Loss of Endothelial Annexin A1 Aggravates Inflammation‐Induched Vascular Aging

Abstract Chronic inflammation is increasingly considered as the most important component of vascular aging, contributing to the progression of age‐related cardiovascular diseases. To delay the process of vascular aging, anti‐inflammation may be an effective measure. The anti‐inflammatory factor annexin A1 (ANXA1) is shown to participate in several age‐related diseases; however, its function during vascular aging remains unclear. Here, an ANXA1 knockout (ANXA1−/−) and an endothelial cell‐specific ANXA1 deletion mouse (ANXA1△EC) model are used to investigate the role of ANXA1 in vascular aging. ANXA1 depletion exacerbates vascular remodeling and dysfunction while upregulates age‐ and inflammation‐related protein expression. Conversely, Ac2‐26 (a mimetic peptide of ANXA1) supplementation reverses this phenomenon. Furthermore, long‐term tumor necrosis factor‐alpha (TNF‐α) induction of human umbilical vein endothelial cells (HUVECs) increases cell senescence. Finally, the senescence‐associated secretory phenotype and senescence‐related protein expression, rates of senescence‐β‐galactosidase positivity, cell cycle arrest, cell migration, and tube formation ability are observed in both ANXA1‐knockdown HUVECs and overexpressed ANXA1‐TNF‐α induced senescent HUVECs. They also explore the impact of formyl peptide receptor 2 (a receptor of ANXA1) in an ANXA1 overexpression inflammatory model. These data provide compelling evidence that age‐related inflammation in arteries contributes to senescent endothelial cells that promote vascular aging.


Blood pressure measurement
Mice were placed in a heating cylinder, which was covered with a net to confine the mice and keep them warm.Inside the sleeve, blood pressure was measured via the tail with a noninvasive tail sphygmomanometer (Softron, China).Measurements were repeated at least 3 times to ensure stability.The mean systolic blood pressure (SBP), mean diastolic blood pressure (DBP), mean blood pressure and HR were obtained from the machine.The PP was calculated as the difference between the mean SBP and mean DBP.

PWV measurement
Mice were anesthetized by intraperitoneal administration of 1% pentobarbital sodium and fixed on the heating platform along with the electrocardiography (ECG) apparatus.
Given the distance, two probes were used simultaneously to acquire Doppler velocity spectrograms for the aortic arch and abdominal aorta separately by a Doppler Flow Velocity System (Indus, USA).The electrocardiographic signal was acquired for measurement of the time interval, and the distance was measured by a ruler.PWV(m/s) =Distance(mm)/Time(ms).

Aortic function assessment
A wire myograph system (DMT620M, ADInstruments, Australia) was used for the assessment of aortic function.The tissue around the vascular rings was removed, and vessels were mounted onto the wire in a gas-filled chamber (95% O2 and 5% CO2) at 37°C.A force of 6 mN was applied to maintain an initial tension recording with LabChart software (version 8).After three equilibration cycles times (20 minutes each), the vessels were subjected to two cycles of contraction by the application of 60 mм high-potassium physiological saline solution.The aortic rings were constricted by the application of PHE (10 -6 м; Sigma-Aldrich, St. Louis, MO, USA) and relaxed by the application of ascending concentrations of ACH, (10 -10 -10 -4 м; Sigma-Aldrich, St. Louis, MO, USA), which simulated endothelial-dependent vasodilation.PHE (10 -10 to 10 -6 м) was used to study the constriction of the vascular rings.To evaluate endothelium-independent vasodilation, Nω-nitro-L-arginine methyl ester (L-NAME; 10 - 5 м; Beyotime, Shanghai, China) was applied before induction of constriction by PHE (10 -6 м), after the cumulative addition of SNP (10 -9 -10 -5 м; Sigma-Aldrich, St. Louis, MO, USA).

Details of animal studies
The heterozygotes were purchased from GemPharmatech (Nanjing, China).The offspring of heterozygotes were identified by PCR with nucleic acid gel imaging using DNA extracted from mouse tails with a one-step mouse genotyping kit (Vazyme, China).
Mice were perfused with normal saline through the left ventricle, and the thoracic aorta was immediately harvested and placed in physiological saline solution (PSS) to preserve endothelial activity.The portion of the thoracic aorta located immediately before the aortic arch was used for the measurement of vascular systolic and diastolic function 20 .The rest of the aorta was fixed with 4% paraformaldehyde overnight.
First, slides containing fixed thoracic aorta tissue were sequentially subjected to HE staining, VVG staining, and incubation with antibodies specific for P53, P21, TNF-α and IL-6 for immunohistochemical staining.Abdominal aortas were harvested in liquid nitrogen and stored at -80°C for western blotting.The other 6 mice per group were dissected for harvesting of the thoracic aorta for SA-β-gal staining.Cardiac tissues, with the exception of the aorta, were harvested, and the cardiac apex was stored at -80°C for western blotting, while the rest of the tissues were fixed with 4% paraformaldehyde.The first slide was used for HE staining, and the second slide was used for CD31 immunohistochemical staining.The median lobe of the liver was fixed for paraffin sectioning, the anterior right lobe was fixed for cryosectioning, and the left lateral lobe was frozen at -80°C for western blotting.The left side of epididymal fat, dorsal brown fat, kidney and dorsal skin were obtained for western blotting, while the corresponding right side were obtained for paraffin sectioning.

Enzyme-linked immunosorbent assay (ELISA)
The concentration of ANXA1 in serum from healthy donors was measured by ELISA kits (ab222868, Abcam, UK).The standard and samples were prepared prior to incubation at room temperature and were incubated for 2 hours in wells.The wells were then washed five times, and a biotinylated anti-ANXA1 antibody was added for 1 hour.
After washing as described above, the wells were incubated with the streptavidin-peroxidase conjugate and chromogen substrate for 30 minutes and 15 minutes, respectively.After stop solution was added, the absorbance at 450 nm was measured using a plate reader (Bio-Rad, Hercules, CA, USA).The concentration of ANXA1 in mouse serum was quantified according to the manufacturer's protocols (37) (MBS9407240, MyBioSource, USA).MCP-1 (MJE00B, R&D Systems, Australia) and CRP ELISA kits (MCRP00, R&D Systems, Australia) were used for quantification according to the manufacturer's instructions.

Histopathological examination
Thoracic aortas fixed with 4% paraformaldehyde overnight were deparaffinized with xylene and hydrated in water with decreasing concentrations of ethanol.A HE kit (Servicebio, Wuhan, China) and VVG staining (Servicebio, Wuhan, China) were used to evaluate the vascular wall thickness and elastin breaks along with the collagen area.
The MT, MA, MT/LD and percentage of collagen area (collagen/MA) were calculated with IPP7.0 software according to the methods described previously(5).

Immunohistochemical staining
Four-micrometer sections of paraffin-embedded thoracic aortas were deparaffinized with xylene and hydrated with decreasing concentrations of ethanol (100% to 50%).

Multiplex immunofluorescence staining
A tyramide signal amplification (TSA) kit (Servicebio, Wuhan, China) was applied for multiplex immunohistochemical staining, which was performed to localize and quantify the expression of ANXA1 in the aortas of mice at different ages.Fourmicrometer sections of paraffin-embedded thoracic aortas were deparaffinized and hydrated according to the steps as described above.After removal from ethylenediaminetetraacetic acid (pH=9.0), the aortic sections were blocked in 3% bovine serum albumin (BSA) for at least 1 hour at room temperature and incubated with the primary antibody at 4°C overnight.A horseradish peroxidase (HRP)conjugated secondary antibody was then applied for 50 minutes at room temperature and was replaced by TSA staining solution.A microwave was used for antibody removal.The other primary antibodies were applied according to the manufacturer's instructions.

Masson Trichrome Assay
4μm sections of paraffin-embedded skin sections were dewaxed to water as previous described.Masson Trichrome staining was performed with Masson Stain Kit (Solarbio, China) according to the manufacturer instructions.Collagen fibers were stained for blue and muscle fibers were stained for red.

Culture and identification of HUVECs
Primary HUVECs were purchased from ScienCell Research Laboratories (8000).
Beginning at passage two, cells were characterized by immunofluorescence with specific antibodies against CD31 (1:100, Abcam, UK).
At 20-30% confluence, the HUVECs were transduced with 2 μL of the sh-ANXA1 lentiviral vector (2×10 8 TU mL -1 ) or 1 μL of the ANXA1 overexpression lentiviral vector (1×10 8 TU mL -1 ) and 20 μL of enhanced infection solution (HitransG A, REVG004, Genechem, Shanghai) to downregulate or upregulate ANXA1 expression, respectively.Fresh medium was replaced 12 hours later.Forty-eight hours after infection, 1 μg mL -1 puromycin (S250J0, BasalMedia) was added to the culture medium, and the concentration was maintained at 0.5 μg/mL for at least 7 days for selection of stably transduced cells.The infection rate was determined by the observation of green fluorescence in nearly 90% of the cells 72 hours after infection.Uninfected cells and empty lentiviral vector were used as controls.Cells from passage 2 to passage 8 were used for transduction.
Thoracic aortas were fixed with 4% paraformaldehyde overnight after harvesting.After washing twice with PBS, the aortas were dipped in fresh SA-β-gal staining solution and incubated at 37°C overnight.The aortas were then washed with PBS three times and photographed by a digital camera (Canon, Japan).The area percentage of positive staining was analyzed by ImageJ 1.51 software.
HUVECs seeded in 6-well plates were washed once with PBS (B320KJ, BasalMedia) to remove cell debris, fixed for 15 minutes in fixative solution and washed 3 times with PBS.The cells were then incubated with fresh SA-β-gal staining solution at 37°C for 12 hours.The percentage of senescent cells was determined under an inverted light microscope (TS100, Nikon, Japan) by random selection of 5 microscopic fields of view at 10× and 20× magnification and was then calculated by ImageJ 1.51 software.

Cell cycle analysis
The cell cycle in HUVECs was analyzed by flow cytometry in an Accuri C6 Plus instrument (BD Biosciences, China) with a cell cycle detection kit (KGA512, KeyGen BioTech, Jiangsu, China) according to the manufacturer's guidance.A total of 1×10 6 cells were harvested and centrifuged for 5 minutes at 2000 rpm.Precooled PBS was used for resuspension.The cells were fixed with 70% alcohol at 4°C overnight after another centrifugation step.The cells were washed with cold PBS twice for 3 minutes each by centrifugation at 1000 rpm and filtered through a 200-mesh strainer.Fresh propidium iodide (PI) staining solution was added to the cells at 4°C for 30 minutes in the dark.A total of 1×10 4 cells were selected at a rate of no more than 60 cells/minute.
Data were analyzed by ModFit 4.1.5software.

Wound healing assay
Six-well plates were marked at intervals of 0.5 cm on the opposite side to delineate the area to be photographed.HUVECs were seeded in the 6-well plates (5×10 5 cells/well) and incubated overnight.A 200 μL pipette tip was used to generate a wound.After washing with PBS, the wound was randomly photographed in 5 areas under an inverted microscope at 4× magnification.Incubation at 37°C was continued, and the wound was photographed at 24 hours and 48 hours.ImageJ 1.51 software was used for analysis of the repopulated area.

Tube formation assay
Matrigel (Corning, USA) was thawed on ice for 2 hours, and all pipettes and plates were precooled before the experiments.Matrigel was diluted 1:1 with ECM and added carefully into a prerinsed 24-well plate (200 μL/well).After incubation for 2 hours at 37°C in an incubator, the Matrigel had solidified, and 45000 trypsinized HUVECs were then seeded onto the Matrigel.After 4-6 hours of incubation in a 37°C incubator, tube formation was observed by microscopy and analyzed by ImageJ 1.51 software by determining the number of tubes.

qRT-PCR
RNA was isolated from HUVECs with a TransZol Up Plus RNA Kit (ER501-01, TransGen Biotech, Beijing, China) following the manufacturers' instructions.
Quantitative RNA was used for cDNA synthesis with cDNA Synthesis SuperMix (11141ES60, Yeasen, Shanghai, China).SYBR Green Master Mix (11202ES08, Yeasen, Shanghai, China) was applied to quantify the mRNA expression of GAPDH, ANXA1, P53, P21, P16, VCAM-1, ICAM-1, Iκ B,SERPINE 1,and TGFβ 2. The CT values were compared with that of GAPDH h using the following formula: Fold change=2 -ΔΔct , Δct=ct (ct value of the target gene)-ct (ct value of the reference gene) and ΔΔct=Δct (ct value of the experimental group)-Δct (ct value of the control group).The primers used to amplify all the genes listed above were designed and synthesized by Sangon Biotech Company (Shanghai, China).

4Figure S1 .
Figure S1.Correlation of ANXA1 expression in serum from healthy humans with

Figure S9 .
Figure S9.Age-related and inflammatory mRNA was elevated in TNF-α group：

Figure
Figure S10.ANXA1 knockdown facilitated the senescence and inhibited the

Figure S11 .
Figure S11.Function compromised was observed in ANXA1 knockdown