfsd2a in the regulation of endothelial cell fitness. It is plausible that the increase of P-gp and Mfsd2a levels in the ERCC1 models is an early protective response to the impaired BBB integrity to aid CNS homeostasis. However, further studies are needed to validate this hypothesis. The observed changes of the BBB in the EC-KO mice is accompanied by IgG leakage and loss of homeostatic marker expression in microglia in the WM. The leakage of blood-derived components such as fibrinogen has been reported to activate microglia in elderly and AD subjects ( 89 ), and to contribute to neuroinflammation and cognitive decline. Indeed, we also observed an increase in ICAM1 + vessels and leukocyte infiltration, including CD8 + T cells, into the WM of EC-KO mice. ICAM1 is a crucial mediator of the immune cell migration cascade and its p53-induced overexpression has been previously found in senescent endothelial cells of atherosclerotic lesions ( 90 ). Moreover, the presence and function of infiltrating peripheral immune cells in the aging brain, AD subjects, and age-related mouse models is increasingly described ( 91 – 94 ). In the aging brain, CD8 + T cells are suspected to interact with CNS cells including microglia and neurons ( 95 ), but their role is still heavily debated ( 96 ). Together these results position endothelial cell aging as one potential starting point of BBB inflammation and immune cell infiltration in the aging brain. Collectively, our data highlight the significance of brain EC aging in BBB dysfunction, a key contributor in the development and progression of several diseases of the CNS. Hence, targeting brain vascular aging may be a promising strategy to alleviate age-related vascular disorders and their neurological implications. Declarations Acknowledgements We acknowledge gratefully René de Vries for his technical assistance in isolating mouse material. We thank Prof. dr. IA Romero and Prof. dr. PO Coureaud for providing the hCMEC/D3 cell line and Peter. J. M. Stroeken for providing the constructs for the Ercc1 knock down. Further, we would like to thank Henrique Nogueira Pinto for differentiating and providing the human BPCs. Lastly, we wish to thank the Microscopy and Cytometry Core Facility for their expertise and support with the microscopy. Conflict of interest The authors declare no competing financial interests. Funding This work was funded by the European Union´s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant (ENTRAIN) (agreement No. 813294) to HdV, Horizon ERC Advanced (ERC-AD 2022: 101097983) to HdV, by a grant from the Dutch Research Council (NWO Vidi grant 91719305 to G.K.), and grants from the Dutch MS Research Foundation (18-1023MS to G.K. and 20-1106MS to M.E.W.). This work was further supported by a grant to NMdW from ZonMw Onderzoeksprogramma Dementie (project nr: 10510022110005). AAJ and AJMR were funded by TKI-LSH grant # EMCLSH19013. IAM is funded by The Dutch Heart Foundation 2021 E. Dekker Grant (03-006-2021-T019). Author contributions CEH performed experiments, analyzed data, designed and conceived the study and wrote the manuscript. DV performed experiments, analyzed data, designed and conceived the study and revised the manuscript. LvdM performed experiments, analyzed data and revised the manuscript. AAJ, HNP, WKF, and BvhH performed experiments. RF and MEW provided material and valuable scientific input, plus revised the manuscript. GK provided material and valuable scientific input, revised the manuscript and obtained funding. IM provided valuable scientific input, contributed to design the study and revised the manuscript. AJMR, NMdW and HEdV supervised the study, contributed to design the study, obtained funding and revised the manuscript. All authors read and approved the publication of this manuscript. Data availability Source data is available upon reasonable request. References Kadry H, Noorani B, Cucullo L. 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CD8+ T cells in neurodegeneration: friend or foe? Molecular Neurodegeneration. 2022;17(1):59. Tables Tables 1 to 2 are available in the Supplementary Files section Additional Declarations There is no duality of interest Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1: Primer details SupplementaryTable2.xlsx Supplementary Table 2: FDR-corrected values of multiplex qPCR on shERCC1 and NTC cells SupplementaryTable3.xlsx Supplementary Table 3: FDR-corrected values of multiplex qPCR on WBH of WT and EC-KO mice SupplementaryFigure1.tif Supplementary Figure 1. ZO-1 expression in shERCC1 and NTC cells and original WB, related to Figure 1 a Representative images of ZO-1 in shERCC1 and NTC cells (scale bar: 25 µm). b Original Western blot images of ERCC1 (left) and GAPDH (right) from brain ECs transduced with the ERCC1 knock down construct (shERCC1) or control (NTC). SupplementaryFigure2.tif Supplementary Figure 2. Mean and maximum length of sprouts, related to Figure 2 a Quantification of mean sprout length, and maximum sprout length in shERCC1 and NTC cells (n=16-20). Each dot represents a biological replicate presented as box plot with median ± quartiles; whiskers extend to minimum and maximum. Statistical comparison of two groups was performed using two-tailed Student’s t-test for normally distributed data, or the Mann-Whitney test for non-normally distributed data. SupplementaryFigure3.tif Supplementary Figure 3. Vascular densities and mural cells in EC-KO and WT brains, related to Figure 4 a Representative images of LAMININ, PDGFRβ and αSMA immunoreactivity of CRTX and HC in WT and EC-KO mice; white arrowheads indicate αSMA + vessels (arteriole, upper panel), yellow arrowheads indicate PDGFRβ + , αSMA - vessels (capillary, lower panel) (scale bar: 50 µm). b Quantification of total cell count (nuclei count), total EC and arterial EC count (percentage of total) in WT and EC-KO CRTX, WM and HC brain tissue, (n=6-7). c Quantification of vascular densities including overall vessel density and arterial density in WT and EC-KO brain tissue, (n=6-7). d Quantification of smooth muscle cell (SMC) count and coverage of arteriole area, (n=6-7). e Quantification of MI of PDGFRβ and αSMA in LAMININ + area of WT and EC-KO mice, (n=6-7). f Area of IgG reactivity measured in grey matter (GM, average CRTX and HC) tissue of WT and EC-KO mice. Data is presented as box plot with median ± quartiles; whiskers extend to minimum and maximum. All data have been statistically tested by unpaired student-t test with Welch’s correction when the variance of the groups was significantly different. SupplementaryFigure4.tif Supplementary Figure 4. Astrocyte reactivity in EC-KO and WT brains, related to Figure 5 a Representative images of GFAP in whole brain slices of EC-KO and WT mice (scale bar: 1000µm). bPresentation of immune cell subsets (CD45, CD8) in WT mouse brain comparing perivascular and parenchymal location. Table1.xlsx Table 1: Details on WT and EC-KO mice Table2.xlsx Table 2: Primary antibody details Cite Share Download PDF Status: Published Journal Publication published 03 Jan, 2025 Read the published version in Cell Death & Disease → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4358616","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":302274256,"identity":"ea95e8b9-2e03-4484-a165-eeca928ce09e","order_by":0,"name":"Cathrin Hansen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYJACZih1AMpPgNLsBLWwgZQaIGlhJqiFx4A4LfwNzAeYC2ru2PNL93z8dOPPHznz9uSnm3lz7BjMcWiROMCWwDzj2LPEmXPObpbObTMwljnzzOw277ZkBstm7FoMgO5h5mE7nGBwI3eDdG6DQeIMiQSQlgMMBodxaeH/wMzz77C9/Y2cx79z/hjUz5BI/0ZACw8DM2/bYcYNEjls0jlsBgkSEjn4bZE4zGZwmLfvcOKMG2lm1rltxoYzeN6U3Zy7LZkHlxb+9uaHj3m+Hbbnn5H8+HbOHzl5Cfb0bTfebrOTMzjegF0PMCQPYJXgwa5+FIyCUTAKRgExAACdFljfEJzIzwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-7504-9413","institution":"Amsterdam UMC","correspondingAuthor":true,"prefix":"","firstName":"Cathrin","middleName":"","lastName":"Hansen","suffix":""},{"id":302274257,"identity":"e78941b0-e397-4c01-8085-5c46b5040679","order_by":1,"name":"Davide Vacondio","email":"","orcid":"","institution":"Amsterdam UMC","correspondingAuthor":false,"prefix":"","firstName":"Davide","middleName":"","lastName":"Vacondio","suffix":""},{"id":302274258,"identity":"64fb10e4-ac96-4bd9-a1ec-11263cb99f38","order_by":2,"name":"Lennart van der Molen","email":"","orcid":"","institution":"Radboud university medical center","correspondingAuthor":false,"prefix":"","firstName":"Lennart","middleName":"van der","lastName":"Molen","suffix":""},{"id":302274259,"identity":"cb4fa4f5-edf9-4732-ae4a-0ab1643d6c0d","order_by":3,"name":"Annika A. 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Enlarged cells are encircled in black; scale bar: 200 µm. \u003cstrong\u003ed\u003c/strong\u003e Representative images of phosphorylated yH2AX in shERCC1 and NTC cells (yH2AX, white); scale bar: 25 µm. Yellow arrowheads indicate yH2AX foci and green arrowhead yH2AX pan-expression. \u003cstrong\u003ee\u003c/strong\u003e\u003cem\u003e \u003c/em\u003emRNA expression of senescent and SASP targets (\u003cem\u003eP21, P16, IL-6, TNFA, IL-1B, ICAM1\u003c/em\u003e) in NTC and shERCC1 cells, n=4. \u003cstrong\u003ef \u003c/strong\u003emRNA expression of BBB transporters \u003cem\u003eP-GP\u003c/em\u003e and \u003cem\u003eMFSD2A\u003c/em\u003e in shERCC1 and NTC cells (n=7). \u003cstrong\u003eg \u003c/strong\u003emRNA expression of BBB junction marker \u003cem\u003eCLND5\u003c/em\u003e, \u003cem\u003eCDH5 \u003c/em\u003eand \u003cem\u003eZO-1 \u003c/em\u003ein shERCC1 and NTC cells (n=7). Data have been normalized to \u003cem\u003eGAPDH\u003c/em\u003eand presented as fold change to NTC values. Each data point represents the mean of a single experiment performed in triplicates. \u003cstrong\u003eh \u003c/strong\u003eRepresentative images of CLDN5 (white arrowheads indicate junctional immunoreactivity) and VE-cad in shERCC1 and NTC cells (scale bar: 25 µm). \u003cstrong\u003ei\u003c/strong\u003e Transendothelial electrical resistance shown over time and quantification of maximal resistance (box plot) in shERCC1 and NTC cells (n=5,6). Data is shown as box plots with median ± quartiles; whiskers extend to minimum and maximum. Statistical comparison of two groups was performed using (paired) two-tailed Student’s t-test for normally distributed data, or the Wilcoxon-test/ Mann-Whitney test for non-normally distributed data. Exact p-values are reported and statistical significance set at p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/ba9b3b726c945823ffd97b3c.png"},{"id":61591657,"identity":"b98ec284-e2e7-4457-9d49-63733abf6d68","added_by":"auto","created_at":"2024-08-01 15:36:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":819728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eshERCC1 cells show enhanced endothelial migration and sprouting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e mRNA expression of angiogenic markers \u003cem\u003eVEGFA\u003c/em\u003e, \u003cem\u003eSNAI2\u003c/em\u003e, \u003cem\u003eDLL4\u003c/em\u003e, \u003cem\u003eNOTCH1\u003c/em\u003e and \u003cem\u003eKDR\u003c/em\u003e in shERCC1 and NTC cells (n=4). \u003cstrong\u003eb \u003c/strong\u003eRepresentative images of CD31 and DLL4 expression in shERCC1 and NTC cells. \u003cstrong\u003ec\u003c/strong\u003e Representative images of scratch-wound assay at t=0 and t=22 hrs in shERCC1 and NTC cells. The pink outline indicates the scratch borders. \u003cstrong\u003ed\u003c/strong\u003eRepresentative images of CD31 in sprouting shERCC1 and NTC cells with the manual analysis of sprouts marked in pink (scale bar: 150 µm). \u003cstrong\u003ee\u003c/strong\u003e Quantification of total number of sprouts, cumulative sprout length, and minimum sprout length per cell type (n=16-20). Each dot represents a biological replicate and for the qPCR an average of technical triplicates, presented as box plots with median ± quartiles; whiskers extend to minimum and maximum. Statistical comparison of two groups was performed using (paired) two-tailed Student’s t-test for normally distributed data, or the Mann-Whitney test for non-normally distributed data. Exact p-values are reported and statistical significance set at p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/049b7bdce2a87fd994a0cab7.png"},{"id":61591658,"identity":"6c419817-764d-4591-ac42-8a3852322cca","added_by":"auto","created_at":"2024-08-01 15:36:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":781405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEC-KO mice display increased number of P21\u003c/strong\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e cells and BBB transporters specifically in the white matter\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eHeatmap visualizes gene expression profile of WBH comparing EC-KO mice with WT mice. Target categories comprise senescence and BBB markers (n=11-14).\u003cstrong\u003e b\u003c/strong\u003e Representative image of P21 (senescent cell identifier) and Lectin immunoreactivity in EC-KO mice brain tissue; yellow arrowhead indicates P21\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e nucleus; white arrowhead indicates P21\u003csup\u003e-\u003c/sup\u003e nucleus (scale bar: 50 µm).\u003cem\u003e \u003c/em\u003e\u003cstrong\u003ec \u003c/strong\u003eQuantification of total vascular P21\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e cells per mm\u003csup\u003e2\u003c/sup\u003e in WT and EC-KO (N=6). \u003cstrong\u003ed\u003c/strong\u003e Representative images of \u0026nbsp;MDR1A and MFSD2A reactivity in cortex (CRTX), white matter (WM) and hippocampus (HC) in WT and EC-KO brains (scale bar: 50 µm). \u003cstrong\u003ee\u003c/strong\u003e Quantification of mean fluorescent intensity (MI) of MDR1A and MFSD2A in Lectin\u003csup\u003e+\u003c/sup\u003e area (transporter expression) and transporter area normalized to Lectin area (transporter density) (n=6). \u003cstrong\u003ef\u003c/strong\u003e Representative images of CLDN5 reactivity in WM of WT and EC-KO mice (scale bar: 25 µm). \u003cstrong\u003eg \u003c/strong\u003eQuantification of CLDN5\u003csup\u003e+\u003c/sup\u003e vessels (CLDN5\u003csup\u003e+\u003c/sup\u003e, Lectin\u003csup\u003e+\u003c/sup\u003e objects) and MI of CLDN5 in Lectin\u003csup\u003e+\u003c/sup\u003e area (n=6-7). Data is shown as box plots with median ± quartiles; whiskers extend to minimum and maximum. All data have been statistically tested by unpaired student-t test with Welch’s correction when the variance of the groups were significantly different or Mann Whitney test for non-parametric datasets. Exact p-values are reported and statistical significance set at p\u0026lt;0.05 (red).\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/691140010703619376075676.png"},{"id":61591659,"identity":"549abf01-cea2-4579-8c39-653ed2b7581d","added_by":"auto","created_at":"2024-08-01 15:36:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":671208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEC-specific Ercc1 deficiency induces angiogenic markers and BBB leakage \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e mRNA expression of \u003cem\u003eCd31, Vegfa, Dll4, Notch1, Kdr, Snai2, Pdgfrb and Angpt2\u003c/em\u003e in WBH\u003cem\u003e \u003c/em\u003eare plotted as box plots to visualize effect size\u003cstrong\u003e \u003c/strong\u003ein EC-KO and WT mice (n=11-14). \u003cstrong\u003eb \u003c/strong\u003eRepresentative image of COLLAGEN IV and SNAI2 immunoreactivity in the WM of\u003cstrong\u003e \u003c/strong\u003eEC-KO and WT mice.\u003cstrong\u003e c \u003c/strong\u003eRepresentative images of LAMININ, PDGFRβ and αSMA immunoreactivity of WM brain tissue in WT and EC-KO mice; white arrowheads indicate αSMA\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e vessels (arteriole, upper panel), yellow arrowheads indicate PDGFRβ\u003csup\u003e+\u003c/sup\u003e, αSMA\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e vessels (capillary, lower panel) (scale bar: 50 µm). \u003cstrong\u003ed,e \u003c/strong\u003eQuantification of capillary ECs and capillary density as well as pericyte number and coverage in EC-KO and WT mice. \u003cstrong\u003ef\u003c/strong\u003e Representative images of IgG immunoreactivity in EC-KO and WT mice and semi-quantification of IgG\u003csup\u003e+\u003c/sup\u003e area (n =5-10; scale bar: 20µm). Data is shown as box plots with median ± quartiles; whiskers extend to minimum and maximum. All data have been statistically tested by unpaired student-t test with Welch’s correction when the variance of the groups were significantly different or Mann Whitney test for non-parametric datasets. Exact p-values are reported and statistical significance set at p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/e280603327a3a88d8791171a.png"},{"id":61592092,"identity":"8be4567f-7adf-4415-989a-cef155daec5c","added_by":"auto","created_at":"2024-08-01 15:44:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":763808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced vascular ICAM1 expression and immune cell infiltration in the white matter of EC-KO mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Multiplex qPCR mRNA on WBH comparing EC-KO and WT mice (n=11-14). \u003cstrong\u003eb\u003c/strong\u003e Representative images of IBA1 immunoreactivity in WM of EC-KO and WT mice (scale bar: 50µm).\u003cstrong\u003e c\u003c/strong\u003e Representative images of P2RY12 immunoreactivity in WM of EC-KO and WT mice (scale bar: 50µm).\u003cstrong\u003e d \u003c/strong\u003eQuantification of P2RY12\u003cstrong\u003e \u003c/strong\u003eMI in EC-KO and WT mice (n=6-7). \u003cstrong\u003ee\u003c/strong\u003e Representative images and quantification of GFAP\u003csup\u003e+\u003c/sup\u003e area covering the vessel (% GFAP in Lectin\u003csup\u003e+\u003c/sup\u003e area) in WM of EC-KO and WT mice (scale bar: 25 µm). \u003cstrong\u003ef\u003c/strong\u003e Representative images of ICAM1\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003evessels in EC-KO and WT mice (scale bar: 30 µm). \u003cstrong\u003eg\u003c/strong\u003e Quantification of ICAM1\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e vessels and vascular ICAM1 expression in both WT and EC-KO WM brain tissue (n=5-7). \u003cstrong\u003eh\u003c/strong\u003e Representative images of CD45, CD8 and Lectin immunoreactivity presenting both perivascular (top panel) and parenchymal (lower panel) location of peripheral immune cells (scale bar: 20 µm). \u003cstrong\u003ei \u003c/strong\u003eQuantification of CD45\u003csup\u003e+\u003c/sup\u003e immune cells per mm\u003csup\u003e2\u003c/sup\u003e in EC-KO and WT mice. Data is shown as box plots with median ± quartiles; whiskers extend to minimum and maximum. All data have been statistically tested by unpaired student-t test with Welch’s correction when the variance of the groups were significantly different or Mann Whitney test for non-parametric datasets. Exact p-values are reported and statistical significance set at p\u0026lt;0.05 (red).\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/582be97adc8b6a5f53d9c069.png"},{"id":72949458,"identity":"4e2b3b9c-13cb-4c52-8f5c-b50c278743ae","added_by":"auto","created_at":"2025-01-04 08:07:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6880379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/47114816-7e85-4efe-9264-6c4ec4b59782.pdf"},{"id":61591044,"identity":"2d0c7598-d044-4c73-b7bb-ba32062cf26b","added_by":"auto","created_at":"2024-08-01 15:28:03","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1: Primer details\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/9ae91397e08047ee2c302031.xlsx"},{"id":61591661,"identity":"dfddd1c1-b212-4298-b3b7-a0ddaf8cd0fb","added_by":"auto","created_at":"2024-08-01 15:36:03","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2: FDR-corrected values of multiplex qPCR on shERCC1 and NTC cells\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/8f91df1b1f65f9e26a989beb.xlsx"},{"id":61591052,"identity":"eb67e226-754f-4324-99b3-24b98940fe51","added_by":"auto","created_at":"2024-08-01 15:28:03","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 3: FDR-corrected values of multiplex qPCR on WBH of WT and EC-KO mice\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/ff20c164e739617b50555bf9.xlsx"},{"id":61591056,"identity":"264e7f61-5a66-4a11-bd77-d72224ec10e8","added_by":"auto","created_at":"2024-08-01 15:28:04","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17407496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. ZO-1 expression in shERCC1 and NTC cells and original WB, related to Figure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eRepresentative images of ZO-1 in shERCC1 and NTC cells (scale bar: 25 µm). \u003cstrong\u003eb \u003c/strong\u003eOriginal Western blot images of ERCC1 (left) and GAPDH (right) from brain ECs transduced with the ERCC1 knock down construct (shERCC1) or control (NTC).\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/b1dee9f96a826770ca441aa0.tif"},{"id":61591054,"identity":"13d5137e-6c19-4c17-98b5-61d4d1e5269a","added_by":"auto","created_at":"2024-08-01 15:28:04","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6357172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Mean and maximum length of sprouts, related to Figure 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Quantification of mean sprout length, and maximum sprout length in shERCC1 and NTC cells (n=16-20). Each dot represents a biological replicate presented as box plot with median ± quartiles; whiskers extend to minimum and maximum. Statistical comparison of two groups was performed using two-tailed Student’s t-test for normally distributed data, or the Mann-Whitney test for non-normally distributed data.\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/3dc8d01d8ebf000a8278d071.tif"},{"id":61591057,"identity":"69072cba-155f-4ce4-84fe-6365f9c417bc","added_by":"auto","created_at":"2024-08-01 15:28:08","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":80972400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Vascular densities and mural cells in EC-KO and WT brains, related to Figure 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eRepresentative images of LAMININ, PDGFRβ and αSMA immunoreactivity of CRTX and HC in WT and EC-KO mice; white arrowheads indicate αSMA\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e vessels (arteriole, upper panel), yellow arrowheads indicate PDGFRβ\u003csup\u003e+\u003c/sup\u003e, αSMA\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e vessels (capillary, lower panel) (scale bar: 50 µm). \u003cstrong\u003eb\u003c/strong\u003e Quantification of total cell count (nuclei count), total EC and arterial EC count (percentage of total) in WT and EC-KO CRTX, WM and HC brain tissue, (n=6-7). \u003cstrong\u003ec \u003c/strong\u003eQuantification of vascular densities including overall vessel density and arterial density in WT and EC-KO brain tissue, (n=6-7). \u003cstrong\u003ed\u003c/strong\u003e Quantification of smooth muscle cell (SMC) count and coverage of arteriole area, (n=6-7). \u003cstrong\u003ee \u003c/strong\u003eQuantification of MI of PDGFRβ and αSMA in LAMININ\u003csup\u003e+ \u003c/sup\u003earea of WT and EC-KO mice, (n=6-7). \u003cstrong\u003ef\u003c/strong\u003e Area of IgG reactivity measured in grey matter (GM, average CRTX and HC) tissue of WT and EC-KO mice. Data is presented as box plot with median ± quartiles; whiskers extend to minimum and maximum. All data have been statistically tested by unpaired student-t test with Welch’s correction when the variance of the groups was significantly different.\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/2f2c3fd18d75cdb2db429a3e.tif"},{"id":61591055,"identity":"72a95138-01ff-4b28-a320-950756de87c2","added_by":"auto","created_at":"2024-08-01 15:28:04","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":15448764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. Astrocyte reactivity in EC-KO and WT brains, related to Figure 5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eRepresentative images of GFAP in whole brain slices of EC-KO and WT mice (scale bar: 1000µm). \u003cstrong\u003eb\u003c/strong\u003ePresentation of immune cell subsets (CD45, CD8) in WT mouse brain comparing perivascular and parenchymal location.\u003c/p\u003e","description":"","filename":"SupplementaryFigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/b42f0b5a7e34c0a6751334f2.tif"},{"id":61591662,"identity":"3be4547a-9651-4d86-8aed-13ace9068c5e","added_by":"auto","created_at":"2024-08-01 15:36:03","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":10563,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1: Details on WT and EC-KO mice\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/6e13a23542591052457b02b3.xlsx"},{"id":61591051,"identity":"07f6009c-d548-44d0-8bb0-f1ae5fc3daa0","added_by":"auto","created_at":"2024-08-01 15:28:03","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":12197,"visible":true,"origin":"","legend":"\u003cp\u003eTable 2: Primary antibody details\u003c/p\u003e","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4358616/v1/927331bbe4dcb05df34355da.xlsx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Endothelial-Ercc1 DNA repair deficiency provokes blood-brain barrier dysfunction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBrain endothelial cells (ECs) line the interior wall of cerebral microvasculature and establish the blood-brain barrier (BBB), which maintains the delicate homeostasis of the central nervous system (CNS). Brain endothelial tight junctions (i.e. Claudin-5) and adherens junction proteins (i.e. VE-cadherin) ensure the BBB-specific paracellular resistance, which prevents uncontrolled entry of blood components and infiltration of peripheral immune cells into the brain (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Additionally, BBB-specific transporters, such as the efflux transporter P-glycoprotein (P-gp) and major facilitator superfamily domain-containing protein 2a (Mfsd2a), regulate the metabolite exchange between CNS and periphery (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), ensuring optimal brain performance.\u003c/p\u003e \u003cp\u003eDuring aging, brain EC fitness and function are severely affected (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), leading to abnormal vascular responsiveness to cerebral blood flow and disruption of the BBB (\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Aging-related alterations in BBB function include reduced integrity, altered transport mechanisms (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and abnormal angiogenesis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Physiological angiogenesis describes the multistep process of new vessel formation from the existing vasculature and is crucial to respond to the tissues oxygen needs (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In elderly, impaired angiogenesis and pathological vascular remodelling is suggested to contribute to microvascular rarefaction and potentially reduced tissue perfusion (\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Although dysfunction of brain ECs is recognized as a significant factor in the onset and progression of age-related neurodegenerative diseases such as stroke and different forms of dementia, including Alzheimer\u0026rsquo;s disease and vascular dementia, the underlying mechanisms remain elusive (\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWith age, cellular repair mechanisms are known to gradually deteriorate, leading to the accumulation of DNA damage and the advancement of cellular aging (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). DNA damage response can induce aging via several mechanisms, including metabolic changes, transcriptional stress and senescence (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Cells can progress into senescence at the end-stage of their replicative capacity, marked by irreversible cell cycle arrest (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Senescent cells are metabolically active and acquire a cell-specific senescence-associated secretory phenotype (SASP) characterized by cytokine (i.e. Interleukins IL-6, IL-1β), chemokine (i.e. CXCL1, CXCL10), vasoactive mediator and growth factor (i.e. VEGF, TGF-β) production. Mouse models employing the deletion of the Excision repair cross complementation group 1 (\u003cem\u003eErcc1\u003c/em\u003e), a DNA repair endonuclease, have been successfully used to study human aging and senescence (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). \u003cem\u003eErcc1\u003c/em\u003e knock out mice (\u003cem\u003eErcc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eΔ/\u0026minus;\u003c/em\u003e\u003c/sup\u003e KO) closely mimic human vascular aging by displaying increased vascular stiffness, extracellular matrix remodelling and reduced vasodilator function (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), thereby supporting the link between DNA damage and age-related vascular impairments. Nevertheless, it remains unclear how specifically endothelial aging affects BBB function.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the role of EC aging in BBB dysfunction and inflammation \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. We report that ERCC1 deficiency in human brain ECs results in SASP expression, reduced BBB function and enhanced endothelial sprouting via a dysregulation of the Dll4-Notch axis. In line, EC-KO mice demonstrate increased angiogenic marker expression as well as higher numbers of endothelial cells and pericytes, specifically in the white matter (WM). EC-KO mice also display BBB leakage, glial reactivity at the vasculature and leukocyte infiltration in WM areas. Together, we suggest that senescent brain ECs accumulate during aging, thereby promoting BBB impairment and excessive sprouting, which in turn might contribute to the pathogenesis of neurodegenerative diseases.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All animal procedures were performed at the Erasmus Laboratory Animal Science Center following the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and approved by the National Animal Care Committee and the administration within Erasmus University Medical Center Rotterdam (protocol number 118-13-03).\u003c/p\u003e \u003cp\u003eEndothelial-specific \u003cem\u003eErcc1\u003c/em\u003e KO animals were bred as described previously (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In brief, the Cre-loxP system was used to generate a conditional mouse model to knock out endonuclease \u003cem\u003eErcc1\u003c/em\u003e in ECs (B6.Cg-Tg(Tek-cre)12Flv/J, The Jackson Laboratory, Bar Harbor, USA). In the resulting litters, \u003cem\u003eTie2\u003c/em\u003ecre+/-:\u003cem\u003eErcc1\u003c/em\u003efl/- mice have an \u003cem\u003eErcc1\u003c/em\u003e KO in ECs, where Cre-recombinase is active (from here on referred to as EC-KO mice). \u003cem\u003eTie2\u003c/em\u003ecre+/-:\u003cem\u003eErcc1\u003c/em\u003efl/+ animals were used as wild type (WT) controls. Mice (male and female) were kept in individually ventilated cages, in a 12-h light/dark cycle with food and water \u003cem\u003ead libitum\u003c/em\u003e. Animals were euthanized around the age of 22 weeks by cardiac perfusion with ice-cold phosphate-buffered saline (PBS), after which brains were collected. Each brain was divided sagittally and one hemisphere snap-frozen, the other post-fixed in 1.6% paraformaldehyde (PFA) for 24 hours followed by incubation in 30% sucrose for 24 hours. Sagittal brain slices (10 \u0026micro;m) were cut (CryoStar NX70, Thermo Fisher Scientific, Waltham, USA) and stored at -80 ⁰C upon use. For details on the animals see Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eCryosections were defrosted, permeabilized and blocked with 5% normal goat serum and 0.1% Triton-X100 in PBS (Sigma-Aldrich, Saint Louis, MO, USA), PBS only or Tris-buffered saline (TBS). Primary antibodies were incubated overnight at 4\u0026deg;C. Tissue slides were incubated for 1 hour at room temperature with secondary antibodies coupled to Alexa Fluor 488, 555 or 647 fluorophores (Molecular Probes, Eugene, OR, USA). The tissue slides were then counterstained with Hoechst (Molecular Probes, Eugene, OR, USA), embedded in Mowiol (in-house) mounting medium, and stored in the dark at 4\u0026deg;C until microscopic evaluation. Antibody details are listed in Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eImmunocytochemistry (ICC) was performed similarly to IHC on human cerebral microvascular endothelial cells (hCMEC/D3) transduced with lentiviral constructs (see \u003cem\u003eLentiviral short hairpin RNA knock down for ERCC1\u003c/em\u003e). Shortly, cells were seeded in 8-well \u0026micro;-slides (#80826, Ibidi, M\u0026uuml;nchen, Germany) and fixed with 1.6% PFA, ice-cold methanol or acetone for 10 min at room temperature and permeabilized for 5 minutes using 0.05% Triton-X100 in PBS. Blocking, primary and secondary antibody incubation occurred as described above. Nuclei were visualized using Hoechst and wells were filled with Mowiol before imaging. Antibody details are listed in Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopy and image acquisition\u003c/h2\u003e \u003cp\u003eImages were acquired with wide field imaging using the Olympus VS200 (Olympus, Tokyo, Japan) slide scanner or confocal imaging using the Leica SP8 confocal microscope (Leica, Wetzlar, Germany) with a 60x or a 63x oil immersion objective, respectively. Regions of interest (ROI) were acquired as z-stacks of 4 or 8 \u0026micro;m and step size of 266 nm or 130 nm. For wide field images, deconvolution was performed using Huygens Professional 21.10 software (Scientific Volume Imaging B.V., Hilversum, The Netherlands). NIS elements (version 5.30.03, Nikon Europe B.V., Amsterdam, The Netherlands), FIJI and QuPath-0.2.3/-0.4.4 were used for automated and manual analysis. Three ROIs per brain region per animal were imaged in the white matter (WM; corpus callosum, dorsal fornix and anterior commissure), the hippocampus (HC, supra and infra-pyramidal molecular layer) and the cortex (CRTX). For the vascular analysis, arterioles were defined as αSMA\u003csup\u003e+\u003c/sup\u003e vessels with smooth muscle cells (SMCs) (αSMA\u003csup\u003e+\u003c/sup\u003e, often PDGFRβ\u003csup\u003e+\u003c/sup\u003e cells) and capillaries as αSMA\u003csup\u003e\u0026minus;\u003c/sup\u003e, PDGFRβ\u003csup\u003e+\u003c/sup\u003e vessels with pericytes (PCs) (PDGFRβ\u003csup\u003e+\u003c/sup\u003e cells) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Immune cell counts were performed blinded and manually by two researchers using QuPath-0.2.3/-0.4.4. Migrated immune cells were defined by their proximity to the vasculature as perivascular (still in contact with the abluminal side of the vessel marker) or parenchymal (minimal 10 \u0026micro;m distanced from the vessel).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHuman brain endothelial cell culture\u003c/h2\u003e \u003cp\u003eThe immortalized hCMEC/D3 cell line was a kind gift provided by Prof.dr. IA Romero (Open University, Milton Keynes, UK) and Prof.dr. PO Coureaud (Universit\u0026eacute; Paris Descartes, France) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Cells were cultured from passages 29 to 39 in endothelial basal medium-2 (EBM-2) supplemented with 2.5% (v/v), heat-inactivated fetal bovine serum, growth supplement kit (#CC-3156, #CC-4147; Lonza, Basel, Switzerland), and 1% (v/v) penicillin-streptomycin (#15140-122; Gibco, Thermo Fisher Scientific, Waltham, USA). hCMEC/D3 cells were grown on bovine skin collagen I-coated culture flasks (#C5533; Sigma-Aldrich) until confluent unless stated otherwise. For culture, cells were maintained at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e and routinely screened for the presence of mycoplasma.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLentiviral short hairpin RNA knock down of\u003c/b\u003e \u003cb\u003eERCC1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eShort hairpin RNAs were used to knock down \u003cem\u003eERCC1\u003c/em\u003e (shERCC1) expression in hCMEC/D3 as previously described (\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Sub-confluent HEK 293T cells were co-transfected with the specific expression plasmids and packaging plasmids (pMDLg/pRRE, pRSV-Rev and pMD2G) using calcium phosphate as transfection reagent. Infectious lentiviral particle-containing supernatant was collected after 48 hours, concentrated using Amicon Ultra15 filters (UFC910024; Merck, Darmstadt, Germany) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C upon further use. hCMEC/D3 cells were transduced at passage 30 by adding the concentrated supernatant 4\u0026ndash;6 hours after seeding and stable cell lines were selected 24 hours later using puromycin treatment (2 ng/ml, P7255; Sigma Aldrich). The knock down efficiency was assessed using quantitative real-time PCR (qRT-PCR) and Western blot. Constructs (TRCN0000049920) with 84% knock down efficiency were used for subsequent experiments. shERCC1 encodes for 5\u0026rsquo;- CAAGAGAAGATCTGGCCTTAT-3\u0026rsquo;. hCMEC/D3 cells transduced with lentivirus expressing non-targeting shRNA (NTC; SHC002, Sigma-Aldrich) were used as control cells. Transduced shERCC1 and NTC cells were used from passage (P) 1\u0026ndash;7. The assessed gene expression in shERCC1 cells compared to NTC showed the same effect between P1 and P7, with varying effect sizes over the different passages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eInduced pluripotent stem cell-derived brain pericytes\u003c/h2\u003e \u003cp\u003eHuman induced pluripotent stem cells (hiPSC) were differentiated to neural crest (NC)-derived brain pericytes (hiBPC) using previously published protocols (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Briefly, episomal hiPSC line (#A13700, Gibco, Thermo Fisher Scientific, Leusden, The Netherlands) was cultured in mTeSR Plus medium (STEMCELL Technologies, Vancouver, Canada) and grown on vitronectin-coated plates (Invitrogen, Thermo Fisher Scientific). HiPSCs were passaged as single cells, seeded onto Matrigel-coated plates (2 x 105 cells/cm\u003csup\u003e2\u003c/sup\u003e) and cultured for 5 days in NC induction medium, consisting of DMEM/F12 GlutaMAX\u0026trade; (Gibco, Thermo Fisher Scientific), 1\u0026times; B27 (Gibco, Thermo Fisher Scientific), 0.5% bovine serum albumin and 3 \u0026micro;M CHIR 99021 (Tocris, Bristol, United Kingdom). The resulting NC cells were seeded onto 0.1% gelatin-coated plates (2.5 x 104 cells/cm\u003csup\u003e2\u003c/sup\u003e) and cultured for additional 5 days in pericyte medium (ScienCell, Carlsbad, CA, USA). Induced brain pericytes (iBPCs) were characterized by immunocytochemistry (ICC) and RT-qPCR (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). iBPC were used between passages 2\u0026ndash;4 in \u003cem\u003espheroid-based sprouting experiments\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eElectric Cell-substrate Impedance Sensing (ECIS)\u003c/h2\u003e \u003cp\u003eThe transendothelial electrical resistance (TEER) of shERCC1 and NTC cells was assessed using the ECIS\u0026trade; Model 1600R (Applied BioPhysics, Troy, NY) as previously reported (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In short, cells were seeded at a density of 100.000 cells into 8W10\u0026thinsp;+\u0026thinsp;ECIS arrays (#72040, Ibidi). Impedance was measured at multiple frequencies over a time course of 120 hours. To quantify the maximum resistance [ohm], the data at 4000 Hz was normalized to the resistance at time before medium replacement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eScratch-wound and spheroid-based sprouting assay\u003c/h2\u003e \u003cp\u003eFor the scratch-wound assay, NTC and shERCC1 cells were grown to confluence and the scratch was induced diagonally with a plastic pipette tip. Cell migration was imaged for 22 hrs at 10x magnification, bright field, at 37\u0026deg;C with the Nikon Ti2 live cell imaging system (Nikon, Tokyo, Japan). Spheroid-based sprouting assays were performed as previously reported (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In brief, NTC and shERCC1 cells and iBPCs were re-suspended in a ratio of 20:1 in EGM-2 medium supplemented with 0.25% methylcellulose (4.000 cP, Sigma-Aldrich, Saint Louis, MO, USA). Cell suspension was seeded in a 24-well plate and flipped upside down. After 24h, the spheroids were collected and re-suspended in 1,5 mg/ml collagen type-I rat tail mixture (Enzo science, Farmingdale, NY, USA) and re-plated in a 24-well plate upside down until complete polymerization. 30 minutes after polymerization, EGM-2 medium was administered and wells were incubated at 37\u0026deg;C and 20% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e for 5 days. Images were taken using the Nikon LIPSI Ti2 confocal spinning disk imaging system (Nikon, Tokyo, Japan), 10x objective, and adjusted for brightness/contrast in ImageJ. Sprouting number and length were analysed using the ImageJ plugin NeuronJ (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and real-time quantitative polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from mouse whole brain homogenates (WBH) using the RNeasy Lipid Tissue Mini Kit (#174804, Qiagen) and from hCMEC/D3 using TRIzol (#15596-018, Thermo Fisher Scientific). RNA quantity was assessed by Nanophotometer (Implen, Westlake Village, USA). The High-Capacity cDNA Reverse Transcription Kit (#4368813, Thermo Fisher Scientific) was used to synthesize cDNA and transcripts of interest were detected with SYBR Green (#4309155, Thermo Fisher Scientific) using the QuantStudio\u0026trade; 3 Real-Time PCR System (#A28567, Thermo Fisher Scientific). Expression was normalized to housekeeping genes \u003cem\u003eβ-actin\u003c/em\u003e (WBH) and glyceraldehyde 3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e; hCMEC/D3) using the 2\u0026thinsp;\u0026minus;\u0026thinsp;\u003csup\u003eΔΔ\u003c/sup\u003eCT relative quantification method. Primer sequences are summarized in Supplemental Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNuclear fractionation and Western Blot\u003c/h2\u003e \u003cp\u003ehCMEC/D3 cells were washed with cold PBS and lysed on ice with cell lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing protease and phosphatase inhibitors (Roche, Almere, The Netherlands, and Cell Signaling Technology, Boston, MA, USA, respectively). Nuclear fractions were isolated using the NE-PER extraction kit (Thermo Fisher Scientific), following the manufacturer\u0026rsquo;s instructions. All samples were diluted in Laemmli buffer (2x) (BioRad Hercules, CA, USA) (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue) and heated to 95\u0026deg;C for 3\u0026ndash;5 min. Lysates were separated on SDS-PAGE followed by transfer to nitrocellulose for immune blot analysis. Blots were blocked with blocking buffer (Licor, Lincoln, USA) for 1 hour at room temperature. Subsequently, membranes were incubated in blocking buffer containing 0.1% Tween-20 with primary antibodies (Table\u0026nbsp;2) overnight at 4\u0026deg;C and detected and quantified by incubation with IRDye secondary antibodies (1 hour, room temperature) (LI-COR) and imaged by Azure Sapphire Biomolecular Imager (Azure Biosystems, Inc, Sierra CT, Dublin, CA, USA). Original Western blots are depicted in SI.1b.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were performed blinded and data are plotted as box plots with median\u0026thinsp;\u0026plusmn;\u0026thinsp;quartiles and whiskers extend to minimum and maximum values. Statistical tests were performed using GraphPad Prism v9 (GraphPad Software, La Jolla, USA). We used Shapiro-Wilk test for data normality. For comparing two experimental groups, two-tailed Student\u0026rsquo;s t-test was used and non-parametric data was analysed by Mann-Whitney test. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and nominal p-values are reported throughout the manuscript. For multiple Student\u0026rsquo;s t-test Benjamini\u0026ndash;Hochberg correction was performed (q\u0026thinsp;=\u0026thinsp;10%) and the q-values can be found in Supplementary Tables\u0026nbsp;2 and 3. Of note, all significantly different genes survived FDR correction. Test details are indicated in the corresponding Figure legend. For the creation of the gene expression heat map, we used the web-based tool MetaboAnalyst (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.metaboanalyst.ca\u003c/span\u003e\u003cspan address=\"http://www.metaboanalyst.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed: 10/07/2023).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eERCC1 deficiency induces BBB impairment in brain ECs\u003c/h2\u003e \u003cp\u003eTo investigate the effect of aging on brain EC function \u003cem\u003ein vitro\u003c/em\u003e, we generated an accelerated aging model by reducing the expression of Ercc1 (shERCC1) in a human brain EC cell line (hCMEC/D3). shERCC1 cells expressed less \u003cem\u003eERCC1\u003c/em\u003e mRNA (83%, p\u0026thinsp;=\u0026thinsp;0.010, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and showed less ERCC1 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, SI.1b) compared to the non-targeting control cells (NTC). With increasing passage (P) number, shERCC1 cells adopted an enlarged cell size (encircled, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), which is characteristic of senescent cells (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). As a measure of DNA damage, we evaluated the phosphorylation of histone H2AX (yH2AX) (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). shERCC1 cells showed yH2AX foci (yellow arrowhead) and some pan-nuclei H2AX phosphorylation (green arrowhead) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Among the tested senescence and SASP markers, we found a significant increase in \u003cem\u003eIL-6\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.004), \u003cem\u003eIL-1B\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.016) and intercellular adhesion molecule 1 (\u003cem\u003eICAM1\u003c/em\u003e) (p\u0026thinsp;=\u0026thinsp;0.047) mRNA expression in shERCC1 cells compared to NTC, while no significant differences were observed in \u003cem\u003eTNFA\u003c/em\u003e, CDKN1A and CDKN2A expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we investigated the expression of BBB transporters and junction components upon silencing Ercc1. shERCC1 cells showed an increased expression of the transporters \u003cem\u003ePGP\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.003) and \u003cem\u003eMFSD2A\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.016) compared to NTC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Junctional markers like claudin5 (Clnd5) and VE-cadherin (VE-Cad, \u003cem\u003eCDH5\u003c/em\u003e) were decreased in shERCC1 cells, both in their RNA expression (\u003cem\u003eCLDN5;\u003c/em\u003e p\u0026thinsp;=\u0026thinsp;0.0156, \u003cem\u003eCDH5;\u003c/em\u003e p\u0026thinsp;=\u0026thinsp;0.0010) and protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg,h). Zona occludens-1 (ZO-1, \u003cem\u003eTJP1\u003c/em\u003e) did not differ between conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, SI. 1a). In line with decreased CLDN5 and VE-Cad levels, shERCC1 cells displayed a significantly reduced barrier resistance compared to NTC cells (p\u0026thinsp;=\u0026thinsp;0.017) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). Together, these results indicate that ERCC1 knock down induces DNA damage accumulation and BBB dysfunction in brain ECs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eERCC1 deficiency enhances migration and sprouting of brain ECs\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe reduction in BBB markers such as Cldn5 can underlie a (transient) loss of EC identity, which has been associated, among others, with angiogenesis and vascular remodeling (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Thus, we evaluated the delta like canonical notch ligand 4 (Dll4)-Notch1 axis, which is fundamental in the regulation of EC sprouting angiogenesis (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). \u003cem\u003eNOTCH1\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0009) and \u003cem\u003eDLL4\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0313) mRNA expression were decreased in shERCC1 cells compared to NTCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), and DLL4 density was reduced in shERCC1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Next, we assessed the mRNA expression of \u003cem\u003eVEGFA\u003c/em\u003e and kinase insert domain receptor (\u003cem\u003eKDR\u003c/em\u003e, gene encoding vascular endothelial growth factor receptor 2), which are pivotal in the regulation of the Dll4-Notch1 pathway. We observed a significant increase of \u003cem\u003eVEGFA\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.016) and a decreasing trend for \u003cem\u003eKDR\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.059) in shERCC1 cells compared to NTCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Lastly, we assessed the mRNA expression of \u003cem\u003eSNAI2\u003c/em\u003e, a transcription factor which has been shown to directly regulate DLL4 expression in ECs (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). We report a significant increase in \u003cem\u003eSNAI2\u003c/em\u003e mRNA expression (p\u0026thinsp;=\u0026thinsp;0.016) in shERCC1 cells compared to NTC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo functionally assess the dysregulated Notch pathway in shERCC1 cells, we performed a scratch-wound assay and a sprouting assay. shERCC1 cells closed the scratch significantly faster than the NTC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Further, shERCC1 cells showed a significant increase in the number of sprouts (p\u0026thinsp;=\u0026thinsp;0.007) and cumulative sprout length (p\u0026thinsp;=\u0026thinsp;0.007) compared to NTC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed,e). The minimum length of the sprouts was significantly decreased in shERCC1 cells compared to NTC cells (p\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). No change was observed in the mean or maximum sprout length (SI.2a). Together these data indicate a dysregulated Dll4-Notch1 axis in shERCC1 cells, which may explain their impaired angiogenic capacity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEC-specific\u003c/b\u003e \u003cb\u003eErcc1\u003c/b\u003e \u003cb\u003eKO mice show senescence and increased BBB transporters in white matter tissue\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo study the impact of EC-specific accelerated cellular aging on brain homeostasis \u003cem\u003ein vivo\u003c/em\u003e, we utilized EC\u003cem\u003e-\u003c/em\u003eKO mice (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). First, we examined the senescence profile of EC-KO mice compared to WT mice by using multiplex qPCR on whole brain homogenates (WBH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). \u003cem\u003eErcc1\u003c/em\u003e mRNA expression was reduced in EC-KO compared to WT brains (p\u0026thinsp;=\u0026thinsp;0.0001). The mRNA expression of the senescence markers \u003cem\u003eCdkn1a\u003c/em\u003e (encoding P21) (p\u0026thinsp;=\u0026thinsp;0.002), \u003cem\u003eTnfa\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.002) and \u003cem\u003eIcam1\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.004) were increased in EC-KO brains, and \u003cem\u003eIl-6\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.06) showed a similar trend, while C\u003cem\u003edkn2a\u003c/em\u003e (encoding P16) and \u003cem\u003eIl-1b\u003c/em\u003e did not differ between genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Immunohistochemical analysis also showed enhanced levels of P21 in EC-KO mice (p\u0026thinsp;=\u0026thinsp;0.021) compared to WT mice which co-localized with Lectin, an endothelial cell marker (p\u0026thinsp;=\u0026thinsp;0.021; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the properties of the BBB in the EC-KO mice, we next investigated the mRNA expression of BBB-associated markers in WBH. We found a significant increase in \u003cem\u003eCldn5\u003c/em\u003e and \u003cem\u003eCdh5\u003c/em\u003e mRNA expression in EC-KO mice compared to WT (p\u0026thinsp;=\u0026thinsp;0.009 and p\u0026thinsp;=\u0026thinsp;0.0009, respectively), while \u003cem\u003eLama1, Tjp1, Mdr1a\u003c/em\u003e (encoding P-GP), and \u003cem\u003eMfsd2a\u003c/em\u003e were unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). We then examined P-GP and MFSD2A levels in the different brain regions of EC-KO and WT using immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). We found an increase of P-GP (p\u0026thinsp;=\u0026thinsp;0.046) and MFSD2A (p\u0026thinsp;=\u0026thinsp;0.003) immunoreactivity (fluorescent mean intensity (MI)) in the white matter (WM) and a trend towards increased P-GP expression in the hippocampus (HC) (p\u0026thinsp;=\u0026thinsp;0.102) of EC-KO mice compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Concomitantly, we found higher transporter coverage (reactivity area/Lectin area) of the vasculature (P-GP (p\u0026thinsp;=\u0026thinsp;0.039); MFSD2A (p\u0026thinsp;=\u0026thinsp;0.013)) in the WM of EC-KO mice. No differences were found in the cortex (CRTX) or HC. Focusing from now on the WM, we analyzed CLDN5 levels via immunohistochemistry. No differences were observed when comparing the number of CLDN5\u003csup\u003e+\u003c/sup\u003e vessels and the MI of CLDN5 within the vasculature between EC-KO and WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef,g). In summary, endothelial specific \u003cem\u003eErcc1\u003c/em\u003e-mediated aging increases P21\u003csup\u003e+\u003c/sup\u003e cells and BBB transporter levels specifically in the WM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEC-KO mice increase angiogenic marker expression and show BBB leakage\u003c/h2\u003e \u003cp\u003eFollowing our findings in shERCC1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we studied angiogenesis-related markers in the WBH of EC-KO and WT mice. We observed an increase of \u003cem\u003eKdr\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.026), platelet-derived growth factor receptor beta (\u003cem\u003ePdgfrb)\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.030), angiopoietin2 (\u003cem\u003eAngpt2\u003c/em\u003e) (p\u0026thinsp;=\u0026thinsp;0.001) and a positive trend for \u003cem\u003eCd31\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.075) in EC-KO mice compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). \u003cem\u003eVegfa, Dll4, Notch1\u003c/em\u003e and \u003cem\u003eSnai2\u003c/em\u003e mRNA did not change between the experimental groups, but we observed an increase in SNAI2 on protein level in the WM vasculature of EC-KO mice compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince we found an increase in angiogenic markers in EC-KO mice, we next investigated potential changes in the vascular architecture in EC-KO and WT mouse brains. We used a triple immunostaining with PDGFRβ, alpha-smooth muscle actin (αSMA) and LAMININ, to discriminate capillaries from arterioles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, SI. 4a) (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The capillary ECs density was increased in the WM of EC-KO compared to WT mice (p\u0026thinsp;=\u0026thinsp;0.043), but not in CRTX or HC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In contrast, the absolute cell count and percentage of total ECs or arterial ECs did not differ between genotypes in all regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, SI. 4b). Furthermore, the capillary density in the WM of EC-KO mice showed an increasing trend (p\u0026thinsp;=\u0026thinsp;0.088), while overall vascular density and arterial density did not change between EC-KO in WT in all three brain regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, SI. 4c). Finally, the number of PCs was significantly higher in the WM (p\u0026thinsp;=\u0026thinsp;0.027) of EC-KO mice as well as in the HC (p\u0026thinsp;=\u0026thinsp;0.028), while the PC coverage of the endothelium was unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). No differences were detected in the number and coverage of smooth muscle cells (SMCs), nor in the mean expression levels of PDGFRβ and αSMA between EC-KO and WT (SI. 4d,e). Lastly, we assessed if the vascular changes resulted in BBB leakage. We found an increased IgG reactivity in the WM of EC-KO animals compared to WT (p\u0026thinsp;=\u0026thinsp;0.040) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), while IgG reactivity did not differ between genotypes in the grey matter (average CRTX and HC) (SI. 4f). Taken together, these data indicate that EC-KO mice display microvascular changes in the WM which results in local BBB leakage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEC-KO mice display an inflamed BBB and immune cell infiltration in the white matter\u003c/h2\u003e \u003cp\u003eBased on the observed IgG leakage, specifically in the WM of EC-KO mice, we next focused on the possible presence of local inflammation. In the whole brain lysates, we found a significant increase of \u003cem\u003eP2ry12\u003c/em\u003e mRNA (p\u0026thinsp;=\u0026thinsp;0.027), a homeostatic marker for microglia, and a similar trend for \u003cem\u003eGfap\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.06), a marker for reactive astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In WM tissue, we observed more IBA1\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e cells in EC-KO mice compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Furthermore, we found a decreased P2RY12 levels in the WM of EC-KO mice (p\u0026thinsp;=\u0026thinsp;0.012) compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,d), which may indicate microglia activation. Of note, the GFAP-vessel co-localization was higher in the WM of EC-KO (P\u0026thinsp;=\u0026thinsp;0.006) compared to WT. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, SI. 4a). Next, we analyzed BBB inflammation by the vascular expression of ICAM1. EC-KO mice displayed more ICAM1\u003csup\u003e+\u003c/sup\u003e vessels (p\u0026thinsp;=\u0026thinsp;0.001) and a trend towards higher vascular ICAM1 levels (p\u0026thinsp;=\u0026thinsp;0.052) in the WM compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef,g). Using CD45 to identify leukocytes and CD8 for cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells specifically (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), we found an increased density of immune cells in the WM of EC-KO mice (p\u0026thinsp;=\u0026thinsp;0.018) compared to WT. Almost half of the cells were in the parenchymal tissue similarly in WT and EC-KO brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). 27% of parenchymal cells were CD8\u003csup\u003e+\u003c/sup\u003e T cells in the EC-KO mice compared to 9% of the perivascular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). In the WT brains, all parenchymal Cd45\u003csup\u003e+\u003c/sup\u003e cells were positive for Cd8 (total count: 2 cells), while all perivascular cells were Cd8\u003csup\u003e\u0026minus;\u003c/sup\u003e (SI. 4b). In summary, our findings show that endothelial aging coincides with BBB inflammation and increased peripheral immune cells migration into the brain, highlighting a key role of endothelial cells in CNS aging and subsequent inflammation, specifically in the WM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreclinical and clinical studies indicate that aging is a critical factor inducing endothelial dysfunction (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In line, brain endothelial dysfunction is frequently found during healthy brain aging as well as in neurological disorders such as stroke and Alzheimer\u0026rsquo;s disease (\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). However, the role of brain endothelial aging and senescence in BBB impairment remains largely unknown. In this study, we evaluated the consequences of ERCC1 deficiency, a model for accelerated aging, in brain ECs \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. We show that ERCC1-deficient brain ECs display reduced BBB integrity, increased transporter expression and more endothelial sprouting. We validated our \u003cem\u003ein vitro\u003c/em\u003e findings in EC-KO mice, which display a higher expression of angiogenic genes and more capillary ECs and pericytes, specifically in the WM. Furthermore, the WM of the EC-KO animals demonstrates IgG leakage and increased glial cell reactivity near the vasculature, which coincided with immune cell infiltration in the brain parenchyma. Together, our work highlights the effect of endothelial cell aging on BBB dysfunction, angiogenesis and local inflammation.\u003c/p\u003e \u003cp\u003eIn this study, shERCC1 display classical hallmarks of DNA damage and cellular aging including H2AX phosphorylation and increased SASP component expression (i.e. \u003cem\u003eIL-1B\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eVEGFA\u003c/em\u003e). Conversely, expression of \u003cem\u003eCDKN1A\u003c/em\u003e (P21) and \u003cem\u003eCDKN2A\u003c/em\u003e (P16), known senescence markers (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), was unaffected in shERCC1 cells. SASP and P21/P16 elevation are not always concomitant (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), and the presence of the latter is not a pre-requisite for cell aging as exemplified by studies in post-mitotic cardiomyocytes (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Furthermore, in our set-up, the high proliferative capacity of hCMEC/D3 cells combined with ERCC1 deficiency, may highlight cellular aging features directly associated to DNA damage-related cell stress, while overshadowing the P21/P16 expression present in the few senescent cells with halted cell cycle. Lastly, VEGF, highly expressed in the shERCC1 cells, is known to negatively regulate P21/P16 expression (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), which may explain the similarity with NTC cells. The cellular stress of shERCC1 cells is also accompanied by a reduced expression of Cldn5 and VE-cad, resulting in an impaired barrier integrity, as seen during aging \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Together, these results substantiate the role of DNA damage in inducing cell aging, and highlight the effect of brain EC aging on BBB function.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e, EC-KO mice displayed increased vascular P21 expression compared to WT animals. However, not all brain ECs were P21\u003csup\u003e+\u003c/sup\u003e, which aligns with previous reports on this model suggesting partial efficiency of the Cre-lox system in deleting \u003cem\u003eErcc1\u003c/em\u003e (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). P21 expression is increased in the brain endothelium of elderly compared to young individuals (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) and MRI studies positively associate age and enhanced BBB permeability in healthy elderly suggesting the possible effect of aging on BBB dysfunction (\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). In our study, EC-KO mice displayed increased IgG leakage, possibly underlying reduced BBB resistance resulting from EC aging, which is in line with our \u003cem\u003ein vitro\u003c/em\u003e findings on BBB dysfunction. Furthermore, we observed an increase in \u003cem\u003eCldn5\u003c/em\u003e and \u003cem\u003eCdh5\u003c/em\u003e expression in the whole brain homogenate of EC-KO animals compared to WT, but no changes in \u003cem\u003eCldn5\u003c/em\u003e expression in the WM vessels. Interestingly, we show an increased number of brain EC in WM capillaries, which could partially explain the increase in junction mRNA expression. In sum, our findings present an \u003cem\u003ein vivo\u003c/em\u003e model for accelerated vascular aging, which recapitulates some of the features, including reduced BBB integrity, observed during healthy cerebrovascular aging in humans.\u003c/p\u003e \u003cp\u003eIn EC-KO mice, the increased number of ECs in WM brain capillaries was concomitant with an enhanced expression of angiogenic markers (\u003cem\u003eAngpt2\u003c/em\u003e, \u003cem\u003eKdr\u003c/em\u003e), suggesting increased EC sprouting. Furthermore, we observed a general increase in vascular SNAI2 expression in the WM on EC-KO animals. High Snai2 expression has been previously shown to directly impair the Dll4-Notch1-axis, resulting in angiogenesis characterized by dysfunctional vessels (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Similarly, shERCC1 cells displayed increased \u003cem\u003eSNAI2\u003c/em\u003e and reduced \u003cem\u003eDLL4\u003c/em\u003e and \u003cem\u003eNOTCH1\u003c/em\u003e expression together with increased sprout number. These findings may indicate a reactivation of the angiogenic program in the WM of EC-KO mice sustained by Snai2. In line with our findings, previous studies found increased angiogenic markers (i.e. Angpt2) in brain ECs isolated from the corpus callosum of aged mice compared to younger animals (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Interestingly, in our study we found the major changes in the WM of EC-KO mice, while the CRTX and HC seemed to be less affected, suggesting a regional susceptibility to EC aging. In humans, the WM has been previously shown to be more susceptible to age-related pathologies including vascular dementia (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). A possible explanation may lie in the inherent lower capillary density of the WM, which makes this area more sensitive to hypoxic insults, a known trigger for angiogenesis via different pathways including Snai2 upregulation (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan additionalcitationids=\"CR70 CR71 CR72\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). Eventually, studies in aged mice and elderly show vessel rarefaction and reduced vessel length, which may be the result of dysfunctional angiogenesis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR75 CR76 CR77\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). Together our data suggest that DNA-damage in brain ECs may sustain dysfunctional angiogenesis via dysregulated Dll4-Notch1 signalling, and that the WM is more susceptible for this process. However, more research is warranted to fully comprehend the mechanisms underlying brain vasculature maintenance and remodelling during aging.\u003c/p\u003e \u003cp\u003eWith age, a decrease in BBB transporter expression is observed (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e). However, both our endothelial aging models show increased transporter (P-gp and Mfsd2a) expression, specifically in the WM of EC-KO mice. P-gp expression can be primarily regulated by inflammation and oxidative stress, as evidenced by increased P-gp levels in stroke and seizure studies (\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e). Further, other senescent mouse models showed higher P-gp brain vasculature expression, postulating a protective role for senescent cells in toxin efflux from the aging brain (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e). Similarly, the increase in Mfsd2a might also be protective. Mfsd2a limits vesicle-mediated transcytosis, which is crucial to maintain BBB integrity, as shown by barrier leakage in Mfsd2a KO mice (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e). Under homeostatic conditions, increased Mfsd2a expression induces characteristics of cellular aging, while Mfsd2a overexpression alleviates tissue damage after acute brain injury (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). These evidences highlight the multifaceted role of Mfsd2a in the regulation of endothelial cell fitness. It is plausible that the increase of P-gp and Mfsd2a levels in the ERCC1 models is an early protective response to the impaired BBB integrity to aid CNS homeostasis. However, further studies are needed to validate this hypothesis.\u003c/p\u003e \u003cp\u003eThe observed changes of the BBB in the EC-KO mice is accompanied by IgG leakage and loss of homeostatic marker expression in microglia in the WM. The leakage of blood-derived components such as fibrinogen has been reported to activate microglia in elderly and AD subjects (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e), and to contribute to neuroinflammation and cognitive decline. Indeed, we also observed an increase in ICAM1\u003csup\u003e+\u003c/sup\u003e vessels and leukocyte infiltration, including CD8\u003csup\u003e+\u003c/sup\u003e T cells, into the WM of EC-KO mice. ICAM1 is a crucial mediator of the immune cell migration cascade and its p53-induced overexpression has been previously found in senescent endothelial cells of atherosclerotic lesions (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e). Moreover, the presence and function of infiltrating peripheral immune cells in the aging brain, AD subjects, and age-related mouse models is increasingly described (\u003cspan additionalcitationids=\"CR92 CR93\" citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e). In the aging brain, CD8\u003csup\u003e+\u003c/sup\u003e T cells are suspected to interact with CNS cells including microglia and neurons (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e), but their role is still heavily debated (\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e). Together these results position endothelial cell aging as one potential starting point of BBB inflammation and immune cell infiltration in the aging brain.\u003c/p\u003e \u003cp\u003eCollectively, our data highlight the significance of brain EC aging in BBB dysfunction, a key contributor in the development and progression of several diseases of the CNS. Hence, targeting brain vascular aging may be a promising strategy to alleviate age-related vascular disorders and their neurological implications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge gratefully René de Vries for his technical assistance in isolating mouse material. We thank Prof. dr. IA Romero and Prof. dr. PO Coureaud for providing the hCMEC/D3 cell line and Peter. J. M. Stroeken for providing the constructs for the \u003cem\u003eErcc1\u003c/em\u003e knock down. Further, we would like to thank Henrique Nogueira Pinto for differentiating and providing the human BPCs. Lastly, we wish to thank the Microscopy and Cytometry Core Facility for their expertise and support with the microscopy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the European Union´s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant (ENTRAIN) (agreement No. 813294) to HdV, Horizon ERC Advanced (ERC-AD 2022: 101097983) to HdV, by a grant from the Dutch Research Council (NWO Vidi grant 91719305 to G.K.), and grants from the Dutch MS Research Foundation (18-1023MS to G.K. and 20-1106MS to M.E.W.). This work was further supported by a grant to NMdW from ZonMw Onderzoeksprogramma Dementie (project nr: 10510022110005). AAJ and AJMR were funded by TKI-LSH grant # EMCLSH19013. IAM is funded by The Dutch Heart Foundation 2021 E. Dekker Grant (03-006-2021-T019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCEH performed experiments, analyzed data, designed and conceived the study and wrote the manuscript. DV performed experiments, analyzed data, designed and conceived the study and revised the manuscript. LvdM performed experiments, analyzed data and revised the manuscript. AAJ, HNP, WKF, and BvhH performed experiments. RF and MEW provided material and valuable scientific input, plus revised the manuscript. GK provided material and valuable scientific input, revised the manuscript and obtained funding. IM provided valuable scientific input, contributed to design the study and revised the manuscript. AJMR,\u0026nbsp;NMdW\u0026nbsp;and HEdV supervised the study, contributed to design the study, obtained funding and revised the manuscript. All authors read and approved the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSource data is available upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKadry H, Noorani B, Cucullo L. A blood\u0026ndash;brain barrier overview on structure, function, impairment, and biomarkers of integrity. Fluids and Barriers of the CNS. 2020;17(1):69.\u003c/li\u003e\n \u003cli\u003eHan L, Jiang C. Evolution of blood-brain barrier in brain diseases and related systemic nanoscale brain-targeting drug delivery strategies. Acta Pharm Sin B. 2021;11(8):2306-25.\u003c/li\u003e\n \u003cli\u003eDonato AJ, Magerko KA, Lawson BR, Durrant JR, Lesniewski LA, Seals DR. 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Molecular Neurodegeneration. 2022;17(1):59.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Endothelial aging, BBB, ERCC1, senescence, white matter, angiogenesis, immune cell migration","lastPublishedDoi":"10.21203/rs.3.rs-4358616/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4358616/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAging of the brain vasculature plays a key role in the development of neurovascular and neurodegenerative diseases, thereby contributing to cognitive impairment. Among other factors, DNA damage strongly promotes cellular aging, however, the role of genomic instability in brain endothelial cells (EC) and its potential effect on brain homeostasis is still largely unclear. We here investigated how endothelial aging impacts blood-brain barrier (BBB) function by using excision repair cross complementation group 1 (ERCC1)-deficient human brain ECs and an EC-specific \u003cem\u003eErcc1\u003c/em\u003e knock out (EC-KO) mouse model. \u003cem\u003eIn vitro,\u003c/em\u003e ERCC1-deficient brain ECs displayed increased senescence-associated secretory phenotype (SASP) expression, reduced BBB integrity and higher sprouting capacities due to an underlying dysregulation of the Dll4-Notch pathway. In line, EC-KO mice showed more P21\u003csup\u003e+\u003c/sup\u003e cells, augmented expression of angiogenic markers and a concomitant increase in the number of brain ECs and pericytes. Moreover, EC-KO mice displayed BBB leakage and enhanced cell adhesion molecule expression accompanied by peripheral immune cell infiltration into the brain. These findings were confined to the white matter, suggesting a regional susceptibility. Collectively, our results underline the role of endothelial aging as a driver of impaired BBB function, endothelial sprouting and increased immune cell migration into the brain, thereby contributing to impaired brain homeostasis as observed during the aging process.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Endothelial-Ercc1 DNA repair deficiency provokes blood-brain barrier dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-01 15:27:58","doi":"10.21203/rs.3.rs-4358616/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8493d08-5d51-414d-8230-f9f9d3389882","owner":[],"postedDate":"August 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31892150,"name":"Biological sciences/Neuroscience/Neural ageing"},{"id":31892151,"name":"Biological sciences/Physiology/Ageing"}],"tags":[],"updatedAt":"2025-01-04T08:06:54+00:00","versionOfRecord":{"articleIdentity":"rs-4358616","link":"https://doi.org/10.1038/s41419-024-07306-0","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-01-03 05:00:00","publishedOnDateReadable":"January 3rd, 2025"},"versionCreatedAt":"2024-08-01 15:27:58","video":"","vorDoi":"10.1038/s41419-024-07306-0","vorDoiUrl":"https://doi.org/10.1038/s41419-024-07306-0","workflowStages":[]},"version":"v1","identity":"rs-4358616","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4358616","identity":"rs-4358616","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}