FLRT2 plays a critical role in endothelial cell senescence and vascular aging

doi:10.21203/rs.3.rs-2233081/v1

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FLRT2 plays a critical role in endothelial cell senescence and vascular aging

https://doi.org/10.21203/rs.3.rs-2233081/v1

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The roles of fibronectin leucine-rich transmembrane protein 2 (FLRT2) in physiological and pathological processes are poorly known. Here, we identify a novel function of FLRT2 in preventing endothelial cell senescence and vascular aging. We found that FLRT2 expression was lower in cultured senescent endothelial cells as well as in aged rat and human vascular tissues. FLRT2 silencing in human endothelial cells induced senescence through mTORC2, but not mTORC1, AKT, and p53. We uncovered that FLRT2 directly associated with ITGB4 and thereby promoted ITGB4 phosphorylation, while inhibition of ITGB4 significantly mitigated the induction of senescence triggered by FLRT2 depletion. Importantly, FLRT2 silencing in mice promoted vascular aging and overexpression of FLRT2 rescued a premature vascular aging phenotype. We propose that FLRT2 could be targeted therapeutically to prevent senescence-associated vascular aging.

Subject terms: FLRT2, ITGB4, mTORC2, endothelial cell senescence, vascular aging

FLRT2

ITGB4

mTORC2

endothelial cell senescence

vascular aging

Cellular senescence is characterized by indefinite loss of proliferation potential. Replicative senescence occurs following critical telomere shortening after cells undergo extended rounds of division^1,2. Premature senescence is triggered by a range of acute intrinsic and extrinsic signals, such as strong mitotic stimuli, oxidative stress, and DNA damage^3,4. Major signaling pathways driving cellular senescence are the p53/p21 and the RB/p16 tumor suppressive programs⁵. Senescent cells exhibit distinctive characteristics, such as an enlarged and flattened morphology, senescence-associated β-galactosidase (SA-β-Gal) activity, and altered gene expression⁶. Although senescent cells do not divide, they are metabolically active and secrete many proteins to the extracellular space; a vast array of secreted chemokines, cytokines, growth factors, and matrix metalloproteases comprise the senescence-associated secretory phenotype (SASP)⁷. While senescent cells are beneficial during development and wound repair, their accumulation in tissues has been associated with many age-related declines and diseases⁸.

Senescent endothelial cells are linked to age-related vascular dysfunction, which leads to arterial stiffness, atherosclerosis, and stroke^9,10. Proinflammatory or atherosclerotic factors, such as tumor necrosis factor-alpha (TNFα), oxidized low-density lipoprotein (ox-LDL), and/or H₂O₂ can cause damage in human endothelial cells and promote premature senescence^11,12. Furthermore, SASP factors secreted from senescent endothelial cells, such as nitric oxide (NO), matrix metalloproteinases (MMPs), interleukin (IL)1, and IL8, can trigger several cellular processes including inflammation, apoptosis, and changes in cytoskeletal integrity^13,14. Accordingly, senescence of endothelial cells induced by different stimuli can influence vascular function and modulate pathogenesis of age-related vascular disease. Therefore, understanding the molecular mechanisms underlying endothelial cell senescence can help establish strategies for preventing senescence-associated vascular diseases.

FLRT2 is a member of the highly conserved fibronectin leucine-rich repeat transmembrane family of proteins^15,16. Structurally, FLRT2 comprises a short intracellular region, a type-III fibronectin-like domain, and 10 extracellular leucine-rich domains in humans^17–19. FLRT2 has been reported to promote cell proliferation and mediate cell-cell interactions in chondroprogenitor cells²⁰ and acts as the predominant ligand of UNC5B during vascular morphogenesis and placental labyrinth formation²¹. These reports suggest that FLRT2 is involved in cell cycle regulation and/or vascular development, although relatively little research has focused directly on FLRT2.

Here, we report that FLRT2 prevents endothelial cell senescence and vascular aging through a signaling program that includes ITGB4, mTORC2, AKT and p53.

Cell culture

Human umbilical vein endothelial cells (HUVECs), endothelial colony-forming cells (ECFC), human microvascular endothelial cells (HMVECs), and human alveolar epithelium cells (HAECs) were cultured in endothelial cell growth basal medium-2 (Lonza) supplemented with 2% fetal bovine serum, human FGF-β, vascular endothelial growth factor, R3-insulin growth factor-1, human endothelial growth factor, hydrocortisone, ascorbic acid, heparin, gentamicin, and amphotericin-B (Lonza). HMVECs were cultured in microvascular endothelial cell growth medium BulletKit (Lonza).

Reagents and antibodies

Antibodies recognizing pRb, phospho-pRb, cleaved PARP, phospho-SRC (Y416), p21, p16, mTOR, phospho-mTOR (S2448), phospho-mTOR (S2481), phospho-ERK1/2, phospho-AKT (T308), phospho-AKT (S473), phospho-NDRG1 (Thr346), phospho-p70S6K (Thr389), and phospho-SIN1 (Thr86) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies recognizing flotillin-1, cyclin D1, cyclin A, CDK2, CDK4, AKT, phospho-PKCα (Ser657), integrin β1, integrin β3, and integrin β5 were from Santa Cruz (Dallas, TX, USA). Antibodies recognizing clathrin and caveolin-1 were purchased from Biosciences (San Diego, CA, USA). Antibodies recognizing p53, actin, and CD31 antibodies were from Leica Biosystems (Wetzlar, Germany), Abcam (Richmond, BC, Canada), and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Antibodies recognizing integrin β4, integrin α6, Raptor, Rictor, and FLRT2 were from Abcam (Cambridge, UK), while an antibody recognizing phospho-integrin β4 (Y1510) was from Assay Biotechnology (Sunnyvale, CA, USA). The anti-integrin β4 antibody (clone ASC-8) for blocking integrin β4 activity was purchased from Millipore (Charlottesville, VA, USA). Rapamycin, LY294002, and PD098059 were purchased from Millipore. Torin was purchased from Selleckchem (Houston, TX, USA).

SA-β-Gal staining

Cells were washed with 1X phosphate-buffered saline (PBS), fixed in 3.7% formaldehyde for 5 min, and washed with PBS. The fixed cells were then incubated with a solution containing 1 mg/mL of 5-bromo-4-chloro-3-indolyl B-D-galactoside (X-Gal), 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl₂ for 16 h at 37°C. The cells were then washed and imaged using a microscope (Olympus CKX41; Olympus Shinjuku, Tokyo, Japan).

Human arterial tissue samples

Human vascular tissue sections of the splenic arteries were obtained from atherosclerosis patients at Yeungnam University Hospital (Daegu, Republic of Korea) aged 1–90 years (grouped in 10-year age intervals; n = 20 for each group) consenting to surgical excision from 1995 to 2012 with Internal Review Board approval (YUMC 2015-01-017).

Immunoblotting analysis

The cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitors (Sigma-Aldrich, St Louis, MO, USA). Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred to a nitrocellulose membrane, which was then blocked with 3% non-fat dried milk or 3% bovine serum albumin (BSA) and incubated with the primary antibodies at 4°C overnight. Next, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. The protein bands were developed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific) and visualized after exposure to X-ray film (Agfa Gevaert NV, Mortsel, Antwerp, Belgium).

Immunoprecipitation

The cell lysates were prepared using a 1% NP-40 lysis buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% NP-40) containing protease inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich). The lysates were precleared with Sepharose beads coupled with Protein G or A (Sigma-Aldrich), the indicated antibodies added, and tumbled with Sepharose beads coupled with Protein G or A. The immune complexes were then subjected to SDS-PAGE, followed by immunoblotting.

Cell viability

Cells were seeded in 60-mm dishes, cultured for 24 h, and treated with conditioned media (CM) as indicated in the figures for 3 days. Next, the cells were trypsinized and harvested. Cell viability was measured using the trypan blue exclusion assay; cell suspensions were diluted (1:1) with 0.4% trypan blue (GIBCO, Grand Island, NY, USA) and counted using a hemocytometer under a microscope (Olympus CKX41; Olympus).

Cell cycle analysis

Cells were harvested by trypsinization, fixed in 95% ethanol, washed with PBS, resuspended in 1 mL PBS containing 1 mg/mL RNase and 50 mg/mL propidium iodide (Sigma-Aldrich), and incubated in the dark for 30 min. The distribution across cell cycle compartments was analyzed using a fluorescence-activated cell sorting (FACS) Calibur flow cytometer (BD Biosciences, San Diego, CA, USA).

BrdU incorporation assay

Cell proliferation was measured using a BrdU cell proliferation assay kit (Roche Applied Science) following the manufacturer’s instructions. BrdU incorporation was determined by measuring the absorbance at 450 nm using a spectrophotometer.

RNA interference

Cells were transfected with siRNA duplexes using RNAiMAX (Invitrogen, Karlsruhe, Germany). The sequences of the siRNAs (Bioneer Inc., Daejeon, Korea) were as follows:

Con Si: 5′-CCUACGCCACCAAUUUCGUdTdT-3′

FLRT2 Si: 5′-GAGCUUGGUAAAUGUCACUdTdT-3′

FLRT2 Si #2: 5′-CACUCUACACUAUACAGUAdTdT-3′

FLRT2 Si #3: 5′-GCAUUUCUAAAGGCCAUUUdTdT-3′

Mouse FLRT2 Si: 5′-[Cyanine5]CACUCUACACUAUACAGUAdTdT-3′

p53 Si: 5′-CACUACAACUACAUGUGUAdTdT-3′

p21 Si: 5′-CUGUACUGUUCUGUGUCCUdTdT-3′

FGFR Si: 5′-CUCACUGUGGAGUAUCCAUdTdT-3′

SRC Si: 5′-GGCUGAGGAGUGGUAUUUUdTdT-3′

AKT Si: 5′-GACAACCGCCAUCCAGACUdTdT-3′

Raptor Si: 5′-CACCTCACTTTATTTCCATdTdT-3′

Rictor Si: 5′-ACUUGUGAAGAAUCGUAUCdTdT-3′

EPS8L2 Si: 5′-GAGCUUUCUCUGGCCAAGUdTdT-3′

PLVAP Si: 5′-GUGACAACACACACCAUGAdTdT-3′

GPC1 Si: 5′-GUGCUUUGCCUGGCUACAUdTdT-3′

SDC1 Si: 5′-GACUUGGAAGACUCCAUGAdTdT-3′

CACNG6 Si: 5′-CGGCUCUCCCUCUUUCAUAdTdT-3′

EPHB6 Si: 5′-GGUGAAUGUAAGACGCUGUUdTdT-3′

PLCD3 Si: 5′-CCUGGAGAAGACAGUAGAAdTdT-3′

ITGB1 Si: 5′-CAGACAUCAUUCCAAUUGUdTdT-3′

ITGB3 Si: 5′-CAGAUGUCAUUCCAUAUCAdTdT-3′

ITGB4 Si: 5′-GACUUCGUGUGCGGACAGUdTdT-3′

ITGB5 Si: 5′-CUGUUGAAGGUACAUCGUUdTdT-3′

Plasmid transfection

Plasmids were transfected using Lipofectamine 2000 reagent (Invitrogen, Karlsruhe, Germany), following the manufacturer’s instructions. The plasmids expressing pRK5 beta4 and pRK5 beta4 1355 T (ΔCYT) were purchased from Addgene (Cambridge, MA, USA). The plasmids expressing FLRT2 (NM_013231) Human Tagged ORF Clone and FLRT2 (NM_201518) Mouse Tagged ORF Clone were purchased from OriGene (Rockville, MD, USA).

RT-qPCR: reverse transcription (RT) followed by real-time quantitative polymerase chain reaction (qPCR)

Total RNA was extracted using the TRIzol reagent (Invitrogen), following the manufacturer’s instructions and resuspended in diethyl pyrocarbonate-treated water. The RNA was subjected to reverse transcription (RT) using a complementary DNA (cDNA) synthesis kit (Bio-Medical Science Co. Ltd, Daejeon, Korea). The cDNA was then subjected to real-time quantitative (q)PCR analysis using pre-designed gene-specific primers purchased from Bioneer Co., Ltd. (Daejeon, Korea) and iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories).

RNA sequencing data processing and analysis

Total RNA was isolated from HUVECs using TRIzol reagent, resuspended in RNase-free water, and used for RNA-seq analysis by Macrogen (Seoul, Korea). The expression level of each transcript was quantified as fragments per kilobase of transcripts per million mapped reads (FPKMs). The thresholds were set using the absolute value of twofold change to determine significant differences in gene expression. The DAVID gene annotation tool (version 6.8; http://david.ncifcrf.gov/) was used for gene ontology analysis of the genes of interest.

Tube formation assay

Matrigel (BD Biosciences) was polymerized (200 µL/well of a 48-well tissue culture plate) for 30 min at 37°C. Trypsinized HUVECs (4×10⁴) were resuspended in 200 µL of CM and seeded into each well. The cells were cultured for 16 h and images of the tubes were captured using a microscope. The number of branch points and branches was counted, and the tube area was measured using the Fuji Multi Gauge V2.3 software (Fuji, Tokyo, Japan).

Immunofluorescence staining

The cells were fixed with 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked using 3% BSA in PBS for 1 h. Next, the cells were incubated with the primary and fluorescein-conjugated secondary antibodies for 1 h at room temperature. Immunofluorescence was examined using an LSM 510 META laser scanning microscope (ZEISS, Germany).

The Duolink Proximity Ligation Assay

To assess possible physical interaction between FLRT2 and ITGB4, HUVECs were subjected to Duo-link assay with anti-FLRT2 (Abcam) and anti-ITGB4 (Abcam) as primary antibodies, following the manufacturer’s instruction (Sigma-Aldrich). Fluorescent images of the cells were observed under an LSM 510 META laser scanning microscope.

Isolation and characterization of lipid rafts

HUVECs were lysed with 1 mL of lysis buffer (1% Triton X-100, 25 mM HEPES (pH 6.5), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail). Equal amounts of proteins were subjected to discontinuous sucrose gradient ultracentrifugation (40%, 30%, and 5%) using an SW41 Ti rotor (39,000 rpm) for 16 h at 4°C. The sucrose gradients were fractionated into 13 fractions, including the pellet. An opaque buoyant band corresponding to the lipid rafts was collected from the interface between the 30 and 5% sucrose gradients. Aliquots of fractions collected from the bottom of the gradient were analyzed using immunoblotting. The protein concentration was determined using the bicinchoninic acid method (Thermo Fisher Scientific), following the manufacturer’s instructions.

Immunohistochemistry

The tissues were cryosectioned into 20 µm-thick sections, fixed with 3.7% formaldehyde, and permeabilized with 0.01% Triton-X100 for 15 min. The samples were then blocked with 3% BSA for 1 h at room temperature and incubated with rat anti-CD31 (Thermo Fisher Scientific) and anti-FLRT2 (Abcam) diluted (1:50) in blocking solution overnight at 4°C. The sections were washed with PBS and incubated with Alexa Fluor 594 goat anti-hamster IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and Alexa Fluor 488 goat anti-rabbit (Jackson ImmunoResearch Laboratories) diluted (1:50) in blocking solution for 1 h at room temperature. The sections were washed thrice with PBS and mounted on microscopy slides.

Animal experiments

Fifty µg of Cy5–tagged mouse FLRT2 Si or Flag-tagged FLRT2 plasmid were mixed with in vivo-jetPEI® (Polyplus transfection;Illkirch, France), following the manufacturer’s instructions. Cy5–tagged mouse FLRT2 Si was used to assess siRNA distribution in mouse aorta (thoracic) or vein (vena cava). The solution was vortexed gently and left for 15 min at room temperature. The mixture was injected into the tail veins of 8-week-old male mice four times over 14 days, and the treated mice were sacrificed for immunoblotting, SA-β-Gal staining, and immunofluorescence. All animal care and experimental procedures were approved by the INHA University-Institutional Animal Care and Use Committee (INHAIACUC) (Approval Number INHA-200917-722-1).

Statistical analysis

All data are expressed as mean ± standard deviation. The data were analyzed using one-way analysis of variance or Student’s t-test. The differences were considered significant at p < 0.05.

FLRT2 levels decline in senescent endothelial cells and in aged vascular tissues

To investigate a possible association between FLRT2 and endothelial cell senescence, we first examined the expression levels of FLRT2 in replicatively senescent human umbilical vein endothelial cells (HUVEC), endothelial colony-forming cells (ECFC), and human microvascular endothelial cells (HMVEC). Senescence of each endothelial cell type was confirmed by assessing characteristic morphological changes, higher levels of senescence marker proteins (p53, p21, and unphosphorylated pRb), and increased SA-β-Gal activity (Fig. 1a-c). Importantly, relative to proliferating cells, FLRT2 expression levels declined in all three paradigms of senescent endothelial cells. To begin to study if there might be similar trends in FLRT2 expression in older tissues, where senescent cells accumulate, we examined FLRT2 expression levels in rat and human vascular tissues with increasing age. As shown (Fig. 1d), FLRT2 levels decreased dramatically in the aortas of old rats (24 months old) compared to those of young rats (6 months old). In human vascular tissues, p53 and p21 levels increased (Supplementary Fig. 1a), while FLRT2 levels decreased in older tissues, especially in persons > 50 years old (Fig. 1e and Supplementary Fig. 1b). These findings indicate that the expression levels of FLRT2 decline both with senescence in culture and with aging, suggesting connections between the loss of FLRT2 and endothelial cell senescence.

FLRT2 knockdown induces premature senescence of endothelial cells

To examine the potential role of FLRT2 in endothelial cell senescence, we transfected HUVECs with a small interfering RNA (siRNA) directed at FLRT2 mRNA (FLRT2 Si). HUVECs in which FLRT2 was silenced proliferated more slowly than control cells (transfected with Con Si) (Fig. 2a) and expressed proteins consistent with a phenotype of senescence, including hypophosphorylated pRb and elevated p53 and p21, increased SA-β-Gal positivity, and decreased DNA replication as evidenced by the relative levels of cells in proliferative cell cycle phases (S and G2/M) and the reduced incorporation of 5-bromo-2′-deoxy-uridine (BrdU) (Fig. 2a-e). In keeping with earlier reports that senescence decreased the ability of endothelial cells to form tubes in culture²², the tube-forming ability of FLRT2 was also markedly attenuated in FLRT2-silenced cells (Supplementary Fig. 2a). At the same time, FLRT2 knockdown increased the levels of secreted SASP factors (Supplementary Fig. 2b). To rule out the possibility of off-target effects by FLRT2 Si, we transfected cells with other FLRT2 Si RNAs (FLRT2 Si #2 and #3) and found similar effects on endothelial cell senescence (Supplementary Fig. 3). Given the observed rise in p53 and p21 in cells with silenced FLRT2, we studied their involvement in endothelial cell senescence. We discovered that lowering the levels of p53 or p21 abrogated the senescence program elicited by silencing FLRT2 in HUVECs (Supplementary Fig. 4). These findings support a model wherein FLRT2 depletion induces cellular senescence in endothelial cells in a p53/p21-dependent fashion.

FLRT2 regulates endothelial cell senescence through the mTORC2-AKT-p53 pathway

To further investigate the signaling pathways through which loss of FLRT2 triggers senescence, FLRT2-silenced cells were treated with several kinase inhibitors that have been implicated in senescence: LY294002 (a PI3K inhibitor), Rapamycin (an mTORC1 inhibitor), Torin (an mTORC1/2 inhibitor), and PD098059 (an ERK inhibitor). Compared with control cells, treatment with LY29004 or Torin reduced the levels of p53 and p21, as well as SA-β-Gal activity (Supplementary Fig. 5a, b). We then asked whether activation of mTOR and AKT is required for the induction of cell senescence after silencing FLRT2. The effect of FLRT2 silencing on the activities of mTORC1 and mTORC2 in HUVECs is shown in Fig. 2F. Silencing FLRT2 triggered the phosphorylation of AKT at serine (S473), mTORC2 complex (S2481) and its substrates, PKCα and NDRG1; by contrast, phosphorylation remained at basal levels for AKT at T308, mTORC1 complex (S2448) and its substrate, S6K (Fig. 2f). Since the functions of mTORC1 and mTORC2 are closely linked to those of Raptor and Rictor, respectively^{23, 24}, we evaluated the phenotype of cells in which FLRT2 was silenced jointly with Raptor or Rictor. Silencing FLRT2 and Rictor (a subunit of mTORC2) decreased AKT phosphorylation at S473, elevated p53 and p21, and increased SA-β-Gal activity (Fig. 2g, h). On the other hand, the senescence phenotypes of cells in which FLRT2 and Raptor (a subunit of mTORC1) were silenced were slightly diminished compared to those of cells in which only FLRT2 was silenced.

The proteins in the FLRT family have been reported to physically interact with FGF receptors and modulate the FGF-extracellular signal-regulated kinase (ERK) signaling pathway^17,25. Therefore, to investigate whether the FGFR signaling pathway is involved in senescence induced by silencing FLRT2, we examined the roles of FGFR and its downstream signaling molecules (SRC, ERK, and AKT). We detected a dramatic increase in AKT phosphorylation at S473, but no increase in FGFR1 or ERK phosphorylation (Supplementary Fig. 6a). When cells were subjected to double siRNA transfections to reduce FLRT2 along with FGFR1, SRC or AKT, the rise in p53 and p21, as well as SA-β-Gal activity seen after FLRT2 silencing was only abrogated if both FLRT2 and AKT were silenced simultaneously (Supplementary Fig. 6b-e). By contrast, p53 and p21 remained elevated if FGFR1 or SRC were jointly silenced along with FLRT2 Si alone. These results indicate that AKT, but not the FGFR pathway, is likely implicated in the induction of senescence by silencing FLRT2.

We previously found that in PTEN-loss-induced cellular senescence (PICS), mTOR kinase directly binds to p53 and phosphorylates it at serine 15, resulting in p53 activation and p21 accumulation²⁶. Thus, we examined if mTOR physically associated with p53 cells rendered senescent by silencing FLRT2. Immunoprecipitation analysis using antibodies against Raptor or Rictor confirmed an increase in p53 binding to mTORC2 (but not to mTORC1) (Fig. 2i). Together, these findings suggest that mTORC2 binds to and critically activates p53 levels in endothelial cells rendered senescent by silencing FLRT2.

Endothelial cell senescence induced by silencing FLRT2 is associated with enhanced ITGB4 activation

To connect at the molecular level FLRT2 and mTORC2 in this senescence paradigm, we analyzed the transcriptome of cells after FLRT2 silencing. As shown, 366 transcripts were > twofold increased after silencing FLRT2 compared with the control silenced group (Supplementary Fig. 7a). Gene set enrichment analysis was performed using a modified Fisher’s exact test, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that the genes elevated after FLRT2 silencing were significantly enriched for components of PI3K-AKT signaling, cell cycle, p53 signaling, and cellular senescence-related pathways (Supplementary Fig. 7a). From among the top 50 upregulated genes, the levels of eight mRNAs encoding proteins that localized in the plasma membrane (GeneCards confidence = 5) (Supplementary Fig. 7b) were confirmed by reverse transcription (RT) followed by real-time quantitative (q)PCR analysis (Supplementary Fig. 7c). Their involvement in the signaling underlying FLRT2 depletion-induced cellular senescence was then examined by reducing them individually in HUVECs using specific siRNAs (Fig. 3a). We found that both AKT activation and the rise in p53 and p21 observed in cells rendered senescent by FLRT2 silencing were reversed by silencing ITGB4. Further experiments indicated that ITGB4 was phosphorylated at tyrosine 1510 (Y1510) in the FLRT2-silenced cells (Fig. 3b, c), and that FLRT2 and ITGB4 physically interacted in FLAG-tagged FLRT2-overexpressing cells (Fig. 3d). Consistent with these observations, we found colocalization of FLRT2 and ITGB4 by immunofluorescence staining of HUVECs (Fig. 3e), and verified the interaction between FLRT2 and ITGB4 by using the Duo-link assay (Materials and Methods, Fig. 3f). Moreover, increased ITGB4 phosphorylation at Y1510 was observed in lipid rafts of the plasma membrane of FLRT2-silenced cells (Fig. 3g). These results suggest that FLRT2 associates with and prevents the activation of ITGB4 in lipid rafts and further suggest that FLRT2 depletion may promote cellular senescence by facilitating the activation of ITGB4.

To investigate the involvement of other β-integrins in senescence induced by silencing FLRT2, we individually silenced integrins ITGB1, -3, and − 5, which are known to be expressed in endothelial cells²⁷. As shown, only silencing ITGB4 significantly suppressed the increase in levels and/or activity of mTORC2, AKT, p53, p21, and SA-β-gal (Fig. 4a, b) triggered by FLRT2 depletion, as well as the increased abundance of mRNAs encoding the SASP factors IL1A (IL-1α), IL6, IL8, and MMP3 (Fig. 4c) in cells rendered senescent by silencing FLRT2. Treatment with increasing doses of ASC-8, an ITGB4 inhibitor, progressively decreased the phosphorylation of AKT and the accumulation of p53 and p21 in FLRT2-silenced HUVECs (Fig. 4d), and markedly reduced cell senescence elicited by silencing FLRT2 (Fig. 4e).

Next, cells in which ITGB4 and FLRT2 were silenced were transfected with vectors that expressed wild-type (WT) ITGB4 or mutant ITGB4 ΔCYT (which lacked the tyrosine phosphorylation site present in the cytoplasmic tail²⁸) as shown in Fig. 4F. In these cells, ITGB4 WT induced AKT phosphorylation and expression of p53 and p21, but ITGB4 ΔCYT did not (Fig. 4f). We also transfected ITGB4 WT and ITGB4 ΔCYT into human aortic endothelial cells (HAEC), which do not express endogenous ITGB4²⁹. In these cells, transfection with ITGB4 WT markedly increased AKT, p53, and p21 and other senescence phenotypes in HAEC, while ITGB4 ΔCYT expression did not (Supplementary Fig. 8). To examine whether ITGB4 might require the physical interaction between mTORC2 and p53 in FLRT2 depletion-induced cellular senescence, we immunoprecipitated Rictor in the presence or absence of ITGB4 in FLRT2-silenced cells (Fig. 4g). We found that Rictor physically associated with p53 in FLRT2-silenced cells in the presence of ITGB4, while the binding of Rictor to p53 was completely inhibited in cells in which FLRT2 and ITGB4 were silenced, indicating that the binding of p53 to mTORC2 is mediated by ITGB4. These findings provide evidence that ITGB4 is a key player in the program of endothelial cell senescence triggered by FLRT2 silencing, and that mTORC2, AKT, and p53 signaling pathways are downstream effectors in this paradigm.

Knockdown of FLRT2 induces senescence in mouse aorta and vein endothelial cells

To begin to explore the biological significance of our findings in vivo, we examined the effects of FLRT2 knockdown on senescence phenotypes in mouse vascular tissues. Using a transient silencing approach³⁰, we reduced aortic FLRT2 expression by injecting Cy5-tagged FLRT2 Si (or Con Si in control groups) along with an in vivo transfection reagent (Materials and Methods), into the tail veins of mice (Fig. 5a). After lowering FLRT2 levels, aorta tissues revealed increased phosphorylation of ITGB4 at Y1510, mTOR at S2481, and AKT at S473, and higher abundance of p53 and p21 (Fig. 5a), along with higher levels of SA-β-Gal signal, as compared with the Con Si group (Fig. 5b). Delivery of Cy5-tagged FLRT2 Si to the vasculature and uptake by the endothelium was confirmed by CD31 immunostaining (Fig. 5c). We confirmed by fluorescence microscopy that FLRT2 silencing induced the phosphorylation of ITGB4, mTORC2, and AKT, and increased the expression levels of p53 and p21 in arterial endothelial cells (Fig. 5d). In addition, in the veins of mice injected with FLRT2 Si, we observed increased SA-β-Gal activity and elevated signaling through ITGB4, mTORC2, AKT, p53 and p21 (Supplementary Fig. 9). Moreover, the endothelial cell senescence traits induced by FLRT2 silencing (which elevated phosphorylation of ITGB4 at Y1510, mTOR at S2481, and AKT at S473, increased p53 and p21 levels and SA-β-Gal activity, and decreased cell proliferation) were diminished by FLRT2 overexpression in culture (Fig. 6a-c). To test whether overexpression of FLRT2 rescues the FLRT2 Si-induced senescence in vivo, we injected Cy5-tagged FLRT2 Si which is specific to the FLRT2 3’UTR (to avoid silencing the ectopic FLRT2 mRNA) or Con Si with either FLRT2 expression vector or empty vector into the tail veins of mice along with an in vivo transfection reagent. Consequently, when we overexpressed FLRT2 in FLRT2-silenced mice which were generated using in vivo-jetPEI® (Polyplus transfection; Illkirch, France), we found that the senescence phenotypes, including increased SA-β-Gal activity, phosphorylation of AKT at S473, and expression levels of p53/p21 showing in FLRT2-silenced mice, were rescued in the aortas of mice (Fig. 6d, e). Immunostaining of P-AKT, p53, and p21 were also evidently decreased to the control levels in aorta tissues of mice (Fig. 6f). Collectively, our findings lend strong support to a model whereby FLRT2 regulates endothelial cell senescence through a signaling paradigm involving ITGB4, mTORC2, AKT, p53, and p21 in cultured endothelial cells and mouse vascular tissue.

Vascular endothelial cells can undergo premature senescence upon exposure to agents that are risk factors for vascular diseases. Exposure of endothelial cells to low or high glucose promotes the expression of senescence markers, such as the SA-β-Gal, p16, and iNOS, and decreases the expression of eNOS^{31, 32}. Other stimuli, such as ox-LDL, homocysteine, ceramide, and Ang II promote endothelial cell senescence through various mechanisms^{33– 37}. For example, Ang II induces endothelial cell senescence by activating the mitogen-activated protein kinases³⁷, and ox-LDL promotes HUVEC senescence by lowering SIRT1 production ³⁸. The induction of senescence increases the susceptibility of endothelial cells to pathological stresses, which results in endothelial injury and consequently promotes age-related vascular diseases, such as cardiovascular diseases (coronary artery disease, atherosclerosis, and hypertension), peripheral vascular diseases, diabetic retinopathy, renal vascular diseases, and microvascular diseases^{33, 39}.

There is limited information about the functional roles of FLRT2 in cultured cells and in mice, although there is evidence that it is implicated in cardiovascular function. Ablation of FLRT2 in mouse embryos disrupted heart formation and led to cardiac insufficiency⁴⁰, while other studies further supported a role for FLRT2 in vascular function based on observations that FLRT2 is expressed in endothelial cells in the placental labyrinth, and that mice lacking FLRT2 in endothelial cells exhibited embryonic lethality with systemic congestion and hypoxia ²¹. Here, we first showed that FLRT2 expression declined in senescent endothelial cells and in aged rat and human arterial tissues (Fig. 1). We then observed that depleting FLRT2 in endothelial cells induced cellular senescence (Fig. 2).

During mouse embryogenesis, FLRT proteins reportedly regulate FGF signaling in many tissues by interacting with a signaling receptor²⁵. We previously reported that signaling through FGFR and AKT promotes the expression of p53 and p21 during senescence in cancer cells⁴¹. Here, however, we observed that FGFR1 and downstream signaling molecules SRC and ERK were not involved in FLRT2 depletion-induced endothelial cell senescence (Supplementary Fig. 6). Silencing ITGB4 markedly mitigated the senescent phenotype of HUVECs, indicating that ITGB4 mediates senescence in this cell type⁴². The activity of mTORC2 increases in HUVECs undergoing replicative senescence and H₂O₂-induced premature senescence, with a concomitant reduction in the expression levels of NRF2 mRNA ⁴³.

We recently reported that the activation of ITGB4 is essential for radiation-induced cellular senescence in A549 lung cancer cells⁴⁴ and that mTOR kinase is a key activator of p53 in senescence triggered by loss of PTEN in cancer cells [26]. However, the underlying mechanism through which ITGB4 and mTORC2 contribute to endothelial cell senescence is not well understood. In this study, we identified ITGB4 as an important intermediary molecule for the activation of mTORC2, but not mTORC1, in endothelial senescence and vascular aging (Fig. 2–4). Our data suggest that among ITGB family members, ITGB4 plays a specific role in mediating endothelial cell senescence by linking signals from the transmembrane protein, FLRT2, to the mTORC2- AKT-p53 signaling paradigm. A physical association between FLRT2 and ITGB4 in lipid rafts of the plasma membrane critically prevented ITGB4 phosphorylation and thereby maintained ITGB4 in an inactive state (Fig. 3). If FLRT2 did not properly inhibit ITGB4 activation, persistent activation of signaling through mTORC2-AKT-p53 induced endothelial cell senescence, as shown in aortic and venous tissues of the FLRT2 knockdown mouse model (Fig. 5 and Supplementary Fig. 9), and by the rescue of the effects of FLRT2 loss on endothelial cell senescence and vascular aging when FLRT2 was overexpressed (Fig. 6). Our findings indicate that an interaction between FLRT2 and ITGB4 in lipid rafts prevents endothelial cell senescence and vascular aging by acting through mTORC2, AKT, p53, and p21. Therefore, we propose that therapeutic regimens capable of regulating upstream molecules FLRT2 and ITGB4 in this endothelial cell senescence paradigm, could be developed as a novel strategy for the prevention of vascular aging and age-related vascular diseases.

Acknowledgements

Carotid arteries of 6 and 24 months-old rats were a kind gift from Prof. Hae Young Chung (Pusan National University, Pusan, Korea). This work was supported by grants to JSL from RCIC (2021R1A5A2031612) and the Basic Research Science Program (2020R1A2B5B02002375, 2021R1A6A3A01086396) through the National Research Foundation (NRF) funded by the Korean government (MSIT).

Author contributions

Investigation, H.J.H; Writing - original draft, H.J.H., and J.-S.L.; Writing - review & editing, H.J.H., J.-S.L., D.K., J.-R.K., Y.-G.K., H.J.P., A.B.H., and M.G; Validation, H.J.H.; Visualization, H.J.H.; Conceptualization, D.K., J.-R.K., Y.-G.K., and H.J.P.; Resources, J.H.C., and J.-R.K.; Data curation, J.S-.L.; Supervision, M.G., and J.-S.L.; Project administration, J.S-.L.; Funding acquisition, J.-S.L.

Competing interests

The authors declare no competing interests.

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FLRT2 plays a critical role in endothelial cell senescence and vascular aging

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Abstract

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Introduction

Materials And Methods

Cell culture

Reagents and antibodies

SA-β-Gal staining

Human arterial tissue samples

Immunoblotting analysis

Immunoprecipitation

Cell viability

Cell cycle analysis

BrdU incorporation assay

RNA interference

Plasmid transfection

RT-qPCR: reverse transcription (RT) followed by real-time quantitative polymerase chain reaction (qPCR)

RNA sequencing data processing and analysis

Tube formation assay

Immunofluorescence staining

The Duolink Proximity Ligation Assay

Isolation and characterization of lipid rafts

Immunohistochemistry

Animal experiments

Statistical analysis

Results

FLRT2 levels decline in senescent endothelial cells and in aged vascular tissues

FLRT2 knockdown induces premature senescence of endothelial cells

FLRT2 regulates endothelial cell senescence through the mTORC2-AKT-p53 pathway

Endothelial cell senescence induced by silencing FLRT2 is associated with enhanced ITGB4 activation

Knockdown of FLRT2 induces senescence in mouse aorta and vein endothelial cells

Discussion

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References

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