Long-term Administration of CU06-1004 Ameliorates Cerebrovascular Aging and BBB Injury in Aging Mouse Model: A Randomized Control Trial

doi:10.21203/rs.3.rs-1845446/v2

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Research Article

Long-term Administration of CU06-1004 Ameliorates Cerebrovascular Aging and BBB Injury in Aging Mouse Model: A Randomized Control Trial

https://doi.org/10.21203/rs.3.rs-1845446/v2

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Journal Publication

published 01 Feb, 2023

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Background: Age-related changes in the cerebrovasculature, including blood-brain barrier (BBB) disruption and vascular dementia are emerging as potential risks for many neurodegenerative diseases. Therefore, endothelial cells that constitute the cerebrovasculature play a key role in preventing brain injury. Our previous study showed that CU06-1004, endothelial cell dysfunction blocker, prevented vascular leakage and enhanced vascular integrity in ischemic reperfusion injury and normalization of tumor vasculature. Here, we evaluate the effects of CU06-1004 on age-related decline in cerebrovascular function of aged mice brain.

Results: In this study, we investigated the protective effects of CU06-1004 on reducing oxidative stress-induced damage in human brain microvascular endothelial cells (HBMECs). HBMECs were treated with hydrogen peroxide (H₂O₂) to establish an oxidative stress-induced cellular injury model. Pretreatment with CU06-1004 considerably reduced oxidative stress-induced cytotoxicity, ROS generation, senescence-associated β-galactosidase activity, and senescence markers in HBMECs. Additionally, pretreatment with CU06-1004 decreased the expression levels of inflammatory proteins, compared to H₂O₂treatment alone. Based on the cytoprotective effect of CU06-1004 in HBMECs, we further examined the vascular protective effects of CU06-1004 on cerebrovascular aging in aged mice. Long-term administration of CU06-1004 alleviated age-associated cerebral microvascular rarefaction and cerebrovascular senescence in the aged mouse brain. CU06-1004 supplementation also reduced extravasation of plasma IgG by improving BBB integrity in the aged mouse brain. This improvement in BBB integrity was associated with reduced neuronal injury and cognition memory dysfunction in aged mice. A series of behavioral tests revealed improved motor and cognitive function in aged mice that received CU06-1004.

Conclusion: These findings suggest CU06-1004 has promise as a therapeutic for delaying age-related cerebrovascular impairment and improving cognitive function in old age.

CU06-1004

Blood-brain barrier

Aging

Brain microvascular endothelial cell (BMEC)

Reactive oxygen species (ROS)

Cerebrovasculature

Inflammation

Neurodegenerative disorders

The blood-brain barrier (BBB) is a physical barrier composed of brain microvascular endothelial cells (BMECs) that limits the movement of substances from circulating blood into the brain, maintaining brain homeostasis. The BMECs play an important role in BBB integrity by forming tight junctions in the cerebral blood vessels (1, 2).

However, as aging progresses, the BMECs are damaged by various stimuli and environmental factors, losing the ability to proliferate, migrate, and repair damage (3, 4). The damaged BMECs then become senescent, an irreversible growth arrest. As senescent cells accumulate in organs, the release of high levels of inflammatory cytokines, matrix metalloproteinases, and immune regulators is induced. This results in the surrounding microenvironment changing to a senescence-associated secretory phenotype (SASP). Development of the SASP is thought to be the main cause of aged-related diseases, and this phenotype can develop if only 2 ~ 3% of the endothelial cells become senescent. Therefore, Senescence of BMECs in the cerebrovascular system could have major implications for cerebrovascular diseases and neurodegenerative disorders, including BBB disruption, mild cognitive impairment, and vascular dementia (5–7). Accumulating evidence has demonstrated oxidative stress and inflammation are the primary factors driving cellular senescence (8, 9). Inflammatory cytokines secreted by senescent cells trigger further inflammation and senescence in surrounding tissue. Additionally, increased presence of inflammatory cytokines reduces levels of endogenous antioxidative enzymes and induces accumulation of reactive oxygen species (ROS) in tissue. Accumulation of ROS in the cerebrovascular system, which is particularly sensitive to oxidative stress, leads to BBB disruption mediated by oxidative stress (10–12). For these reasons, many studies have shown the structure and function of the BBB deteriorates during aging process, leading to increased permeability of BBB (13–15). Disruption of the BBB is associated with the loss of motor neurons, neuroinflammation, and cognitive impairment (16). However, few pharmaceutical interventions have been identified as therapeutic candidates for preserving BBB functionality and preventing cerebrovascular aging (17–19).

CU06-1004 is a small molecule known to activate the cAMP/Rac/cortactin pathway, strengthening the tight junction barrier in endothelial cells and blocking hyperpermeability (20, 21). Acute CU06-1004 treatment for ischemia/reperfusion-induced BBB injury lowered cerebral edema and astrocyte end-foot disruption by stabilizing endothelial cell junctions (22).

Based on these previous findings, we conducted this study to investigate the effects of CU06-1004 on age-related decline in cerebrovascular function of aged mice brain. To investigate the role and potential molecular mechanisms of CU06-1004 in the aged brain, we used both an oxidative stress injury cell model, induced by H₂O₂, and a natural aging mouse model. Our results showed CU06-1004 inhibited oxidative stress-induced HBMEC senescence and inflammation through NF-kB signaling suppression. Furthermore, we observed the novel finding that long-term oral administration of CU06-1004 improved age-associated cerebral microvascular rarefaction in aged mice. Notably, treatment with CU06-1004 increased the expression of tight junction, which was important for BBB maintenance in cerebral microvasculature. Consequently, CU06-1004 treatment attenuated neuropathological changes in the aged brain. We also found CU06-1004 treatment rescued impaired cognition deficits and enhanced muscle function in 23-month-old-mice. In summary, our results demonstrated CU06-1004 can effectively ameliorate age-associated cerebrovascular aging and brain injury, suggesting CU06-1004 has potential as an effective drug to protect against age-related cerebrovascular diseases.

Drug treatment

CU06-1004 was synthesized as described previously (20). Briefly, CU06-1004 was synthesized via tetrahydropyran deprotection and subsequent glycosidation with 4,6-di-O-acetyl-2,3-didieoxyhex-2-enopyran in the presence of an acid. A working solution of CU06-1004 (10 µg/µl) was prepared in dimethyl sulfoxide (DMSO, Sigma, # D2650) for in vitro experiments. The 72-week-old mice were divided into two groups, old-vehicle (n = 15) and CU06-1004 (10 mg/kg) (n = 15). CU06-1004 was dissolved in olive oil (Sigma, # O1514) for oral administration. Both treatments were orally administered 6 days a week using a Zonde needle (100 µl, Jeung Do Bio & Plant Co, # JD-S124) for 6 months, from 18 to 24 months of age.

Experimental Animals

Male C57BL/6J mice (72-week-old) were purchased from Charles River Laboratories Japan (Yokohama, Kanagawa, Japan). Additionally, 6-week-old male C57BL/6J mice (DBL, Korea) were used as young mice and 24-month-old male C57BL/6J mice were used as aged mice. All mice were housed under controlled conditions (24°C ± 1°C, 12 h light/dark cycles, 55% humidity, and specific-pathogen-free) and provided with free access to food and water. All animal facilities and experiments were performed in accordance with the Korean Food and Drug Administration guidelines. All procedures were approved by the Institutional Animal Care and Use Committee at Yonsei University (permit number: IACUC-A-202010-1154-01).

Primary Cultures Of Human Brain Microvascular Endothelial Cells (Hbmecs)

Human brain microvascular endothelial cells (HBMECs) were purchased from ScienCell (Cat. No. 1000) and cultured in EGM-2 media (Lonza, CC-3156). The media was supplemented with the EGM-2 SingleQuots™ kit (Lonza, CC-4176), 20% FBS, and 1% penicillin/streptomycin (P/S, Cat. No. 0503). Cells were routinely passaged at 80−90%⁠ confluency, and cells between passages 3 and 6 were used for experiments. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Measurement Of Cell Viability

Colorimetric 3-(4,5-dimetylthialzol-2-yl)-2,5-diphenyltertrazolium bromide (MTT, Thermo Fisher Scientific, #M6494) assay was used to measure cell viability. MTT is reduced to formazan by mitochondrial dehydrogenases, and the absorbance (570 nm) is directly proportional to viable cell count. HBMECs were seeded into a gelatin-coated 24-well plate at 1 ⋅ 10⁵ cells/well and incubated at 37°C in EGM-2 medium overnight. The following day the cells were treated with either CU06-1004 or H₂O₂. The cells were then washed with 1x PBS and incubated for 4 h at 37°C with MTT solution (0.1 mg/ml) for evaluating cell viability. After the 4h incubation, the MTT solution was removed and a 50:50 solution of dimethyl sulfoxide and ethanol was added (200 µl/well) to solubilize formazan crystals. Absorbance was detected at a wavelength of 540 nm and cell viability was calculated as a percentage of absorbance detected from the control cells.

Rna Isolation And Reverse Transcription Polymerase Chain Reaction (Rt-pcr)

RNA isolation and reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from HBMECs using easy-BLUE™ (iNtRON, #17061). Total RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega Corporation, #M1701) in the presence of oligo(dT) primers and dNTP. The following temperature protocol was used for reverse transcription: Denaturation at 70°C for 5 min, annealing at 25°C for 10 min, and extension at 42°C for 50 min. The following primers were used for PCR: p21: 5’-GCTTCATGCCAGCTACTTCC-3’(forward), 5’-CCCTTCAAAGTGCCATCTGT-3’ (reverse), p16: 5’-CCTCGTGCTGATGCTACTGA-3’(forward), 5’-CATCATCATGACCTGGTCTTCT-3’ (reverse), GAPDH: 5′-CCACCCATGGCAAATTCC-3′(forward), 5′-TCGCTCCTGGAAGATGGTG-3′(reverse). All results were normalized to GAPDH expression levels.

Measurement Of Intracellular Reactive Oxygen Species (Ros)

The formation of ROS was measured using a ROS-sensitive dye, 2’,7’-dichlorodihydrofluorescein diacetate (H₂-DCFDA, Invitrogen, #D399), as an indicator. HBMECs were seeded at 1 ⋅ 10⁴ cells/well in a black, clear bottom 96-well plate containing 100 µl of culture medium and then incubated at 5% CO₂ and 37°C overnight. The following day, HBMECs were starved of media for 2 h and then pretreated with CU06-1004 (10 µg/ml) for 1 h. The media were removed, and the cells were washed twice with PBS. This was followed by stimulation of ROS development via incubation with 100 µM H₂O₂ for 2 h. The cells were then incubated with 10 µM H₂-DCFDA for 30 min at 37°C. The fluorescent product formation was quantified with a spectrofluorometer at 485/520 nm. The fluorescent cells were then washed twice with PBS and observed using a fluorescence microscope (Microscope, Olympus DX51; Camera, Olympus DP72).

Senescence-associated-β-galactosidase (SA-β-gal) staining.

The samples were fixed with 3.7% formaldehyde for 10 min and washed with cold 1x PBS for 15 min at room temperature. Samples were washed twice more with PBS and then incubated at 37°C without CO₂ for 24h with senescence-associated β-gal staining solution, containing 1mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, MERCK, #B4252), 5mM potassium ferrocyanide, 5mM potassium ferricyanide, 150Mm NaCl, 2mM MgCl₂. 0.01% Nonidet-P-40. After the 24h incubation, samples were washed with PBS and then observed for aging by degree of blue color development (23). Staining and imaging were observed under a phase-contrast microscope (Nikon, Japan).

Quantitative immunofluorescent microscopy of cerebral immunoglobulin G extravasation.

The integrity of the BBB was determined by detection of cerebral perivascular extravasation of the plasma protein immunoglobulin G (IgG), a widely used and established method (24). Brain tissues were immersion-fixed in 4% paraformaldehyde for 24 h and immersed in 15% and 30% sucrose each day. The tissues were then frozen in OCT compound and stored at -80 °C. Brain cryosections of 25 µm were placed on Polysine™-coated microscope slides (Leica, #3800050CL). The sections were prefixed in acetone for 30 min at -70°C. Unspecific binding was blocked with 10% goat serum in PBS for 30 min. Goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, #A28175) at a concentration of 1:50 antibody diluent solution was applied to the sections and sections were incubated at 4°C for 20 h. After washing the sections with 0.2% TBST and 1 x PBS, the sections were mounted with mounting solution (DAKO, #S3023). The immunofluorescent images were taken using a Confocal 980 (LSM 980 META; Carl-Zeiss). For each cortex and hippocampus region 5–6 images were randomly taken from each brain section, and all images were used for subsequent quantitative analysis.

Quantitation Of Il-6 And Tnf-α By Elisa

Blood samples through cardiac puncture were obtained in SST tube (Becton Dickinson, # BD365967) and incubated for 30min at RT. Then, the murine serum was collected following sample centrifugation for 10 min at 1500 rpm. The serum concentrations of IL-6 and TNF-α were determined using Quantikine ELISA Kit (R&D systems, #M6000B, #MTA00B) according to the manufacturer protocol.

Histology And Immunohistochemical Analysis

Following 6 months of drug administration, the 24-month-old mice were anesthetized using avertin (2, 2, 2-Tribromoethanol, Sigma Aldrich, #T48402), 250 mg/kg of body weight, and perfused with 0.9% saline solution into the apex of the left ventricle. The brain tissue was removed and fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4°C. Following overnight fixing, brain tissue was incubated in 15% sucrose overnight at 4°C and then transferred to 30% sucrose at 4°C until the tissue sank. Fixed tissue was encapsulated with Tissue-Tek OCT embedding medium for 30 min at room temperature, transferred to an embedding mold filled with OCT, frozen on dry ice, and stored at -70°C. Frozen sections (25-µm-thick) were cut at -20°C, and slides were stored at -80°C until stained for immunofluorescence. The sections were prefixed in acetone for 30 min at -70°C and air dried. The OCT was washed off with running tap water. The sections were incubated in blocking solution for 1 hour at room temperature and then incubated overnight at 4°C in CD31 primary antibody (1:200; Abcam, #ab24590), GFAP (1:200; Millipore, #MAB360), claudin-5 (1:200; Invitrogen, #35-2500), and occludin (1:200; Invitrogen, #711500). After incubation, sections were washed 3 times with 0.2% Triton X-100 in PBS (10 min/wash), and further incubated separately in 488-conjugated secondary antibody (1:400; Invitrogen, #A28175), 594-conjugated secondary antibody (1:400; Invitrogen, #A21297), and 4′,6-diamidino-2-phenylindole (DAPI; 1:1000, Duolink, #D9542). The sections were analyzed using a confocal microscope (LSM 880 META; Carl-Zeiss).

Western Blot Analysis

Western blotting was performed as previously described (25). Briefly, HBMECs were lysed using RIPA buffer (100 mM Tris-Cl, 5 mM EDTA, 50 mM NaCl, 50 mM β-Glycero-phosphate, 50 mM NaF, 0.1 mM Na₃VO_4, 0.5% NP-40, 1% Triton X-100, and 0.5% Sodium deoxycholate) at 4°C. Sample protein concentration was quantified using the SMART™ BCA Protein Assay Kit (iNtRON, #21071). Next, 25 µg of cell lysates were separated by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin in 0.1% TBST and probed with primary antibodies. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Life Science) secondary antibodies. β-actin was used as the loading control. The primary antibodies were obtained from Cell Signaling Technology and were used at a 1:1000 dilution: phospho-IκB-α (#9242), IκB-α (#2859). The other primary antibodies used were ICAM-1 (1:1000, Santa Cruz Biotechnology, #SC-8439), VCAM-1 (1:1000, Santa Cruz Biotechnology, #SC-13160), COX-2 (1:1000, Santa Cruze Biotechnology, #SC-376861) and β-actin (1:2000, Thermo Fisher Scientific, #MA5-15739).

Behavior Test

Wire hang test

This test evaluated the forelimb strength of mice. The apparatus consisted of a stainless-steel wire (90 cm length, 2 mm in diameter), secured horizontally between two vertical stands, 30 cm above a soft padded surface. The wire hang test was conducted at 23 months of age. The mouse was forced to grasp the central position of the wire with its forepaws, and the time it took to fall from the wire to the pad was measured. When the time was over 150 s the mouse was released from the wire, and the time was recorded as 150 s. The trial was conducted three times for each mouse and the averaged value was used for evaluation. The resting time between consecutive attempts was 3 min.

Rotarod Test

This test assessed motor coordination by placing animals on a Rotarod device (Four Lane Rotarod; Ugo Basile, Italy, #MSW-007) that consists of an accelerating rod that the mouse must balance on. If a mouse loses its balance and falls the rod automatically stops and records the time to fall as well as the speed at fall. Prior to the first test, mice were habituated to the testing system until they were able to stay on the rod at a constant speed of 2 rpm for approximately 1 min. During testing, each animal was exposed to the apparatus three times for 300 s per trial. The initial speed of the Rotarod was set to 4 rpm and increased to 50 rpm over 300 s. When the mouse fell the session was over, and the Ugo Basile program stopped the timer (26).

T-maze Alteration

Spatial working memory was assessed using a simple T-maze test (27). Each trial consisted of a sample run and a choice run. In the sample run, one of the goal arms was blocked, forcing the mouse to enter the other goal arm (e.g., the left arm). Prior to beginning the choice run, a 30 s delay was introduced between trials. The blocks were removed, and the mouse was given a free choice of either arm in the choice run. Even without a reward, driven by curiosity, mice usually selected the previously unvisited arm (e.g., the right arm). The animal was considered to have made the correct choice (+) if it visited the previously unsampled arm but incorrect (-) if it visited the previously sampled arm. A total of 10 free choices made by the mice were measured and the percentage of correct arm choices during the choice trials was calculated. Each arm of the T-maze was cleaned between sessions using ethanol to remove any olfactory cues, which may have affected the behavior of the next mouse tested.

Statistical analysis

GraphPad Prism 8 software (GraphPad Software, La Jolla, CA) was used for statistical analyses.

Statistical significance was determined by using as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM) and P values less than 0.05 were considered statistically significant. All experiments were performed at least three times, and representative results are shown.

CU06-1004 reduces H ₂ O ₂ -induced inhibition of HBMEC growth.

Prior to investigating the protective properties of CU06-1004 (Fig. 1A) against H₂O₂ treatment, the cytotoxic potential of CU06-1004 was examined on HBMEC. The MTT assay indicated CU06-1004 did not show any cytotoxic effects at concentrations ranging from 1 to 20 µg/ml (1, 2, 5, 10, and 20 µg/ml). Conversely, HBMEC growth was significantly increased in a dose-dependent manner (Fig. 1B). Further MTT analyses revealed that while 50 µM H₂O₂ did not inhibit cell growth, 100 µM H₂O₂ significantly inhibited the growth of HBMECs (Fig. 1C). Notably however, CU06-1004 pretreatment effectively reversed H₂O₂-induced inhibition of cell growth (Fig. 1D). These results demonstrated CU06-1004’s ability to prevent H₂O₂-induced inhibition of HBMEC growth.

CU06-1004 inhibits H ₂ O ₂ -induced ROS generation and alleviates the inflammatory response of HBMECs.

To elucidate the possible mechanisms by which CU06-1004 prevented inhibition of HBMEC growth we used H₂-DCFDA. H₂-DCFDA is a cell-permeable fluorescent dye which fluoresces upon oxidation by ROS, and it was used to determine the effect of CU06-1004 on H₂O₂-induced oxidative stress in HBMECs. Exposure of HBMECs to H₂O₂ for 2 h significantly enhanced ROS generation compared to untreated HBMECs. However, pretreatment with CU06-1004 for 1 h significantly inhibited the H₂O₂-induced increase in ROS generation in a dose-dependent manner (Fig. 2A-B).

Many studies have determined oxidative stress induces an inflammatory response either directly or indirectly, and oxidative stress and inflammation are primary mechanisms related to onset of age-related vascular endothelial dysfunction (28–30). Therefore, to investigate whether the inflammatory response induced by oxidative stress was alleviated by CU06-1004, we determined the expression of inflammatory proteins in H₂O₂‑treated HBMECs. As shown in Fig. 2C-G, CU06-1004 treatment effectively inhibited the by H₂O₂-induced expression of ICAM-1 and VCAM-1, as well as the expression of COX-2, an inflammation mediating enzyme. Furthermore, NF-κB, as a transcription factor, is considered a major mediator of the inflammatory response. Notably, the level of phosphorylated IκBα, an indicator of NF-κB activity, was significantly reduced in HBMECs treated with CU06-1004. Together, these results suggest CU06-1004 has a protective effect from ROS-induced damage by inhibiting the inflammatory response in HBMECs.

CU06-1004 reverses H ₂ O ₂ -induced senescence in HBMECs.

To investigate the effect of CU06-1004 on HBMECs with senescent phenotype, cells were treated with 100 µM H₂O₂ to induce cellular senescence (Fig. 3A). At 3 days post-H₂O₂ exposure the presence of senescent HBMECs was confirmed by enlarged shape and cytoplasmic granularity of exposed cells. After 5 days, The SA-β-Gal staining assay was used to detect the level of HBMEC senescence induced by H₂O₂. A 3-fold increase in percentage of SA-β-Gal⁺ cells was observed in H₂O₂-treated group compared to control cells. However, pretreatment with CU06-1004 significantly inhibited this effect and reduced the percentage of SA-β-Gal⁺ cells to 2-fold compared to control cells (Fig. 3B). Senescence is a state of permanent cellular arrest that is established and maintained by the expression of cyclin-dependent kinase inhibitors (CKIs). As p16^INK4a and p21 are known to mediate permanent cell cycle arrest (31, 32), we determined mRNA levels associated with these genes by RT-PCR. The expression of p16 ^INK4a and p21 was downregulated in the CU06-1004 treatment group, confirming the anti-senescence effect of CU06-1004 in HBMECs (Fig. 3C-E).

CU06-1004 alleviates age-associated cerebral microvascular rarefaction and vascular aging in aged mice.

Based on results showing the anti-inflammatory and anti-senescence effects of CU06-1004 on HBMECs, we further examined whether long-term administration of CU06-1004 (10 mg/kg via oral gavage) to 18-month-old mice (late middle-age) had a protective effect on cerebrovascular aging. First, we measured the maximal cortical diameter and the ratio of brain to body weight at the postmortem examination. The mean cortical diameter was decreased by 8% in 24-month-old mice (late age) compared to 6-week-old mice (young age). However, no differences were detected between the old-vehicle and old-1004 group. (Fig. 4A-B). Additionally, the brain to body weight ratio was significantly reduced in 24-month-old mice (late age) but was less reduced in old-1004 group (Fig. 4C). Next, to examine brain microvasculature changes in aging, the brain vasculature patterning in the cortex and hippocampus of aged mice brain was analyzed through immunofluorescent staining of endothelial markers. There was significant reduction in capillary vessel density in old mice microvasculature compared to young mice microvasculature. However, the old-1004 group, old mice treated with CU06-1004, showed greater capillary vessel density and higher numbers of branch points, compared to old mice receiving only the vehicle, old-vehicle. This indicates CU06-1004 improves vessel maintenance and inhibits cerebral microvascular rarefaction in aged mice (Fig. 4D-E). Additionally, SA-β-Gal⁺ cells were clearly observed in vessels located in the brain cortex of aged mice, but cell prevalence decreased in the old-1004 group (Fig. 4F). These results suggest long-term administration of CU06-1004 protects against aging-induced microvascular rarefaction and vascular aging.

The disruption of BBB integrity during aging could be prevented by CU06-1004.

We then investigated whether age-induced cerebral microvascular rarefaction affects the BBB integrity. The integrity of the BBB was determined by detection of cerebral extravasation of the plasma protein immunoglobulin G (IgG). As shown in Fig. 5A-B, IgG was almost absent from the parenchyma of the cortex and hippocampus of young mice but was highly abundant in aged mice parenchyma. Notably, this abundance was significantly decreased in the old-1004 group. As BBB permeability is highly dependent on cerebrovascular endothelial tight junctions, we next examined the integrity of these junctions in the brains of young and aged mice. Compared with young mice, aged mice (old-vehicle) cerebral vessels expressed less claudin-5 and occludin proteins. Notably, claudin-5 was upregulated in aged mice that received CU06-1004 treatment (old-1004). However, there was no difference in expression levels of occludin between the aged mice groups (Fig. 5C-D). We used electron microscopy to further examine the minor changes of tight junction complexes responsible for cerebrovascular leakage. In the young mice, ultrastructural analysis showed seamless tight junctions within a smooth endothelial layer surrounded by astrocyte endfeet. Although it was confirmed that the capillary wall became thicker and the astocytic endfeet was considerably swollen in the old mice, the tight junctional complex was less damaged in old-1004 group than old-vehicle (Figure S1). These results indicate aging accelerates the onset of BBB dysfunction and long-term administration of CU06-1004 could prevent damage to BBB integrity associated with aging.

CU06-1004 attenuates neuropathological changes in the aged brain.

Studies have shown BBB dysfunction amplifies neuroinflammation and may act as a key process in the development of neuroinflammation (33). Therefore, we determined astrocyte activation in brain tissues of young and aged mice, as this is a widely accepted hallmark of neuroinflammation in aged mice brain. Histopathological alterations were evaluated using immunohistochemistry and immunofluorescence staining. As shown in Fig. 6A-B, cytoplasmic staining showing GFAP-positive (activated) astrocytes in brain sections of the hippocampus was significantly increased in aged mice compared to young mice. Double immunofluorescence staining showed increased GFAP activation in the hippocampus of aged mice (old-vehicle), suggesting that aging causes upregulation of activated astrocytes. Notably, we found long-term administration of CU06-1004 reduced systemic TNF-α and IL-6 levels, which appeared to increase with aging (Fig. 6C). Additionally, we analyzed expression levels of inflammatory proteins from brain tissue extracts. In brain tissue, the expression levels of proteins such as ICAM-1, VCAM-1, and COX-2 were lower in aged mice that received CU06-1004 (old-1004) than in aged mice that did not (old-vehicle) (Fig. 6D-G). Collectively, these results indicate long-term administration of CU06-1004 exerts anti-inflammatory and neuroprotective effects in aged mice.

CU06-1004 treatment in late middle-age improved motor function and recognition memory dysfunction.

Next, we quantified the neuronal nuclear protein A60-positive (NeuN⁺) cells in the brains of aged mice. The number of NeuN⁺ cells was significantly reduced and incompact in the cortex and the CA1 region of the hippocampus in aged mouse brain compared to young mouse brain. However, NeuN⁺ cell numbers and compactness were restored following CU06-1004 treatment (old-1004), demonstrating the efficacy of CU06-1004 in protecting against neuronal damage in the aged brain (Fig. 7A-B). We then examined whether CU06-1004 treated aged mice showed behavioral and cognitive recovery (Fig. 7C). Motor function and working memory activities were performed with old-vehicle and old-1004 mice at 23 months. The Rotarod test and wire hang test are classic methods for evaluating motor coordination of the limbs and balance in aged animals. As shown in Fig. 7D, both old-vehicle and old-1004 groups exhibited shorter average time to fall off the accelerating rotating rod compared to the young group, but no significant difference was detected between the old-vehicle and old-1004 group. In the wire hang test, the old-1004 group showed a marked increase in hanging time (about by 3-fold) compared to the average hanging time of the old-vehicle group, indicating CU06-1004 enhances motor coordination and forelimb muscle strength (Fig. 7E). The T-maze test was used as a spontaneous alternation task for assessing spatial working memory. Aged mice demonstrated a significantly lower percentage of correct spontaneous alternation choices, indicating an impairment in working memory. However, the increase in percentage of correct spontaneous alternation choices between old-vehicle and old-1004 groups indicated a significant effect of CU06-1004 treatment (Fig. 7F). These results suggest long-term administration of CU06-1004 reduces neuromuscular strength impairment caused by aging and damage to spatial working memory caused by neuronal cell damage.

Aging is a biological process in which the structure and function of all organs progressively deteriorate over time (34). Aging is also a major risk factors for developing various vascular diseases, including, cardiovascular diseases, strokes, eye diseases, and neurodegenerative diseases. Similarly, the vascular system, which supplies oxygen and nutrients throughout the body, is affected by the aging process and becomes more susceptible to diseases in the aged population. Therefore, it is very important to develop novel therapies that can slow the aging process and more effectively treat aging-related diseases (35).

CU06-1004, an endothelial cell dysfunction blocker, has been shown to prevent vascular leakage and enhance vascular integrity in ischemic reperfusion injury and normalization of tumor vasculature. However, the mechanisms underlying the role of CU06-1004 in oxidative stress-induced HBMEC senescence, inflammation, and age-related cerebrovascular dysfunctions remain unknown. In this study, the brains of aged mice showed higher SA-β-galactosidase activity than young mice brains. Capillaries of the young mice brains were interconnected in tubular structures while the capillaries of the aged mice brain were fragmented and disconnected in both the cortex and hippocampal regions. This implied that BMECs that had become senescent, a state of irreversible cell growth inhibition, contributes to the decrease in cerebral capillary density during aging (Fig. 4). Mice that are 18−24 months of age are used to represent humans that are 56−69 years of age. Additionally, humans aged 55−85 years have shown a significant decrease in microvasculature density in brain tissue, similar to that observed in Alzheimer’s disease patients. In normal aging, cerebrovascular loss causes chronic hypoperfusion to the brain, eventually leading to cognitive impairment and vascular dementia. Therefore, maintaining cerebrovascular homeostasis is important for preventing cerebrovascular aging and brain pathology. Moreover, we observed cerebral microvascular rarefaction in aged brain tissue causes incomplete BBB integrity, which in turn leads to exceedingly high trans-endothelial permeability and increased passive extravasation of plasma IgG.

Here we show long-term administration of CU06-1004 in aged mice alleviates age-associated cerebral microvascular rarefaction and inhibits the leakage of plasma IgG into the brain parenchyma by suppressing cellular senescence and upregulating stability of claudin-5, the most enriched tight junction protein in the aged mouse brain (Fig. 5).

It is also known that BBB integrity is strongly affected by oxidative stress. Increased ROS production contributes to cerebral endothelium dysfunction and increased permeability of the BBB (36). Additionally, cerebral endothelial cells have high concentrations of mitochondria, increasing the risk of oxidative damage in cells (37). The oxidation-inflammatory theory of aging also proposes that age-associated oxidative stress is a driving factor of cellular senescence (9). Consistent with previous studies, we observed H₂O₂-induced generation of excessive free radicals activated HBMEC senescence and led to cells exhibiting classical SASPs characteristics such as an enlarged cell shape, cytoplasmic granularity, and increased SA-β-galactosidase activity. Furthermore, it was observed that H₂O₂ exposure activated cell cycle inhibition pathways, p16^INK4a/p21, and strongly suppressed cell proliferative capacity. Alternatively, HBMECs supplemented with CU06-1004 were characterized by attenuated SA-β-galactosidase activity and marked downregulation of inflammatory proteins associated with SASP, potentially due to CU06-1004-mediated NF-κB inhibition. Additionally, CU06-1004 treatment appeared to prevent senescence-associated cell cycle arrest by inhibiting cell cycle suppressors p16^INK4a and p21. This was then confirmed, as HBMECs treated with CU06-1004 showed improved proliferative capacity following H₂O₂ exposure compared to control cells (Fig. 3). Overall, these results indicated CU06-1004 inhibited the development of oxidative stress-induced senescence-associated features and the inflammatory response in HBMECs.

As chronic systemic inflammation increases with aging, cerebrovasculature becomes damaged due to proinflammatory cytokines and proinflammatory molecules (38–41). Chronic systemic inflammation is characterized by low-grade and persistent inflammation, leading to tissue degeneration. Additionally, chronic low-grade inflammation contributes to various age-related pathologies in aging tissue, including tissues of the nervous and musculoskeletal systems (28, 42, 43). Notably, we confirmed that long-term administration of CU06-1004 reduced the degree of systemic inflammation caused by plasma concentrations of proinflammatory cytokines, including TNF-α and IL-6. These results suggest prevention of vascular damage by CU06-1004 may inhibit inflammation in the brain as well as other tissues (Fig. 6). We also showed that aged mice treated with CU06-1004 had improved muscle strength and recognition memory in behavior tests (Fig. 7). These findings emphasize the importance of the BBB in maintaining normal function of the central nervous system, thereby resisting neuronal injury, and improving cognitive function.

In conclusion, this study showed cerebrovascular aging may contribute to age-related cerebrovascular damage and neuroinflammation in the aged brain. Additionally, it was confirmed that CU06-1004 protects the endothelial cells of cerebral blood vessels, HBMECs, against oxidative stress-induced senescence and inflammation through ROS scavenging, leading to reduced cytotoxicity. Long-term administration of CU06-1004 in aged mice alleviates symptoms associated with motor and cognitive deficits, including, cerebral microvascular rarefaction, neuronal losses, and chronic neuroinflammation. Collectively, these results suggest CU06-1004 could be a useful therapeutic for preventing cerebrovascular aging and age-associated brain injury.

Collectively, our data showed that CU06-1004, a known endothelial dysfunction blocker, acts as a neuroprotective against age-related cerebrovascular impairment by exerting anti-senescence and anti-inflammatory effects in HBMECs. And long-term administration of CU06-1004 alleviated age-associated cerebral microvascular rarefaction and cerebrovascular aging thereby improving BBB integrity, and BBB integrity was associated with reduced neuronal injury, reduced cognition memory dysfunction, and improved motor and cognitive function in aged mice. These findings suggest supplementation of CU06-1004 has great promise as a therapeutic for delaying age-related cerebrovascular impairment and improving cognitive function in old age.

BBB

Blood-brain barrier

HBMEC Human brain microvascular endothelial cell

IL-6

Interleukin-6

TNF-α

Tumor necrosis factor alpha

ICAM-1

Intercellular adhesion

VCAM-1

Vascular adhesion molecule-1

NF-ĸB

Nuclear factor-kappa B

IkBα

nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha)

COX-2

Cyclooxygenase-2

ROS

Reactive oxygen species

IgG

Immunoglobulin G

SASP

Senescence-associated secretory phenotype

GFAP

Glial fibrillary acidic protein

CD31

Cluster of differentiation 31

NeuN

Neuronal nuclear protein

Ethics approval and consent to participate

This study was approved by the Institutional Animal Care and Use Committee of Yonsei University (approval number; IACUC-A-202010-1154-01).

Consent for publication

Not applicable.

Availability of data and materials

Not applicable.

Competing interests

The authors declare that they have no competing interests. Mice behavior test graphic was created with BioRender (http://biorender.com)

Funding

Not applicable.

Authors’ contributions

We thank CURACLE Co., Ltd. For providing us with the CU06-1004. HJ designed the project and planned the experiments. HJ performed all experiments and quantifications. HJ, MY and SY performed behavior tests of mice. HJ and YGK discussed the results and wrote the manuscript. HJ, MY, HZ and YM contributed to proofreading the manuscript. YGK supervised and corrected manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

FigureS1.tif
Figure S1. Electron micrographic images of cerebrovasculature in young and aged mice. Electron microscopy (TEM) was used to observe the BBB ultrastructure of the young, old-vehicle and old-1004 groups. EC; endothelial cell, VL; vessel lumen, TJ; tight junction, AC; astrocyte, PC; pericyte. Black arrows point to brain endothelial tight junctions. (A-B) Intact BBB in blood vessels (VL) embedded in closed tight junctions (TJ) between brain endothelial cells of young mice brain. Scale bar = 2 µm. (A1-B1) High-magnification images of the red area boxed in (A-B), highlighting endothelial tight junction with black arrows. Scale bar = 500 nm. (C-F) Disrupted BBB in blood vessels (VL), including thicker capillary walls and swelled astrocytic endfeet in old-vehicle and old-1004 groups. Scale bar = 2 µm. (C1-F1) High-magnifications images of the red area boxed in (C-F), showing discontinuous and increased gap between the cerebrovascular TJ reflecting disrupted BBB in old-vehicle and old-1004 groups. Scale bar = 500 nm. (G) Junctional complex average width (mm) was quantitatively analyzed by measuring the average width between TJ of the TEM images on the young, old-vehicle and old-1004 groups (n = 9~12 per group). All data are presented as the mean ± SD, ***P < 0.001.
FigureS2.tif

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Long-term Administration of CU06-1004 Ameliorates Cerebrovascular Aging and BBB Injury in Aging Mouse Model: A Randomized Control Trial

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Abstract

Figures

Background

Materials And Methods

Drug treatment

Experimental Animals

Primary Cultures Of Human Brain Microvascular Endothelial Cells (Hbmecs)

Measurement Of Cell Viability

Rna Isolation And Reverse Transcription Polymerase Chain Reaction (Rt-pcr)

Measurement Of Intracellular Reactive Oxygen Species (Ros)

Quantitation Of Il-6 And Tnf-α By Elisa

Histology And Immunohistochemical Analysis

Western Blot Analysis

Behavior Test

Wire hang test

Rotarod Test

T-maze Alteration

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 2