Caveolin-1 Alleviates Angiotensin Induced Endothelial Injury and Cerebral Microbleed Via Mitochondrial Protection

Cheng-Cheng Li Third Military Medical University: Army Medical University Xiao-Qin Tang Third Military Medical University: Army Medical University Jie Wang Third Military Medical University: Army Medical University Hai-jun Duan Third Military Medical University: Army Medical University Min Xia Third Military Medical University: Army Medical University Chao Guo Third Military Medical University: Army Medical University Yu-jie Chen Third Military Medical University: Army Medical University Bo Wang Third Military Medical University: Army Medical University Yong-jie Chen Third Military Medical University: Army Medical University Yi Yin Third Military Medical University: Army Medical University Wei-Xiang Chen Third Military Medical University: Army Medical University Hua Feng (  fenghua8888@vip.163.com ) Army Medical University https://orcid.org/0000-0003-4489-9217


Introduction
Cerebral small vessel disease (CSVD) is a neglected but very serious disease in modern society that is characterized by cognitive dysfunction, microbleeds, and white matter hyperintensities [1]. CSVD is related to many diseases, such as stroke, Alzheimer's disease (AD), vascular dementia, and depression [2][3][4]. The incidence of CSVD in people over 60 years old is 6-10 times higher than that of large vessel stroke [4]. Although abundant clinical data on CSVD are available, relatively few basic studies have been conducted. One of the reasons is that no animal model coincides with the pathological manifestations of CSVD. In my previous study, we found that mice infused with Ang for 28 days lead to CSVD phenotype coincided with the early clinical manifestation of the patients [5]. Due to a lack of understanding of its pathogenesis, effective treatment methods have not been developed. Stroke caused by CSVD accounts for 25% of cases, and CSVD increases stroke accidents and worsens the outcome [6,7].
Microbleeds are an independent risk factor for stroke and result from the exudation of red blood cells caused by endothelial cell injury and iron deposition in brain tissue. Therefore, microbleeds are a characteristic of vascular injury. Microbleeds are closely related to cognitive impairment [8], which is a consequence of endothelial dysfunction. The causes of cognitive impairment in individuals with CSVD are mainly vascular factors, such as hypertension, ageing, obesity, and diabetes.
Endothelial cell dysfunction is considered the rst stage of CSVD [9]. Damage to endothelial cells leads to blood-brain barrier (BBB) leakage, promotes in ammatory cell in ltration into the brain parenchyma, and triggers neuroin ammatory reactions [10]. Endothelial dysfunction seriously affects the function of vascular nerve coupling and cognitive function [11]. The brain is one of the organs that consumes the largest amount of energy and accounts for 20% of oxygen consumption, especially in the learning and memory period [12]. Based on accumulating evidence, mitochondrial dysfunction plays a crucial role in endothelial cell injury in patients with cardiovascular and cerebrovascular diseases [13,14]. Although endothelial cells consume less energy than cardiomyocytes and skeletal muscle cells, they also need a large amount of energy to maintain cognitive function. The mitochondrial content of endothelial cells that comprise the BBB is 2-4 times higher than that of endothelial cells in other locations to control the BBB integrity [15]. Furthermore, the production of mitochondrial reactive oxygen species (ROS) induced by mitochondrial dysfunction are an important factor contributing to endothelial dysfunction [16]. Mitochondrial membrane potential (MMP) depolarization results in excess mitochondrial ROS production [17]. As reported in our previous study, the mitochondrial protective drug nicotinamide riboside rescues CSVD, which provides evidence that targeted mitochondrial therapy is a feasible strategy [5].
Therefore, we speculate that mitochondria in endothelial cells play a critical role in the process of CSVD.
Cav-1 is the main protein in caveolae, a membrane structure that invaginates into a ask shape on the cell membrane. Caveolae are rich in sphingomyelin and cholesterol, known as lipid rafts [18,19].
Their size is approximately 50-100 nm, and evidence has shown that vesicle transport is abundant in endothelial cells [20]. Caveolae-mediated vesicle transport is involved in the transport and signal transduction of many macromolecules [21]. Cav-1 not only dominates the function of the BBB but is also critical for maintaining cognitive function [22,20]. The induction of Cav-1 expression effectively improves the cognitive function of individuals with AD, stroke, diabetes, and other diseases [23][24][25]. Cav-1 is involved in the release of NO from vascular endothelial cells [26] and is related to the synthesis of neurotrophic factors. Both of these processes are associated to the function of the neurovascular unit. The dysfunction of the neurovascular unit leads to the dysfunction of neurovascular coupling and eventually leads to cognitive impairment [27]. Cav-1 is signi cantly related to mitochondrial function [28]. The absence of Cav-1 leads to mitochondrial dysfunction, MMP depolarization and premature cell ageing [29]. However, the function and role of Cav-1 in the mitochondria of endothelial cells remain unclear, and thus we plan to investigate microbleeds and cognitive function in a model of CSVD by regulating Cav-1. We suspect that Cav-1 is essential for mitochondria to protect endothelial function.

Results
Ang induces cerebral microbleeds and cognitive dysfunction in mice.
We tested the cognitive function of mice after modelling by performing the nesting experiment, and the nesting ability of mice in the Ang group was signi cantly decreased compared with the control group ( Fig.1 A-B). We used Prussian blue staining to detect cerebral microbleeds in mice after 4 weeks of perfusion of Ang . The microbleeds in the cortex signi cantly increased ( Fig.1 C-D). Endothelial dysfunction is associated with cognitive function and microbleeds. We examined abnormal endothelial cells around blood vessels using immuno uorescence staining to provide support for this concept. Propidium Iodide (PI) staining showed that endothelial nucleus was wrinkled signi cantly in Ang group ( Fig.1 E-F). The sequence of endothelial dysfunction is BBB disruption. The immuno uorescence data showed that the brin leaked from blood vessels in Ang mice ( Fig.1 G-H).
Ang increases the expression of Cav-1 in blood vessels.
Cav-1, which is considered an important molecule for neurovascular coupling function, is expressed at high levels in endothelial cells. We observed that Cav-1 was mainly co-labeled with blood vessels by immuno uorescence, and the uorescence intensity increased in Ang group compared with control group (Fig.2 C-D). Western blot results con rmed that the expression of Cav-1 was signi cantly upregulated ( Fig.2 A-B). We speculate that Cav-1 might play an essential role in the process of CSVD.
We rst analyzed the RNA-Seq data from the endothelial cell line bEnd.3 after Cav-1 knockdown in vitro to explore the role of Cav-1 in endothelial cells. In addition to the classical effects on cytoplasm and cell membrane, the GO (gene ontology) term was enriched in mitochondrial function (Fig3. A). KEGG enrichment revealed that Cav-1 knockdown mainly changed the function about cellular community, cell growth and cell death, which provided a view to focus mitochondrial function (Fig3. B). We screened the rst 16 differentially expressed genes. The heatmap showed that Ccn1, Nptx1, Cav-1, and Stfa2 were the most signi cantly altered genes (Fig3. C). Then, we analyzed the genes whose expression was signi cantly changed at log2>2. The results showed that 25% of the genes were related to mitochondrial function (Fig3. D). Similarly, the genes related to the mitochondrion showed the greatest differences in expression, accounting for 75%.

Cav-1 Knockdown induces mitochondria oxidative injury
We detected mitochondrial function after Cav-1 knockdown by performing live cell staining to obtain more insights into the relationship between mitochondria and Cav-1. Western blot analysis con rmed Cav-1 activation decreases cognitive dysfunction and cerebral microbleeds induced by Ang We injected the Cav-1 activating peptide AP-Cav-1 into mice to further determine the role of Cav-1 in CSVD in vivo. After treatment for 28 days, we performed nesting experiments and found that the nesting function of the mice in the AP-Cav-1 group was signi cantly improved compared with that of the Ang group (Fig6. A-B). Additionally, using Prussian blue staining, we found that the microbleeds of the mice treated with AP-Cav-1 were decreased compared with those of the Ang group (

Discussion
With the increasing development of social economics, the incidence of CSVD has hardly increased. In this study, we identi ed a protective role for Cav-1 in CSVD by maintaining the mitochondrial function of endothelial cells. In an in vitro study, we found that Cav-1 knockdown leads to mitochondrial abnormalities. After overexpression of Cav-1 in endothelial cells, we observed that the mitochondrial dysfunction induced by Ang was signi cantly reversed. Overall, Cav-1 is an endogenous molecule that protects against mitochondrial dysfunction and endothelial dysfunction in CSVD.
Microbleeds and cognitive impairment are considered two important pathological manifestations of CSVD, and microbleeds are the precursor of future bleeding [30]. Vascular dementia accounts for 45% of all types of dementia, and cognitive impairment can cause and aggravate AD [31]. The common cause of these two different pathological processes is endothelial cell dysfunction. We focused on endothelial cells and observed microbleeds, cognitive dysfunction, and endothelial dysfunction in this model. In the present study, the expression of Cav-1, which was originally expressed at relatively high levels in endothelial cells, was upregulated substantially in the CSVD model. First, we postulated that the upregulation of Cav-1 caused endothelial cell damage, but when we knocked down Cav-1, endothelial dysfunction was more serious. We propose that the upregulation of Cav-1 expression exerted an endogenous protective effect.
Clinical data have indicated that microbleeds are robustly relevant to stroke and are a potential risk factor for intracerebral [32]. Both microbleeds and cognitive dysfunction suggest endothelial dysfunction.
Although clinical trials have been carried out on some drugs, such as antihypertensive therapy, statin therapy and antiplatelet therapy, treatments for hypertension do not improve cognitive impairment [33]. Furthermore, treatment with statins and antiplatelet drugs has the risk of bleeding transformation [34][35][36][37]. Although these treatments are all methods to protect endothelial cells, their ineffectiveness may be due to incorrect treatment targets. A better treatment for CSVD is urgently needed.
Here, we observed signi cantly increased expression of Cav-1 in endothelial cells. Cav-1 has been reported to be closely related to cognitive function in many diseases; for example, Cav-1 de ciency causes tentacle reaction abnormalities, which cooperate with eNOS function [22]. Overexpression of Cav-1 in the hippocampus of adult or old rats signi cantly improves cognitive function. Upon Cav-1 activation, the TrkB receptor is expressed in the hippocampus and relocated to the cell membrane for its activation, which improves cognitive function [38]. On the other hand, depletion of Cav-1 upregulated the expression of the amyloid precursor protein and BACE-1, increasing the β-amyloid Aβ42/40 ratio and hyperphosphorylated tau species, which coincide with the pathological manifestations of AD [23]. In previous studies, researchers found that the function of Cav-1 is associated with mitochondrial function. In broblasts, Cav-1 knockdown or knockout decreased mitochondrial respiration, reduced the activity of complex I, inactivated SIRT1, and decreased the NAD+/NADH ratio, which contributed to premature cell senescence [29]. AP-Cav-1, known as the CSD peptide (amino acids 82 to 101 of Cav-1), has been applied to individuals with many diseases and exerts protective effects. In the present study, CSVD mice treated with AP-Cav-1 for 28 days exhibited improved cognitive function in the nesting test and decreased the microbleeds in the brain. Furthermore, after the intervention, the number of PI-labelled endothelial cells were decreased. The CSD peptide treatment might be a new treatment strategy for CSVD, but further clinical study is needed to con rm this hypothesis.
Endothelial dysfunction initiates CSVD [39]. Endothelial dysfunction mainly in uences cognitive function and BBB integration. Moreover, Cav-1 is critical for mitochondrial function. Cognitive dysfunction is an energy metabolism disorder. According to a previous study, Cav-1 and eNOS dominate neurovascular coupling, which requires abundant energy. Using two-photon microscopy, Brian W. Chow et al. found that Cav-1 is one of the two proteins that regulates blood ow changes in the tentacle reaction [22]. In the present study, we found that Cav-1 plays a critical role in endothelial mitochondrial function, which might explain why Cav-1 plays a crucial role in cognitive function. On the other hand, BBB maintenance is supported by retaining Cav-1-mediated vesical transport. Here, knockdown with Cav-1 led to an abnormal MMP and increased mitochondrial ROS. A recent study reported that treadmill excise increases the expression of Cav-1 to protect mitochondrial function and improve the outcome of disease models. By analyzing the RNA-Seq data, we found that Cav-1 de ciency mostly in uenced the mitochondrial process. Furthermore, 75% of the molecules related to mitochondrial function were signi cantly altered. In a subsequent study, Cav-1 overexpression reduced Ang -induced MMP depolarization and decreased mitochondrial ROS generation. However, we did not explore the molecular mechanism underlying the effects of Cav-1 on mitochondria. We will explore the molecular mechanism of changes in mitochondria mediated by Cav-1 in future studies.

Conclusions
We found for the rst time that the expression of Cav-1, an endogenous protective molecule, was upregulated in a CSVD model. We con rmed that Cav-1 was associated with cognitive function and microbleeds. Mechanistically, Cav-1 was critical for maintaining mitochondrial function. We infer that Cav-1 plays a key role in CSVD. It may be a target for the treatment of CSVD, and further clinical data are needed to prove this hypothesis.

Animals
Adult male C57BL/6 mice were used in this study. The mouse model was induced by subcutaneously implanting osmotic minipumps (ALZET® Osmotic Pumps, DURECT Corporation, Cupertino, CA 95041) as previously described [40]. Brie y, the hair of anaesthetized mice was removed from the back, and the skin was disinfected with iodophor and cut with scissors. The osmotic minipumps containing Ang (1000 ng/kg/min, purchased from Absin, Shanghai Hai, China, abs811565) or saline were implanted for 28 days, and the skin was sutured. AP-Cav-1(2mg/kg, synthesized by Sino Biological) was injected intraperitoneally every two days for 28 days. The experimental procedures were approved by the Laboratory Animal Care and Use Committee of the Army Medical University, China. The mice were fed with food and water under a 12 hours light / dark cycle.

Immunoblotting
Western blot methods were performed as previously described [5]. Brie y, extracted brain protein was loaded onto an SDS-PAGE gel and then transferred to a polyvinylidene di uoride (PVDF) membrane. Membranes were blocked with 5% BSA at room temperature for 2 hours. Then, membranes were incubated with primary antibodies overnight at 4°C. After that, the membranes were washed with TBS containing 0.1% Tween-20 for 3 times and then incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Immunoreactive bands were detected using a chemiluminescence reagent kit (Thermo Scienti c, IL, USA, 34580). The primary antibodies used were as follows: Cav-1 (rabbit, 1:1000 CST, 3267S) and GAPDH (mouse, 1:1000, Proteintech 60004-1-Ig). An HRPconjugated anti-rabbit secondary antibody (rabbit, 1:5000, Proteintech SA00001-2) was used. Images were photographed and analysed using Image Lab software (Image Lab 3.0; Bio-Rad).

RNA-Seq
The protocol is from LC-Bio. Brie y, total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA, 10296010) according to the manufacturer's procedure. The total RNA quantity and purity were analyzed with a Bioanalyzer 2100 and RNA 6000 Nano Lab Chip Kit (Agilent, CA, USA) with a RIN number >7.0. Approximately 10 µg of total RNA extracted from a speci c adipose tissue type was subjected to isolation of poly(A) mRNA with poly-T oligo-conjugated magnetic beads (Invitrogen). Following puri cation, the mRNA was fragmented into small pieces using divalent cations at an elevated temperature. Then, the cleaved RNA fragments were reverse transcribed to create the nal cDNA library in accordance with the protocol for the mRNA Seq sample preparation kit (Illumina, San Diego, USA), and the average insert size of the paired-end libraries was 300 bp (±50 bp). Then, we performed paired-end sequencing on an Illumina sequencing platform. provided by manufacturer. In brief, 1*10 4 cells were seeded onto 96-well plates. Then cells were treated with Ang in 1μm for 12 or 24 h. After that, cells were exposed in 10μl CCK-8 for 3 h. The absorbance at 450 nm was measured by microplate.

Mitochondrial Membrane Potential detection
The mitochondrial membrane potential was detected using tetramethylrhodamine (TMRM, Invitrogen, I34361). bEnd.3 cells (1*10 5 ) were plated in a dish for confocal microscopy. After removing the medium, the cells were incubated with TMRM (25 nm) for 30 min at 37°C, and the nuclei were stained with Hoechst (Invitrogen, R37165). The samples were observed with a laser-scanning confocal microscope (Zeiss, LSM780, Germany) and analyzed using ZEN2012 software.

Mitochondrial ROS detection
Mitochondrial ROS were measured using MitoSOX Red (Molecular Probes, USA, M36008), a mitochondrial superoxide indicator. At the end of the experiment, the medium was removed, the cells were washed with PBS and stained with 5 μM MitoSOX Red for 10 min in a humidi ed atmosphere of 5% CO2 at 37°C, and the nuclei were stained with Hoechst (Invitrogen). After washes with PBS, cell sampling was performed using a confocal microscope (Zeiss LSM 780, Germany).

Behavioral test
Nesting experiment: The nesting experiment was performed as described previously [42]. On the 27th day, a 10 cm × 10 cm square cotton cloth was prepared and placed in the feeding cage (each mouse was placed in a cage separately) at 7 pm. The utilization rate of cotton cloth and nesting situation of mice were observed in the morning of the next day. According to the scoring standard, the scores were 0, 1, 2, 3, 4 and 5.

Statistical Analysis
All results are presented as the means ± standard errors of the means (SEM). Prism 6 software was used for statistical analysis, and one-way ANOVA and Tukey's test were used to analyze data from the three groups. Signi cant differences are denoted as * P < 0.05, ** P < 0.01 and *** P < 0.001; NS, not signi cant.

Declarations
Corresponding author Correspondence to Hua Feng.

Ethics declarations
The experimental procedures were approved by the Laboratory Animal Care and Use Committee of the Army Medical University and performed with the guidelines of Animal Use and Care of the National Institutes of Health.

Informed Consent
Not applicable.

Con icts of interest
The authors declare no competing interests.

Consent for publication
Not applicable.

Data Availability
Data are available on reasonable request