Laurent Grosse

Nicole Wagner

Alexander Emelyanov

Clement Molina

Sandra Lacas-Gervais

Kay-Dietrich Wagner

Dmitry V Bulavin
email: dmitry.bulavin@unice.fr

Defined p16 High senescent cell types are indispensable for mouse healthspan

Keywords: Senescence, p16, liver sinusoid endothelial cells, aging, lineage tracing, vascular endothelial cells, liver sinusoids, fibrosis

The accumulation of senescent cells can drive many age-associated phenotypes and pathologies. Consequently, it has been proposed that removing senescent cells might extend lifespan. Here we generated two knock-in mouse models targeting the bestcharacterized marker of senescence, p16 Ink4a . Using a genetic lineage tracing approach, we found that age-induced p16 High senescence is a slow process that manifests around 10-12 months of age. The majority of p16 High cells were vascular endothelial cells mostly in liver sinusoids (LSECs), and to lesser extent macrophages and adipocytes. In turn, continuous or acute elimination of p16 High senescent cells disrupted blood-tissue barriers with subsequent liver and peri-vascular tissue fibrosis and health deterioration. Our data show that senescent LSECs are not replaced after removal and have important structural and functional roles in the aging organism. In turn, delaying senescence or replacement of senescent LSECs could represent a powerful tool in slowing down aging.

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Many chronic diseases manifest with increasing age and tend to prevail in the elderly [START_REF] Campisi | Aging, cellular senescence, and cancer[END_REF][START_REF] Hodes | Disease drivers of aging[END_REF]. The risk for geriatric syndromes, such as frailty, immobility, mild cognitive impairment and incontinence, also significantly increases with aging [START_REF] Lebrasseur | Cellular Senescence and the Biology of Aging, Disease, and Frailty[END_REF]. Substantial evidence has demonstrated that the accumulation of senescent cells can drive many of these age-associated phenotypes and pathologies [START_REF] Childs | Senescent cells: an emerging target for diseases of ageing[END_REF][START_REF] Mchugh | Senescence and aging: Causes, consequences, and therapeutic avenues[END_REF]. For example, senescent cells accumulate in adipose tissue of patients with diabetes and age-related metabolic dysfunction [START_REF] Tchkonia | Cellular senescence and the senescent secretory phenotype: therapeutic opportunities[END_REF], in osteoarthritic joints [START_REF] Jeon | Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment[END_REF][START_REF] Xu | Transplanted Senescent Cells Induce an Osteoarthritis-Like Condition in Mice[END_REF], in the aorta in vascular hyporeactivity and atherosclerosis [START_REF] Roos | Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice[END_REF], and in the lungs in idiopathic pulmonary fibrosis [START_REF] Schafer | Cellular senescence mediates fibrotic pulmonary disease[END_REF]. While selective elimination of these senescent cells confers notable benefits in some tissues, recent studies have also described beneficial roles for senescent cells, raising the question of the differential roles of these cells in various tissues [START_REF] Demaria | An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA[END_REF][START_REF] Munoz-Espin | Programmed cell senescence during mammalian embryonic development[END_REF][START_REF] Storer | Senescence is a developmental mechanism that contributes to embryonic growth and patterning[END_REF]. Thus, how senescence mechanistically or dynamically contributes to the aging process remains largely unknown.

Cellular senescence is a well-defined phenotype primarily characterized by irreversible cell-cycle arrest in response to continuous proliferation or DNA damage [START_REF] Herranz | Mechanisms and functions of cellular senescence[END_REF]. This arrest may be accompanied by apoptosis resistance, increased protein synthesis, metabolic shifts with increased glycolysis, decreased fatty acid oxidation, increased reactive oxygen species generation, and acquisition of a senescence-associated secretory phenotype (SASP) [START_REF] Lebrasseur | Cellular Senescence and the Biology of Aging, Disease, and Frailty[END_REF][START_REF] Tchkonia | Cellular senescence and the senescent secretory phenotype: therapeutic opportunities[END_REF]. This SASP entails the secretion of multiple active substances, pro-inflammatory mediators, and extracellular matrix-damaging molecules, and as such, can ultimately contribute to enhanced inflammation and worsening of several pathological conditions. For example, transplantation of a small number of senescent cells around the knee joint was sufficient to cause osteoarthritis while injection of senescent cells into adipose tissue shortened lifespan [START_REF] Xu | Transplanted Senescent Cells Induce an Osteoarthritis-Like Condition in Mice[END_REF].

Senescence-ablator mouse models have pioneered the field of in vivo senescence studies. With the use of one such model, known as the INK-ATTAC mouse, that is based on a 2,617-bp fragment of the p16 Ink4a gene promoter [START_REF] Baker | Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders[END_REF], it has been proposed that removal of p16-expressing cells results in life extension in mice [START_REF] Baker | Naturally occurring p16(Ink4a)positive cells shorten healthy lifespan[END_REF]. The concern however is whether the reporter construct with a part of the p16 genomic sequence fully resembles endogenous p16 gene expression, especially with aging. This is further supported by the fact that some p16-expressing cells are not efficiently removed by the INK-ATTAC system in several tissues including the liver, colon, and T lymphocytes [START_REF] Baker | Naturally occurring p16(Ink4a)positive cells shorten healthy lifespan[END_REF]. Thus, it is unclear whether there are important senescent cell types in tissues where the INK-ATTAC system does not work and what is the impact of their removal on health span.

Results

Novel mouse model for continuous labeling and elimination of p16 High senescent cells

To accurately monitor p16 Ink4a gene expression, a well-defined marker of senescence, we generated a knock-in mouse line whereby we integrated a targeting cassette in the last exon of the endogenous p16 Ink4a gene (Figure S1). Integrating the expression cassette into the last exon allowed us to preserve most of the p16 Ink4a genomic sequence and the binding sites for the many regulators of p16 transcription. Polycomb proteins are among important negative regulators that bind both the promoter and intronic regions of the p16 Ink4a gene [START_REF] Bracken | The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells[END_REF][START_REF] Kotake | pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene[END_REF]. This in turn is essential to prevent unwanted reactivation of the reporter cassette considering the high number of potential senescence-unrelated regulators of p16 transcription [START_REF] Frescas | Murine mesenchymal cells that express elevated levels of the CDK inhibitor p16(Ink4a) in vivo are not necessarily senescent[END_REF][START_REF] Hall | p16(Ink4a) and senescence-associated betagalactosidase can be induced in macrophages as part of a reversible response to physiological stimuli[END_REF].

To avoid reliance on efficiency of any activating compound as well as timing of senescence induction, we chose to use a constitutively active CRE recombinase. We integrated CRE recombinase, Thymidine Kinase (TK) and a fluorescent reporter (dtTomato) at the end of the 3 rd exon by fusing it with p16 Ink4a mRNA through self-cleaving peptide sequences that would produce separate proteins after translation (Figure S1).

Retrospectively, introduction of a fluorescent reporter in the targeting cassette appeared to be impractical due to low expression of p16 mRNA in vivo; similar was reported by others [START_REF] Demaria | An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA[END_REF]. In addition, inclusion of ganciclovir failed to remove p16 High senescent cells also most likely due to a low level of p16-driven TK expression (data not shown). Once generated, we crossed the resulting p16-Cre knock-in mice with two reporter lines: Rosa26-mTmG (tracer line) that would continuously label p16 expressing cells and Rosa26-DTA (ablator line) that would continuously eliminate p16 expressing cells.

Importantly, the DTA toxicity is lost once DTA is released from a cell providing a very selective approach to eliminate cells expressing it. Our original concern with using a genetic approach to continuously eliminate p16 High cells was that senescent cells were recently detected during embryogenesis [START_REF] Munoz-Espin | Programmed cell senescence during mammalian embryonic development[END_REF][START_REF] Storer | Senescence is a developmental mechanism that contributes to embryonic growth and patterning[END_REF]. However, senescence during embryogenesis was shown to activate a p16 Ink4a -independent program and we further found that p16-Cre mice crossed on a R26-DTA background produced double transgenic pups with expected 25% Mendelian ratio. This finding allowed us to use p16-Cre/R26-DTA mice at adult stages to study the effects of continuously eliminating p16expressing cells. For all further experiments, only heterozygous p16-Cre and heterozygous reporter mice were used.

To validate our model, we first established dermal fibroblasts (DFs) from p16-Cre/R26-mTmG and p16-Cre/R26-DTA newborn mice and passaged them until replicative senescence. We found that by passage 7, ~30% of p16-Cre/R26-mTmG DFs were EGFPpositive (Figure 1A). We next FACS-sorted p16-EGFP-positive cells and found that they were significantly enriched for p16 Ink4a mRNA expression when compared to EGFP-negative cells and majority of these cells were positive for a senescence marker, SA-β gal (Figure 1B&C).

We further observed a robust accumulation of p16 Ink4a mRNA by Real-Time PCR with passaging of p16-Cre/R26-mTmG, but not p16-Cre/R26-DTA DFs; the levels of p19 Arf mRNAs were similar between the genotypes while we observed no p21 Waf1 mRNAs accumulation in DFs with replicative senescence (Figure 1D). Importantly, at passage 5, the level of p16 Ink4a mRNA in p16-Cre/R26-DTA DFs was around 4.7-fold higher than at passage 1 (Figure 1D), suggesting that the DTA system operates when at least this level of induction is reached. Thus, our system is potentially ignoring transient and under-threshold levels of p16 induction while efficiently marking or eliminating senescent cells in which the p16 Ink4a mRNA levels are significantly elevated. We further confirmed that the level of CRE and TK mRNAs (expressed from a single knock-in allele) as expected was ~2-fold lower than the level of p16 mRNA (expressed from both wild-type and knock-in alleles) in passage 7 DFs (Figure 1E) and in different tissues (Figure 1F) obtained from 1-year-old heterozygous p16-Cre/R26-mTmG mice.

Identification of p16 High senescent cells in vivo

To maximize the chances of regenerative replacement, we speculated that removing the first emerging senescent cells, in a relatively young organism, should create the most favorable conditions for their replacement. It is more feasible to replace low numbers of senescent cells in young organisms than high numbers in older organisms, where the regenerative functions of many tissues are markedly declining with age. We therefore monitored p16-Cre knock-in mice carrying a constitutively active Cre recombinase and a Rosa26-DTA reporter for healthy aging. Surprisingly, we found that by 12 months-of-age, some p16-Cre/R26-DTA mice became increasingly sick, some developed untreatable skin ulcerations, other -became morbid and required euthanasia (Figure S2A&B).

As this finding was in sharp contrast to our expectations, we next aimed to determine the underlying cause of this morbidity. No significant changes were observed in the total blood count (Figure S2C), serum biochemistry (Figure S2D), and H&E-stained tissues from p16-Cre/R26-DTA mice (Figure S2E). Only trichrome-stained tissues showed noticeable changes in p16-Cre/R26-DTA mice (Figure S2F). To determine senescent cell type(s) whose removal could be a reason for changes observed with trichrome staining, we turned to analysis of p16-Cre knock-in mice crossed with a Rosa26-mTmG reporter. Our analysis of EGFP-positive cells revealed only rare cells in all studied tissues in 2-month-old mice (Figure 2A). In 12-month-old mice, we detected an increase in the number of EGFP-positive cells in the White Adipose Tissue (WAT), lung and heart, while only a slight increase was observed in other analyzed tissues when compared to 2-month-old mice (Figure 2A, Figure S3A). The highest number of EGFP-positive cells however was observed in the liver, suggesting that p16 High cells emerge with age in significant numbers in the liver and to a lesser degree in other tissues, including the lung, WAT and heart. Using p16-specific antibody (Figure S2G), we observed strong co-localization between p16 and EGFP in the liver (Figure 2B), which supported that our reporter line efficiently labeled p16 High cells. In addition, p16-Cre knockin mice containing both Rosa26-mTmG and Rosa26-DTA alleles showed almost no EGFPpositive cells (Figure S3B). This finding suggests that p16 has been expressed in these cells for an extended period that was sufficient to activate both reporters.

As the most significant accumulation of p16 High cells was observed in the liver, we focused on this organ for further analyses. Analysis of EGFP-positive cells on liver sections revealed their capillary-like morphology, and further co-staining with an anti-Cd31 and Cd146 antibodies confirmed that the largest fraction (> 70% in 1-year-old mice) were vascular endothelial cells (Figure 2C, Figure S3C). About 30% of EGFP-positive cells in the liver appeared Cd45-positive and further analysis revealed that the majority were F4/80positive macrophages (Figure 2C, Figure S3C). In addition, no co-staining was observed between EGFP-positive cells and desmin (a marker of stellate cells) (Figure S3C). Analyses of other tissues showed that EGFP-positive cells in 1-year-old mice were Cd31-positive including in the heart and lung (Figure S3D). Next, we determined the proliferative rate of liver endothelial cells after 3 days of continuous EdU injections of p16-Cre/R26-mTmG mice. We found that both Cd31-and Cd146-positive cells had low proliferative index with only 2-3% of EdU-positive fractions, which translates into 100-150d doubling time (Figure 2D&E). Importantly, less than 0.01% of p16 High -EGFP-positive cells were EdU-positive suggesting that the cells responsible for marked aging-induced p16 up-regulation are nondividing liver vascular endothelial cells.

Next we verify whether age-induced p16 up-regulation is attributed to liver Cd31positive cells independent of our reporter models. We purified Cd31-positive and Cd31negative cells from the livers of 2-month-old and 2-year-old wild-type mice and analyzed p16 mRNA expression by RT-PCR. We found a significant increase in expression of p16 mRNA in Cd31-positive cells in 2-year-old mice (Figure 3A). While almost all p16 mRNA was detected in the Cd31-positive fraction of 2-month-old mice, a noticeable increase was also observed in the Cd31-negative fraction in 2-year-old mice. These data suggest that while Cd31-positive cells are the main source of the aging-induced increase in p16 mRNA expression in the liver, other cell types also contribute to this up-regulation in older animals.

Cd31-positive cells of the liver are mostly liver sinusoid endothelial cells (LSECs) that contain small nuclei and an extremely thin cytoplasm, which complicates the analysis of these cells on sections. To overcome this technical problem, we enriched Cd31-positive cells using magnetic nanoparticles, flattened them by plating and then immediately analyzed. We found that the Cd31-positive fraction contained <1% SA-β-Gal-positive cells in 2-month-old mice (Figure 3A,lower panel). This number increased to 8-10% in 1-year-old mice and reached >50% in 2-year-old mice. Further analysis of other senescence markers revealed an increase in the number of phospho-γH2AX-positive foci, K9me2 and macroH2A nuclear staining in liver Cd31-positive cells obtained from 2-year-old compared to 2-monthold mice (Figure 3B). We also observed decreased Lamin B1 staining, specifically at the nuclear membrane in 2-year-old liver Cd31-positive cells (Figure 3B). Taken together, these data suggest that murine LSECs undergo significant senescence with aging.

Senescent LSECs exhibit metabolic alterations and enhanced detoxifying function with age

Next, we analyzed different senescence markers in liver Cd31-positive cells obtained from 1-year-old p16-Cre/R26-mTmG mice and found that EGFP-positive cells (representing 8-10% of total Cd31-positive fraction) were mostly SA-β-gal-positive, contained more phospho-γH2AX nuclear foci, and less Lamin B1 when compared to EGFP-negative cells (Figure 3C-E). Of note, purification of LSECs using different protocols including Percoll gradient, Cd31, and Cd146 column purification yielded similar numbers of p16 High -EGFP positive cells (Figure S4A). We further determined that LSECs could be maintained in culture without a loss of viability up to 7 days (Figure S4B).

To investigate p16 High /EGFP-positive cells further, we turned to mRNA-Sequencing analysis. Liver Cd31-positive cells were enriched using magnetic nanoparticles and then FACS-sorted to obtain EGFP-positive and EGFP-negative fractions. We first confirmed that the EGFP-positive fraction contained significantly higher levels of p16 Ink4a RNA than the EGFP-negative fraction (Figure 3F). We also observed that multiple SASP components were up-regulated in p16 High /EGFP-positive cells, including: cytokines (IL1a, IL1b, IL15 and IL18), chemokines (Ccl2, Ccl6, Ccl8, Ccl24, Cxcl9, Cxcl12, Cxcl13 and Cxcl16), and other components of SASP (Mmp12, Timp2, and TSLP) (Figure 3F). This finding further supports that these cells were in a state of cellular senescence.

We then performed a GO and KEGG enrichment analysis of the RNA-Seq data (Figure S4C-E). We found significant up-regulation of enzymes involved in fructose/mannose (1.1.1.21; 1.1.1.14; 2.7.1.1), purine (2.7.1.40; 3.6.1.5; 2.4.2.8; 1.7.1.7; 6.3.4.4, 3.6.1.29, 1.17.1.4, 1.17.3.2, 3.5.4.4), pyrimidine (3.6.1.5, 2.7.4.14, 2.4.2.3, 1.8.1.9) and glutathione (6.3.2.2, 1.8.1.7; 1.1.1.42, 1.11.1.12, 1.11.1.19, 2.5.1.18) metabolism. Furthermore, 22 genes involved in oxidative phosphorylation were up-regulated in p16 High /EGFP-positive cells. In addition, we found that genes involved in ribosome synthesis (total 8 genes), basal transcription factors (TFIIB, TAF4, 5 and 7), DNA replication (Mcm2,4 and 7) and spliceosomes were significantly down-regulated in p16 High /EGFP-positive cells. Altogether, our data show that senescent p16 High LSECs undergo significant metabolic changes.

LSECs are highly specialized endothelial cells that act as a permeable barrier between the blood and hepatocytes. The presence of fenestration, absence of diaphragm and lack of basement membrane make them the most permeable endothelial cells of the mammalian body [START_REF] Deleve | Liver Sinusoidal Endothelial Cell: An Update[END_REF][START_REF] Poisson | Liver sinusoidal endothelial cells: Physiology and role in liver diseases[END_REF]. Most importantly, LSECs have the highest endocytic capacity of the entire organism, rendering them indispensable for clearing soluble macromolecules and small particles through endocytic receptors [START_REF] Li | Role of liver sinusoidal endothelial cells and stabilins in elimination of oxidized lowdensity lipoproteins[END_REF][START_REF] Oie | Liver sinusoidal endothelial cells contribute to the uptake and degradation of entero bacterial viruses[END_REF][START_REF] Sorensen | Liver Sinusoidal Endothelial Cells[END_REF]. This property constitutes the major detoxifying function of LSECs (DeLeve and Maretti-Mira, 2017; [START_REF] Poisson | Liver sinusoidal endothelial cells: Physiology and role in liver diseases[END_REF]. Next, we analyzed the expression of endocytic receptors, including scavenger and mannose receptors. We found that although p16 High /EGFP-positive LSECs were senescent, numerous endocytic receptors were significantly up-regulated (Figure 3G). This finding implies that the detoxifying function is not down-regulated, but rather, enhanced in senescent LSECs from 1-year-old mice. To confirm that we analyzed the uptake of oxidized and acetylated LDL and found that it is significantly increased in p16 High /EGFP-positive LSECs (Figure 3H). It is important to note however that while we see an upregulation of scavenger receptors in p16 High LSECs from 1-year-old mice, once senescence progresses it should induce heterochromatin formation accompanied by transcriptional silencing. To investigate how this could affect the expression of scavenger receptors in older animals, we purified different liver fractions from 2-year-old mice and compared them to 2-month-old mice. We found that numerous receptors that were significantly upregulated in p16 High LSECs from 1year-old mice (Figure 3G), show much lower magnitude of induction in 2-year-old mice (Figure S3E). Thus, an upregulation of scavenger receptors in p16 High LSECs of 1-year-old mice is a transient event that coincides with induction of senescence.

Senescent p16 High Cd31-positive cells are not replaced after removal by similar cell types

We next analyzed the consequences of removing p16 High cells in vivo. We crossed p16-Cre knock-ins with a Rosa26-DTA reporter line and established control (single mutant mice, either p16-Cre or Rosa26-DTA) and experimental (double mutant mice) groups. About 20% of p16-Cre/R26-DTA mice became ill by 12 months-of-age (Figure S2A&B), thus we analyzed the different mouse groups at this age. We first checked the efficiency of the removal of p16-expressing cells. We found no significant differences in p16 expression in multiple tissues from young animals (2 months) of different genotypes (Figure S5A, Figure 4A), while 1-year-old p16-Cre/R26-DTA mice show a strongly reduced p16 staining in multiple tissues (Figure 4A), supporting the efficiency of the DTA system in removing p16expressing cells.

The concept of life extension based on removing senescent cells largely depends on the efficient replacement of senescent cells in the tissues and organs in which they are structurally and functionally important. This paradigm is particularly critical in the liver, where LSECs have a major detoxifying function while their proliferative activity is relatively low (Figures 2D&E,3G&H). As the accumulation of senescent cells starts at a young age with only a few cells, we expected that when removing such a small number of cells in p16-Cre/R26-DTA mice, the relatively young tissue should be able to efficiently replace them if such a mechanism exists. To verify our hypothesis, we stained livers from mice of different genotypes with a Cd31 antibody. We found a significant decrease in Cd31-positivity in the p16-Cre/R26-DTA mice (Figure 4B) arguing that senescent LSECs have not been replaced with other Cd31-positive cells after removal. We then analyzed additional tissues to determine whether the lack of replacement of Cd31-positive cells is limited only to the liver or is a more general phenomenon. Similar to the liver, we found a decrease in the number of Cd31-positive cells in the lung, heart, and kidney (Figure 4B). To understand the potential underlining cause for the lack of Cd31-positive cells replacement in the liver, we analyzed the level of Vegfs and their receptors. We found an aging-induced decline in the expression of multiple Vegfs and their receptors in Cd31-positive fractions obtained from 1 and 2-year-old mice (Figure 4C, Figure S4F). Some evidence has been provided for the bone marrow origin of LSEC progenitors [START_REF] Wang | Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats[END_REF]. Next we investigated the fraction of Cd31/Cd45/Cd133-positive bone marrow cells that was reported to be a progenitor pool for LSECs. We found that there was less than 0.5% of p16 High /EGFP-positive cells in a fraction of Cd31/Cd45/Cd133-positive bone marrow cells while there was no difference in the number of Cd133-positive cells among Cd31/Cd45-positive bone marrow cells between control and p16-Cre/R26-DTA mice (Figure S4G). Altogether, our data suggest that senescent Cd31-positive cells are not replaced by a similar cell type after removal which in part could be explained by their low proliferative activity, and reduced expression of different Vegfs receptors with age.

Continuous removal of p16 High senescent cells in vivo results in liver and systemic peri-vascular fibrosis

To understand the consequences of removal of Cd31-positive cells in the liver, we next turned to Scanning Electron Microscopy. We found that in control mice, the liver sinusoids were lined with LSECs with numerous fenestrations (Figure 5A, Figure S5B). By contrast, notable areas of the liver sinusoids in p16-Cre/R26-DTA mice were lacking LSECs. To investigate this further, we turned to Transmission Electron Microscopy. We found numerous LSECs with clearly visible fenestrations and a distinctive Disse Space filled with numerous villi of hepatocytes in control mice (Figure 5B). By contrast, the Disse Space in p16-Cre/R26-DTA mice was filled with collagen depositions (Figure 5B). To verify this finding, we analyzed collagen deposition in the livers using the Picrosirius Red collagenspecific dye. We found a significant increase in collagen deposition in the livers of p16-Cre/R26-DTA mice while more depositions were observed peri-vascularly in several analyzed tissues (Figure 5C). Altogether, these findings reveal that removal of senescent LSECs and vascular endothelial cells does not activate a cell-replacement mechanism but rather activates another form of tissue damage repair, namely, fibrosis.

Acute removal of p16 High senescent cells in vivo results in deregulation of blood vessel permeability and subsequent fibrosis

Our original prediction was that by removing senescent cells early in life, tissues would have a higher chance of repairing damage compared to older animals, when regenerative capacity markedly declines. Our data, however, have indicated that the earliest cell types that undergo age-induced p16 High senescence in the mouse -Cd31-positive endothelial cellsare not replaced after removal, but instead the damage is repaired via fibrosis. The experimental set up of continuous elimination of senescent cells however is different from acute removal of senescent cells in older organism. As the tissue damage after removal of a large number of senescent cells in older mice could be significant, we speculated that this could also potentially activate a physiological response of cell replacement as one could see with partial hepatectomy (DeLeve and Maretti-Mira, 2017; [START_REF] Poisson | Liver sinusoidal endothelial cells: Physiology and role in liver diseases[END_REF]. This in turn could be different from a continuous removal of single senescent cells from early age that is repaired through fibrosis (Figure 5A-C).

To address whether removing a large number of senescent cells in aged animals activates a proper regenerative response based on cell replacement rather than fibrosis, we generated a Tamoxifen (TAM)-inducible p16 knock-in mouse line (p16-CreERT2) (Figure S6). We then crossed p16-CreERT2 knock-ins with the Rosa26-mTmG and Rosa26-DTA reporters and aged mice of different genotypes until at least 1.5 years before placing them on a Tamoxifen (TAM) diet. We first checked p16-EFGP expression and found that the majority of p16/EGFP-positive cells were positive for Cd31 (Figure 6A). Next, we analyzed the physiological consequences of acute removal of p16 High -expressing cells in the aged 1.5-year-old p16-CreERT2/R26-DTA mice. Removal of p16-expressing cells after TAM exposure resulted in a significant reduction in the number of p16-expressing cells in the livers of p16-CreERT2/R26-DTA mice (Figure 6B). We further observed a significant decrease of Cd31-positive cells in analyzed tissues (Figure 6C), suggesting that Cd31positive cells were not efficiently replaced. As the majority of senescent cells are Cd31positive endothelial cells, their acute removal without proper replacement could disrupt blood-tissue barriers. To verify this, we injected TAM-fed animals with Evans Blue via the tail vein to investigate blood-vessel permeability. After 30 minutes, we sacrificed the animals and analyzed Evans Blue retention by different tissues. We found significant accumulation of Evans Blue in multiple tissues in p16-CreERT2/R26-DTA mice compared to controls (Figure 6D&E).

As a large proportion of LSECs in older mice is positive for SA-β-Gal/p16-EGFP and these cells up-regulate multiple scavenger receptors (Figure 3G), we speculated that their acute removal could change blood LDL and oxidized-LDL (ox-LDL) levels. Using an ELISA assay, we found a significant increase in the concentration of plasma LDL and ox-LDL levels in TAM-treated p16-CreERT2/R26-DTA mice compared to controls (Figure 6F). As ox-LDL is an oxidant and a potent inducer of senescence, we checked whether it activates senescence in LSECs. Indeed, we found that ox-LDL was efficient in inducing SA-β-Galpositive LSECs in vitro (Figure 6G). This finding may suggest that a build-up of plasma ox-LDL after the removal of senescent LSECs could trigger further senescence of LSECs. In this case, continuous removal of senescent cells could markedly worsen the health condition of the animal.

To verify whether the tissue repair response differs when p16 High cells are acutely removed in aged mice, we analyzed fibrosis in 1.5-year-old p16-CreERT2/R26-DTA mice 1 month after receiving a TAM diet. We found high levels of collagen deposition in the liver as well as peri-vascular collagen deposition in several tissues of p16-CreERT2/R26-DTA mice (Figure 6H). Our findings thus revealed that removing senescent Cd31-positive endothelial cells, either step-wise or acutely, does not activate a cell-replacement mechanism, but rather induces tissues fibrosis.

To better understand broader consequences of removal of p16 High senescent cells, we carried out the analysis of serum biochemistry and found an increase in the level of urea (Figure S6E) in p16-CreERt2/R26-DTA mice. Further analysis of total blood count found a profound thrombocytopenia in p16-CreERt2/R26-DTA mice (Figure 6I, Figure S6D). It is important to note that a reduction in the level of platelets (about 15%) was also observed in constitute p16-Cre/R26-DTA mice (Figure S2C) but this decrease did not reach statistical significance.

Thrombocytopenia is the most frequent hematologic abnormality in patients with chronic liver disease [START_REF] Hallen | Coagulation Studies in Liver Disease[END_REF][START_REF] Temel | Serum thrombopoietin levels and its relationship with thrombocytopenia in patients with cirrhosis[END_REF] and has been established as one of the most reliable factors to predict overall mortality in patients of different age groups [START_REF] Golwala | Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), Platelet Count and Plateletcrit (PCT) as predictors of in-hospital paediatric mortality: a case-control Study[END_REF][START_REF] Vanderschueren | Thrombocytopenia and prognosis in intensive care[END_REF]. Considering such an important role of platelets in overall survival, we investigated this further. Thrombopoietin (TPO), one of the main regulators of platelets production, is secreted by the liver and kidney. Next, we analyzed the level of TPO in blood serum and found its significant reduction upon removal of p16 High cells in p16-CreERT2/R26-DTA mice (Figure 6J). Thus, removal of senescent LSECs and accompanied liver fibrosis results in reduced levels of serum TPO and subsequent thrombocytopenia. This in turn could be a negative contributing factor to overall survival of p16-Cre/R26-DTA mice.

Senolytic combination of dasatinib and quercetin efficiently removes p16 High macrophages

The removal of some senescent cells is beneficial for healthy aging and overall lifespan [START_REF] Xu | Senolytics improve physical function and increase lifespan in old age[END_REF]. To investigate this in details using our mouse models, we analyzed the drugs that have been identified to eliminate senescent cells, senolytics [START_REF] Fuhrmann-Stroissnigg | Identification of HSP90 inhibitors as a novel class of senolytics[END_REF][START_REF] Roos | Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice[END_REF][START_REF] Yosef | Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL[END_REF][START_REF] Yousefzadeh | Fisetin is a senotherapeutic that extends health and lifespan[END_REF]. We chose to use the most studied senolytic combination of dasatinib and quercetin (D+Q) to check if they eliminate different p16 High senescent cells. First, we purified Cd31-positive LSECs from the livers of 1-year-old p16-Cre/R26-mTmG mice, plated them and treated with D+Q for 5d. Our analysis revealed that the number of EGFP-positive as well as SA-beta-Gal positive cells was comparable between control and D+Q-treated cells (Figure 7A). Next, we turned to in vivo analysis and gavaged 10-months-old p16-Cre/R26-mTmG mice with PEG400 (vehicle) or with D+Q every week for 5 weeks. Our subsequent analysis revealed that the drug combination primarily removed EGFP/ p16 High F4/80-positive cells but not EGFP/ p16 High LSECs or adipocytes (Figure 7B,C). Further analysis of Cd31-positive cells and fibrosis did not reveal any difference between control and D+Q mice (Figure 7D&E, Figure S7). Thus, a senolytic combination of D+Q efficiently removes p16 High macrophages and is ineffective for p16 High Cd31-positive LSECs and adipocytes.

Discussion

Accumulating senescent cells have been shown to be detrimental in many contexts, and their elimination has been proposed to be advantageous for health and lifespan [START_REF] Baker | Naturally occurring p16(Ink4a)positive cells shorten healthy lifespan[END_REF]. Recent studies, however, have also described beneficial roles for p16 and senescent cells, raising the question of their exact role in multiple tissues and under different conditions. During embryogenesis and skin wound healing, activation of the cellular senescence program seems to be physiologically-relevant and important for tissue development and regeneration [START_REF] Demaria | An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA[END_REF][START_REF] Munoz-Espin | Programmed cell senescence during mammalian embryonic development[END_REF][START_REF] Storer | Senescence is a developmental mechanism that contributes to embryonic growth and patterning[END_REF]. Thus, removal of senescent cells could have both positive and negative impact on the organism.

Cd31/Cd146-positive endothelial cells of the liver, LSECs, are the main cell type responsible for clearing blood-borne macromolecular waste (DeLeve and Maretti-Mira, 2017; [START_REF] Poisson | Liver sinusoidal endothelial cells: Physiology and role in liver diseases[END_REF]. Ninety percent of viruses cleared by LSECs which are capable of eliminating as many as 100 million HIV-like particles per minute. Seventy-five percent of LPS is cleared by LSECs with a speed per minute exceeding 150 times of the LPS level required for septic shock. Scavenger receptor type B 1 (SR-B1, SCARB1), which is highly abundant on LSECs mediates selective uptake of high-density lipoprotein (HDL), controlling cardiovascular risk and all-cause mortality. Among the many toxins that are removed by LSECs, ox-LDL is of special interest as it is the main cause of atherosclerosis and a potent inducer of senescence [START_REF] Oh | Long-Term Treatment of Native LDL Induces Senescence of Cultured Human Endothelial Cells[END_REF]. Besides LSECs, a notable fraction of lipoproteins is removed from the blood by hepatocytes, due to easy access to blood content through fenestrations and the lack of a basement membrane in liver sinusoids (DeLeve and Maretti-Mira, 2017). With age however, both murine and human LSECs undergo aging-induced pseudocapillarizationa process that is accompanied by de-fenestration and establishment of a basement membrane (DeLeve and Maretti-Mira, 2017; [START_REF] Hunt | Manipulating fenestrations in young and old liver sinusoidal endothelial cells[END_REF][START_REF] Mclean | Age-related pseudocapillarization of the human liver[END_REF]. The resulting loss of clearance functions by hepatocytes, however, is well-compensated by LSECs through upregulation of expression of multiple scavenger receptors (Figure 3G). These changes however have detrimental consequences for LSECs, as they are accompanied by enhanced intake of LDL including a strong oxidant ox-LDL, which eventually results in the activation of senescence. As a result, a noticeable fraction of Cd31-positive cells is senescent in old mice (Figure 3A&B). Activation of senescence can confer undesirable consequences, such as upregulation of a SASP, which can further enhance inflammation, driving liver fibrosis and thus contributing to a gradual loss in liver function. In turn, delaying senescence through preventing LSEC defenestration with agents like Nicotinamide mononucleotide (NMN) could positively contribute to health-and life-spans [START_REF] Das | Impairment of an Endothelial NAD(+)-H2S Signaling Network Is a Reversible Cause of Vascular Aging[END_REF][START_REF] Hunt | Manipulating fenestrations in young and old liver sinusoidal endothelial cells[END_REF]. Here, we found that removing senescent cells however may be worse than leaving them to accumulate, as this can have systemic and liver-debilitating consequences. Specifically, acute removal of senescent Cd31-positive cells that are adapted to compensate for some of the lost clearance functions of hepatocytes, results in build up of blood-borne macromolecular waste, including ox-LDL (Figure 6F). This in turn could have significant systemic toxic consequences including further activation of senescence (Figure 6G) instigating a positivefeedback loop that could significantly cripple the animal if senescent cells are being continuingly removed. Locally in the liver, removing senescent LSECs activates a fibrotic response (Figures 5C&6H) which is accompanied by reduced serum TPO levels and thrombocytopenia (Figures6I&J). In turn, thrombocytopenia is the most frequent hematologic abnormality in patients with chronic liver disease [START_REF] Hallen | Coagulation Studies in Liver Disease[END_REF][START_REF] Temel | Serum thrombopoietin levels and its relationship with thrombocytopenia in patients with cirrhosis[END_REF] and has been established as one of the most reliable factors to predict overall mortality in patients of different age groups [START_REF] Golwala | Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), Platelet Count and Plateletcrit (PCT) as predictors of in-hospital paediatric mortality: a case-control Study[END_REF][START_REF] Vanderschueren | Thrombocytopenia and prognosis in intensive care[END_REF]. In addition, removal of senescent vascular endothelial cells results in disruption of blood-tissue barrier in the short-term (Figures 6D&E) that is subsequently resolved in perivascular fibrosis thus affecting different tissues and organs (Figure 6H).

The knowledge about the full repertoire of senescent cells in vivo is critical to understand how their removal might affect healthspan. There is absolutely no doubt that elimination of some senescent cells is beneficial for healthy aging and overall lifespan [START_REF] Xu | Senolytics improve physical function and increase lifespan in old age[END_REF]. One such example is senescent macrophages, that can be cleared by a senolytic combination of dasatinib and quercetin (Figure 7), and is beneficial in a variety of pathological conditions [START_REF] Burd | Monitoring tumorigenesis and senescence in vivo with a p16(INK4a)-luciferase model[END_REF][START_REF] Childs | Senescent intimal foam cells are deleterious at all stages of atherosclerosis[END_REF][START_REF] Duong | Macrophage Depletion in Elderly Mice Improves Response to Tumor Immunotherapy, Increases Anti-tumor T Cell Activity and Reduces Treatment-Induced Cachexia[END_REF]. By contrast, we show here that there are abundant p16 High senescent cell types in the aging organism that are structurally and functionally important while their removal has detrimental consequences. Specifically, we found that senescent vascular endothelial cells are not replaced by non-senescent neighbors, but instead their removal activates another type of regenerative responsefibrosis. On the other hand, finding strategies to delay or even to replace important senescent cell types, such as LSECs, could have a strong, positive impact on healthy aging and overall lifespan.

Limitations of Study

While the exact definition and characterization of senescence remain a point of significant discussion, a hallmark of senescent cells is high expression of p16. Senescence clearance attempts usually take advantage of this fact, however the faithfulness of different reporter alleles could be responsible for a confounding variability in the results. The use of knock-in alleles driven by native p16 locus cis-, trans-and epigenetic regulators, as one described here, could address some of the differences. The important aspect that remains, however, is whether such knock-in alleles themself reflect true p16 expression. In addition, it would be important to understand how the native p16 locus is regulated in senescent cell types uniquely identified with the use of native reporter alleles. This would add further mechanistic details highlighting the importance of having native regulation of p16-driven reporters to label and to eliminate senescent cells. Another critical aspect is a non-selective clearance of senescent cells that could be detrimental to the health of the animals. These results however require further support as this could provide a new perspective on how we think of senescence clearance and whether there are more risks than currently anticipated. (E) Analysis of p16 (exon 1-2, amplifies both wild-type and knock-in alleles), CRE and TK mRNAs (present only in knock-in allele) in heterozygous p16-Cre/R26-mTmG DFs of passage 1 and 7 shows expected 2-fold difference between p16 and CRE/TK mRNA levels, n=6.

(F) Same as in (E) but the level of different mRNA was analyzed in tissues obtained from 1-year-old heterozygous p16-Cre/R26-mTmG mice, n=5. as in the number of cells with more than 4 foci (graph). Analysis of SA-β gal-positive cells (C), phospho-γH2AX (D) (the number of foci per cells is indicated and the representative high magnification images are shown) and Lamin B1(E) (EFGP-positive cells labeled with asterisks contain significantly less Lamin B1) in liver Cd31-positive cells of 1-year-old p16-Cre/R26-mTmG mice. The average number of phospho-γH2AX foci was increased from 0.9 in EGFP-negative to 1.9 in EGFP-positive cells (6 different samples, at least 20 cells per sample were counted).

(F) Analysis of p16 Ink4a mRNA and multiple SASP genes by RNA-Seq in EGFP-positive and -negative fractions of Cd31-positive cells obtained from livers of 1-year-old p16-Cre/R26-mTmG mice, n=3.

(G) Same as (F) for the analysis of different scavenger and mannose-like receptors, n=3.

(H) Analysis of uptake of acetylated and oxidized LDL in EGFP-negative and -positive Cd31-positive cells obtained from the livers of 1-year-old p16-Cre/R26-mTmG mice, n=6.

Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

See also Figure S3. mTmG or Rosa26-DTA). Mice were maintained on a mixed background, both males and females up to 1 year-of-age were used for analysis. For TAM experiments, all mice were maintained on TAM-contained diet for 1 month before the analysis. Mice were housed 4 per cage in a temperature (20-22 0 C) and humidity-controlled room, maintained on a 12-h light/dark cycle (07:00 to 19:00 light on), with standard food (A03, Scientific Animal Food and Engineering, U8200G10R) and water provided ad libitum and environmental enrichments.

Cell Culture

Dermal fibroblasts were obtained from the skin of newborn pups (both male and female pups were used) of defined genotypes after enzymatic digestion with 2.5 mg/ml DispaseII (ThermoFisher) in DMEM overnight at 4°C with following digestion during 1 hour at room temperature with 1 mg/ml Collagenase IV (ThermoFisher). Enzymes were inactivated by dilution with DMEM/10% FBS and cell suspension was serially passed through 100, 70, and 40 m cell strainers to obtain a single cell suspension. The cells were cultured in DMEM high glucose/10% FBS with antibiotic at 37 0 C/5% CO2 until they reached confluency and were frozen in FBS/10% DMSO as passage 1.

For Isolation and culture of Cd31+ endothelial cells, the minced livers (of both sexes) were digested for 45-60min in 10 ml DMEM with 100 g/ml Collagenase A and 10 g/ml DNAse I. After inactivation of enzymes with an equal volume of DMEM/10% FBS, cells were strained through 70m nylon mesh and washed several times with PBS/2% FBS. After the final wash 2x10 8 cells were re-suspended in 270 l PBS/2% FBS and incubated on ice for 20 min with 30 l Cd31 MicroBeads and washed with PBS/2% FBS/2mM EDTA. Cd31+ cells were separated on MACS LS Columns (Miltenyi Biotec #130-042-401) according the manufacturer's recommendations. The separated cells were cultured in Endothelial Cell Growth Medium 2 (PromoCell #C-22111) at 37 0 C/5% CO2.

For isolation of cells with Percoll gradient, minced livers were digested for 45-60min in 10 ml DMEM with 500 μg/ml Collagenase type IV (ThermoFisher # 17104019) and 10 μg/ml DNAseI. After inactivation of enzymes with equal amount of DMEM/10% FBS, cells were strained through 100, 70 and 40μm nylon mesh and washed several times with DMEM/10% FBS. After the final wash, 2x10 8 cells were re-suspended in DMEM/10% FBS and loaded on top of 25/50% Percoll (Sigma # P4937) gradient with following spin at 800g for 30 min at 4 0 C. Cells in interface between the density cushions were collected, washed 5µm and 30µm sections prior to mounting them on slides. Sections were kept at -80° until use. The sections from OCT blocks were washed in PBS and incubated in blocking solution.

Then, sections were incubated in blocking solution with primary antibody for 1h at room temperature or O/N at 4°C: rat anti-Cd31 (1/20, ab28364, Abcam), chicken anti-GFP (1/1000, Abcam), rabbit anti-lamin B1 (1/300, ab16048, Abcam) rabbit anti-phospho-H2AX (1/500, 9718, Cell Signalling), rabbit anti-macroH2A (1/500, ab91528, Abcam), rabbit anti-K9me2 (1/500, ab176882, Abcam). The secondary antibodies were Alexa Fluor 488 anti-chicken, Alexa Fluo 647 anti-rabbit and Alexa Fluor 647 anti-rat conjugates (1/500, Life Technology). Nuclei were counterstained with DAPI included in mounting medium (Vectashield, H-1200). Images were taken using a confocal Zeiss LSM 880 microscope (Zeiss, Göttingen, Germany). Isolated liver Cd31-positive cells were plated on µ-Slide 8-well (80826, Ibidi) and stained 24-48H later. Briefly, cells were fixed in 4%PFA (15 min, room temperature), permeabilized (10 min, 0.5% Triton), blocked (30 min, 5% Goat Serum and 1%BSA) and incubated in blocking solution with first antibody (1 hour, room temperature): chicken anti-GFP (1/1000, Abcam), rabbit anti-Lamin B1 (1/300, ab16048, Abcam) Rabbit anti-phospho-H2AX (1/500, 9718, Cell Signalling), rabbit anti-macroH2A (1/500, ab91528, Abcam), rabbit anti-K9me2 (1/500, ab176882, Abcam). Antibody used for the Cd133 FACS: anti-Prominin-1-PE-Vio770, Miltenyi, cat. 130-102-153; and for Cd45: BV421 Rat Anti-Mouse CD45, BD biosciences, cat.563890. The secondary antibodies were Alexa Fluor 488 antichicken, Alexa Fluor 647 anti-rabbit and Alexa Fluor 647 anti-rat conjugates (1/500, Life Technology). Nuclei were counterstained with DAPI included in mounting medium (Vectashield, H-1200). Images were taken using a confocal Zeiss LSM 880 microscope (Zeiss, Göttingen, Germany) and treated using ImageJ/Fiji software (NIH).

Scanning Electron Microscopy (SEM)

The portal vein perfusion was done with a mixture of fixatives, 2% PFA, 2,5% glutaraldehyde, 2mmol CaCl2 in 0,1 M cacodylate buffer and the sample stayed 24h to 48h in the fixative at 4°C before processing to preparation for TEM and SEM. For SEM analysis, samples were washed with distilled water, and cryoprotected in a solution of 30% glycerol for 1h. The samples were, then plunged into liquid nitrogen, freeze fractured, dehydrated in a graded ethanol series, and finally immersed in hexamethyldisilazane (Carl Roth, Karlsruhe, Germany), and dried at room temperature. Samples were then mounted on aluminium stubs and sputter-coated with a 3-nm gold-palladium coating (Cressington 308EM, UK) prior to analysis with a Field Emission Scanning Electron Microscope (FESEM JEOL 6700F, Japan)

Transmission Electron Microscopy (TEM)

For TEM, the samples were fixed by the same procedure as for SEM. After an adequate fixation, the samples were rinsed in 0.1M cacodylate buffer, and post-fixed for 2h in 1% osmium tetroxide in 0.1 M cacodylate buffer. Samples were then rinsed in distilled water, dehydrated in acetone and lastly embedded in epoxy resin. Contrasted ultrathin sections (70 nm) were analyzed under a JEOL 1400 transmission electron microscope mounted with a Morada Olympus CCD camera.

Cell count and proliferation assay

The dermal fibroblasts from p16-Cre/R26-mTmG and p16-Cre/R26-DTA mice were passaged at density 10 5 cells per 10 cm 2 and plated every 3 days in DMEM high glucose/10% FBS with Pen/Strep antibiotics at 37 °C/5% CO2. Cell counting and estimation of GFP+ cells was performed using Countess™ II FL Automated Cell Counter (ThermoFisher #A32136). Cell sorting was performed on FacsAria 3 Analyzer. Sorted fractions of dermal fibroblast were cultured in standard conditions for 12 hours and analyzed by staining for SA-Gal. Cell proliferation assay of Cd31+ and Cd146+ endothelial cells was performed according to the Cell Proliferation Kit II (XTT) (Sigma # 11465015001).

Evans Blue Retention Analysis

Evans Blue experiments were performed by Evans blue injection (Sigma, 150ul, 0.5% in sterile water) into tail vein. Tissues were collected 30 min later, weighted and incubated in formamide for 48H at 56°C. Evans Blue tissue retention was analyzed in formamide at 610nm and normalized by formamide volume and tissue weight.

Uptake ox-LDL and acLDL analyses

Cultured Cd31 + endothelial cells were starved overnight in Advanced DMEM/F-12 media (ThermoFisher # 12634010) and incubated on ice for 1h before addition of LDLs. Media was re-placed with fresh cold Advanced DMEM/F-12 contained 5g/ml DiI-Ox-LDL (ThermoFisher # L34358) or DiI-AcLDL (Kalen Biomedical #770231-9) and incubated for 4h at 37 0 C/5%CO2 and at 4 0 C as control. Cells were washed with PBS/2% FBS/2mM EDTA several times and cultured in Endothelial Cell Growth Medium 2 at 37 0 C/5% CO2. After 24h, cells were lysed in 0.1%SDS/0.1mM NaOH and fluorescence was measured against of standard curve with Ex535/Em595nm on Infinite 200Pro (Tecan). Protein concentration in cellular lysates was estimated with BC Assay -Protein Assay Kit (Interchim) according to the manufacturer's protocol and used for normalization of uptake.

Measurement of LDL and ox-LDL

For quantitative determination of mouse LDL and ox-LDL concentration in plasma, tail vein blood was collected in EDTA solution and processed using Mouse ox-LDL ELISA Kit and Mouse LDL ELISA kit (Cusabio) according to the manufacturer's protocols.

SA-eta-Gal Staining

Cells were washed with PBS and fixed for 5 min at room temperature in 4% PFA, washed 3 times with PBS and stained for 24-48 hours at 37 °C in staining solution: 40mM Citratesodium phosphate pH6; 5mM K3[Fe(CN)6]; 5mM K4[Fe(CN)6]; 2mM MgCl2; 150mM NaCl; 1mg/ml X-gal.

Gene Expression Analysis

RNA from Dermal Fibroblast was isolated with RNeasy Mini Kit (Quiagen # 74104) according to the manufacturer's protocol. 1 g of total RNA was used for cDNA synthesis using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher # K1621) and 30-60 ng cDNA was used for a PCR reaction. The quantitative PCR was performed with KAPA SYBR® FAST qPCR Kit (KAPABiosystem # KR0389) using the StepOnePlus Real-Time PCR System with the following parameters: enzyme activation 2min at 95°C, and 40 cycles of 3"-95°C, and 45"-60°C. Primers for PCR were designed using Primer-BLAST tool at NCBI with the condition of separation of primer pairs with at least one intron.

RNA-Seq sample preparation

RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA integrity and quantitation were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

The clustering of the index-coded samples was performed on a cBot Cluster Generation System using SR Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions.

After cluster generation, the library preparations were sequenced on an Illumina platform and 50 bp/100 bp single-end reads were generated.

QUANTIFICATION AND STATISTICAL ANALYSIS

RNA-Seq Data Analysis

Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.

Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Bowtie v2.2.3 and single-end clean reads were aligned to the reference genome using TopHat v2.0.12. We selected TopHat as the mapping tool for that TopHat can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other nonsplice mapping tools. For unigene DGE, Bowtie v0.12.9 was used to aligned single-end clean reads to the unigene sequences. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels [START_REF] Trapnell | Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[END_REF]. For unigene DGE, we use RSEM to count the reads numbers mapped to each unigene.

Data Representation and Statistical Analysis

Data presented based on at least three independent experiments, values are means +/-SEM. Comparison of mean values between groups was evaluated by 2-tailed Student's t-test using the GraphPad Prism program. Normal distribution of populations for RNA-Seq analysis was calculated using the Shapiro-Wilk normality test. P-values less than 0.05 were considered significant. Any P-value less than 0.05 was designated with one (*) asterisk; less than 0.01 -with two (**) asterisks, less than 0.001 -with three (***) asterisks. All P values and n are reported in the figure legends. Results are considered significant when p < 0.05.

Figure 1 .

 1 Figure 1. Analysis of p16-Cre knock-in cells. (A) Analysis of p16/EGFP-positive cells at different passages of p16-Cre/R26-mTmG DFs. The quantification was done using FACS analysis. (B) The level of p16 Ink4 mRNA was analyzed in p16/EGFP-negative and p16/EFGPpositive cells at passage 7 after FACS-sorting of p16 Ink4a -Cre/R26-mTmG DFs, n=8. (C) Passage 7 p16-Cre/R26-mTmG DFs were FACS-sorted, negative and positive fractions were plated and stained for SA-β-gal. (D) Analysis of p16 Ink4 , p19 Arf , and p21 Waf1 mRNA by Real-Time PCR in p16-Cre/R26-mTmG and p16-Cre/R26-DTA DFs of different passages, n=6.

Figure 4 .

 4 Figure 4. Reduced p16 expression and vessel density in organs of p16-Cre/R26-DTA mice. (A) Photomicrographs of p16 immunostainings of livers, kidneys, hearts, and lungs from 1-year-old control (R26-DTA) and p16-Cre/R26-DTA mice. Arrows indicate vascular endothelial cells. Quantifications of p16 signal area densities are shown in the graphs. (B) Photomicrographs of Cd31 immunostainings of livers, kidneys, hearts, and lungs from 1-year-old controls (R26-DTA) and p16-Cre/R26-DTA mice, n=6. Quantification of signal area densities are shown in the graphs. Scale bars indicate 50 µm. (C) Analysis of different mVegf and their receptors in Cd31-positive cells obtained from the livers of 2-months-old, 1-year-old, and 2-years-old mice, n=6. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4.

Figure 5 .

 5 Figure 5. Loss of p16 High cells results in perivascular fibrosis in p16-Cre/R26-DTA mice. (A) Scanning Electron microscopy pictures of sinusoidal capillaries from the liver of 1-yearold p16-Cre/R26-DTA mice (right panel) versus R26-DTA mice (left pane, similar results

Figure 7 .

 7 Figure 7. A senolytic combination of dasatinib and quercetin (D+Q) efficiently removes p16 High macrophages. (A) Cd31-positive cells were purified and treated with D+Q for 5 days in vitro. The numbers of EGFP-and SA-beta Gal-positive cells were quantified, n=6. (B) 10-months-old p16-Cre/R26-mTmG mice with gavaged with PEG400 (vehicle) or with D+Q every week for 5 weeks. FACS analysis of EGFP-positive cells showed a strong effect of D+Q on p16 High F4/80-positive macrophages but not Cd31-positive LSECs in the liver, n=4. (C) Same as in (B) but abdominal fat was analyzed for EGFP-positive cells. Note a strong reduction in the number of F4/80-positive p16 High macrophages. The percentage of EGFPpositive adipocytes was analyzed using ImageJ program, n=4. (D) Photomicrographs of Cd31 immunostaining of livers in vehicle and D+Q-treated mice as described in (B) (n=4). Quantifications of Cd31 signal area densities are shown in graphs. (E) Photomicrographs of Picrosirius red stained cross sections of livers in vehicle and D+Q-treated mice as described in (B). Right panel images show higher magnification micrographs with Sirius Red specific fluorescence. Scale bars indicate 50 µm. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Acknowledgements

We acknowledge the University's CCMA Electron Microscopy facility supported by Université de Nice Sophia-Antipolis, Région Provence Alpes-Côte d'Azur, Conseil Départemental 06, and Gis Ibisa and we thank Alyssia Mari for technical help. We are extremely grateful to the IRCAN's facilities: the Molecular and Cellular Core Imaging (PICMI), the Histology, the Cytometry (CYTOMED), the animal house (supported by le Cancéropole PACA, la Région PACA, le Conseil Départementale 06, l'INSERM, ARC, IBiSA, and the Conseil Départemental 06 de la Région PACA). The research in DB lab is supported by the Foundation ARC and Agence Nationale de la Recherche (grant "Senage"), in the KDW lab by ARC, Fondation Recherche Medicale, and Inserm Plan Cancer.

Author Contributions

L.G., N.W., A.E., S.L-G. and K.W. designed, performed and analyzed experiments. C.M. was responsible for the mouse colony and carried out some experiments; D.V.B. secured funding, conceived the project, designed and supervised the generation of knock-in mice and the experiments, and wrote the manuscript with input from all authors.

Declaration of Interests

The authors declare no competing interests.

Data are mean ± SEM. *p < 0.05, **p < 0.01.

See also Figure S1. (F) Analysis of plasma levels of LDL and ox-LDL was carried out TAM-treated 1.5-yearold R26-DTA and p16-CreERT2/R26-DTA mice.

See also Figure S7.

STAR METHODS

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed and will be fulfilled by the Lead Contact, Dmitry V. Bulavin (Dmitry.Bulavin@unice.fr).

Materials Availability

Mouse lines generated in this study (p16-CRE and p16-CRE-ERt2 mice) are available from the lead contact upon request and MTAs are required.

Data and Code Availability

The RNA-Seq data was deposited in the Gene Expression Omnibus database with the accession number GSE125439. All the original images have been deposited with Mendeley (doi: 10.17632/49zsh4b2hg.1).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mouse models

The p16-Cre knock-in mice were generated by Ozgene (Australia) by introducing a F2A-Cre-T2A-TK-E2A-tdTomato cassette at the end of the last exon of the p16 Ink4 gene. The use of self-cleaving peptides (F2A, T2A and E2A) allowed to produce separate proteins. C57BL/6 ES cells were used for targeting and correct integration was confirmed by Southern blot analysis (SFigure 1). The Frt-loxed Neo cassette was removed by crossing the wt/p16-Cre heterozygous line with a homozygous FLP deleter line; offsprings were screened by southern blot analysis. Correctly targeted mice were further bred to generate 2 separate lines to reconfirm all the obtained results. A similar strategy was used to introduce a Tamoxifeninducible version of Cre recombinase. Rosa26-mTmG (mixed C57BL/6J and 129/SvJ background) and Rosa26-DTA (C57BL/6 background) mice have been purchased from the Jackson Laboratory. All animal experiments were performed in compliance with the Animal Care and Use Committee and approved by the ethical review committee. For all experiments only heterozygous mice were used for both p16-Cre knock-ins and the reporters (Rosa26-with DMEM/10% FBS and used for culture and RNA isolation.

METHOD DETAILS

Histological analysis

Three μm paraffin sections were used for histological and immunohistological procedures, including Haematoxylin-Eosin, Trichrome Masson (Merck, Darmstadt, Germany) and collagen-specific Sirius Red F 3B (Waldeck Gmbh, Münster, Germany) staining for visualization of interstitial and perivascular fibrosis. For Cd31 immunohistology, after heat-mediated antigen retrieval and quenching of endogenous peroxidase activity, the antigen was detected using Pecam-1 (CD31) antibody (1:1000, rabbit monoclonal, AB182981, Abcam) and the EnVision™ Peroxidase/DAB Detection System from Dako (Agilent, Les Ulis, France). The p16 antigen was detected using monoclonal mouse antibody (1:300, Abcam) and the Vector M.O.M. immunodetection Kit (Vector Laboratories, PK-2200, Burlingame, CA) according to the manufacturer's instructions. Sections were counterstained with haematoxylin (Sigma). For p16/GFP immune-fluorescent doublelabelling, sections were incubated with mouse blocking reagent (Vector Laboratories) before incubation with the mouse monoclonal p16 antibody (1:300, Abcam) and the polyclonal anti-GFP antibody from rabbit (1:500, ab290, Abcam). Antigens were visualized using donkey Alexa Fluor® 488-Alexa Fluor® 594-coupled secondary antibodies in a 1:150 dilution (Jackson ImmunoResearch, Newmarket, Suffolk, UK).

Nuclei were counterstained with DAPI. Omission of the first antibody served as a negative control. Additionally, some slides were incubated with IgG Isotype controls. Picrosirius Redspecific fluorescence was visualized using a Cy5 filter. Slides were viewed under an epifluorescence microscope (DMLB, Leica, Germany) connected to a digital camera (Spot RT Slider, Diagnostic Instruments, Scotland). Area densities for immunohistological stainings were determined using the ImageJ software. Area density was analyzed on at least three different sections of organ per mouse.

For immunofluorescence, tissues were fixed with 4% Paraformaldehyde (24 hours, 4°C), then transferred to 70% ethanol. Paraffin-embedded tissues were cut into 5µm thickness sections and mounted on slides. Sections were kept at room temperature until the use. For immunofluorescence on cryosection, tissues were fixed with 4% PFA (90 min on ice), washed in PBS, incubated in 30% sucrose (Sigma, S0389) (24-48Hours, 4°C), embedded in OCT (Thermoscientific, 6769006) and kept at -80°C. OCT blocks were cut into