Senescent endothelial cells promote pathogenic neutrophil trafficking in inflamed tissues

Abstract

Cellular senescence is a hallmark of advanced age and a major instigator of numerous inflammatory pathologies. While endothelial cell (EC) senescence is aligned with defective vascular functionality, its impact on fundamental inflammatory responses in vivo at single-cell level remain unclear. To directly investigate the role of EC senescence on dynamics of neutrophil-venular wall interactions, we applied high resolution confocal intravital microscopy to inflamed tissues of an EC-specific progeroid mouse model, characterized by profound indicators of EC senescence. Progerin-expressing ECs supported prolonged neutrophil adhesion and crawling in a cell autonomous manner that additionally mediated neutrophil-dependent microvascular leakage. Transcriptomic and immunofluorescence analysis of inflamed tissues identified elevated levels of EC CXCL1 on progerin-expressing ECs and functional blockade of CXCL1 suppressed the dysregulated neutrophil responses elicited by senescent ECs. Similarly, cultured progerin-expressing human ECs exhibited a senescent phenotype, were pro-inflammatory and prompted increased neutrophil attachment and activation. Collectively, our findings support the concept that senescent ECs drive excessive inflammation and provide new insights into the mode, dynamics, and mechanisms of this response at single-cell level.

Keywords: Endothelium; Inflammation; Neutrophils; Senescence.

Figure 1. Development of mouse model of vascular aging with mosaic distribution of progerin expression in endothelial cells.

(A) Schematic depicting the generation of the Tie2-Cre;Lmna^LCS/LCS;Rosa26^tdTomato/+ mouse whereby progerin and tdTomato fluorescent protein are conditionally expressed in ECs. Progerin and tdTomato-expressing mice are referred to as “Cre⁺” and their littermate negative controls as “Cre^-”. (B) The cremasteric microvasculature of a Cre⁺ mouse was immunostained for PECAM-1 (CD31) and analyzed by confocal microscopy tile scanning, illustrating the mosaic pattern of tdTomato expression; regions with or without tdTomato fluorescence are indicated. Scale bars: 20 µm. (C) Schematic depicting the sequence of the mouse progerin peptide that was used to generate the novel specific rabbit anti-mouse progerin Ab. (D–G) Tissues of Cre⁺ animals were co-immunostained for progerin and other EC markers, as shown. (D, E) Representative confocal images of cremaster muscle post-capillary venules depicting the coupling of progerin and tdTomato (tdTmt) expression (Scale bar: 10 µm) and the quantification of this association (n = 4 mice/group in 1 experiment). (F) Representative confocal images depicting coupling between endothelial cell progerin and tdTomato expression in ear and lung tissues. Scale bars: 10 µm. (G, H) Representative flow cytometry plots depicting the correlation of progerin and tdTomato expression in lung endothelial cells of Cre⁺ mouse and its quantification (n = 3 mice/group in 2 independent experiments). Data information: (E) Data are presented as mean. (H) Data are mean ± SEM. *P < 0.05 and ns: not significant. One-way ANOVA followed by Tukey’s post hoc test. Source data are available online for this figure.

Figure 2. Progerin-expressing endothelial cells are senescent and exhibit a pro-inflammatory phenotype.

(A, B) Mouse ear skin was immunostained for VE-cadherin (to delineate ECs) and γH2AX (DNA damage marker). (A) Representative confocal images depicting the correlation between tdTomato and γH2AX expression in ECs of Cre⁺ mice. Scale bar: 3 µm. (B) Quantification of the number of γH2AX positive foci per EC nucleus, each pair of linked blue and magenta data points represent one Cre⁺ mouse (n = 17 mice in 7 independent experiments). (C–F) Lung ECs from Cre^- and Cre⁺ mice were analyzed for multiple senescence markers by flow cytometry (n = 6 mice/group in 2 independent experiments). (C) Representative flow cytometry histogram plots depicting C12FDG fluorescence intensity and (D) its quantification as mean fluorescence intensity (MFI) per mouse. (E, F) Quantification of the EC mean cell size (FSC-A) and granularity (SSC-A), respectively, of progerin positive and negative lung ECs of Cre⁺ mice as expressed per mouse, relative to ECs of Cre^- animals. (G, H) Cremaster microvascular ECs from Cre^- and Cre⁺ mice were imaged by confocal microscopy and their (G) nuclear surface area and (H) nuclear sphericity were quantified. Each data point represents a single-cell nucleus (n = 3–4 mice/group in 3 independent experiments). (I, J) Icam1 (I) and Vcam1 (J) gene expression levels in progerin positive and negative lung ECs of naïve Cre⁺ mice sorted from the same mice were assayed by RT-qPCR (n = 6–7 mice/group in 3 independent experiments). Data information: (B, D–F, I, J) *P < 0.05; **P < 0.01, ***P < 0.001; ****P < 0.0001. Analysis by two-tailed paired Student’s t-test. (G, H) Data are presented as mean ± SEM. *P < 0.05; ***P < 0.001; ns: not significant. One-way ANOVA followed by Tukey’s post hoc test. Source data are available online for this figure.

Figure 3. Microvascular progerin-expressing ECs support enhanced neutrophil adhesion.

Cremaster microvasculature of Cre^- and Cre⁺ or LyzM-EGFP-ki Cre⁺ mice were acutely inflamed (IL-1β) and immunostained for PECAM-1 (ECs) with tdTomato fluorescence identifying progerin expressing endothelial cells. (A) Representative confocal image showing neutrophil (MRP14⁺) accumulation in close apposition to tdTomato expressing ECs. White boxes delineate magnified ROIs in the right panel. Scale bars: 100 µm. (B) The number of extravasated neutrophils per field of view was quantified in control (PBS) or IL-1β-stimulated Cre^- and Cre⁺ animals (n = 3–4 mice/group in 3 independent experiments). (C–H) Neutrophil behaviors were analyzed and quantified by confocal IVM in LyzM-EGFP-ki Cre⁺ mice. (C) Representative confocal image of a stimulated post-capillary venule showing adhesion of EGFP-expressing neutrophils to tdTmt positive ECs. Scale bar: 10 µm. (D) Neutrophil firm adhesion per progerin negative or positive ECs in the same vessel segment, (E) duration of neutrophil firm adhesion to progerin-positive versus negative ECs in the same venular segment, (F) number of crawling neutrophils per progerin negative or positive EC in the same vessel segment. For (B–D), n = 4 mice in 4 independent experiments. (G) Change in neutrophil crawling speed when moving from progerin negative to progerin-positive ECs, each linked pair of data points represents one neutrophil (n = 4 mice in 4 independent experiments). (H) Representative confocal image depicting a neutrophil crawling track from a progerin-positive (tdTmt⁺) to a progerin-negative (tdTmt^-) endothelial cell. Track color indicates crawling velocity and black arrows show time frames with increased neutrophil velocity. Scale bar: 4 µm. Data information: (B) Data are mean ± SEM. **P < 0.01, ***P < 0.001, ns: not significant. One-way ANOVA test followed by Tukey’s post hoc test. (D–G) *P < 0.05. Two-tailed paired Student’s t-test. Source data are available online for this figure.

Figure 4. EC progerin promotes neutrophil-dependent vascular leakage.

Cremaster microvasculature of Cre^- and Cre⁺ mice were acutely inflamed (IL-1β) and immunostained for PECAM-1 (ECs) and MRP14 (neutrophils) with tdTomato fluorescence identifying progerin expressing endothelial cells. (A–C) Quantification of microvascular leakage calculated as percentage coverage of extravasated fluorescent beads (percentage coverage); (A) in Cre^- vs Cre⁺ control and IL-1β-stimulated tissues (n = 3–5 mice/group in 7 independent experiments); (B) in relation to the percentage of progerin-positive ECs in vessel segments of Cre⁺ mice (n = 4 mice/group in 4 independent experiments); and (C) in IL-1β-stimulated control and neutrophil depleted (post treatment with an isotype control or anti-GR-1 antibody, respectively) Cre^- and Cre⁺ mice (n = 3 mice/group in 3 independent experiments). (D) Vascular leakage in the dorsal skin of Cre^- or Cre⁺ mice injected i.d. with PBS, LTB₄ (1 h) or histamine (30 min) (n = 4–7 mice/group in 7 independent experiments). Data information: Data are mean ± SEM. *P < 0.05; **P < 0.01; ns: not significant. (A, C, D) Two-way ANOVA followed by Tukey’s (A, C) and Šídák’s (D) post hoc test. (B) Two-tailed unpaired Student’s t-test. Source data are available online for this figure.

Figure 5. Mice expressing EC progerin exhibit increased lung inflammation.

Cre^- and Cre⁺ mice were subjected to systemic inflammation using a model of endotoxemia (LPS/PepG). (A) Neutrophil accumulation in lungs of Cre^- and Cre⁺ mice at the indicated times post-stimulation (n = 3–7 mice/group in 4 independent experiments). (B) Lung vascular leakage as quantified by extravasation of i.v. injected fluorescent microbeads (n = 3–7 mice/group in 4 independent experiments). (C) Representative confocal images of lung tissue sections of control and endotoxemic Cre^- and Cre⁺ animals immunostained for PECAM-1 (ECs) and MRP14 (neutrophils). White boxes delineate regions of interest, which are shown magnified in the lower panel. Scale bars: 30 µm. (D) Quantification of the number of neutrophils accumulating on lung ECs of Cre^- mice or on progerin negative versus positive lung ECs of the same Cre⁺ mice (n = 3–4 mice/group in 5 independent experiments). (E) Correlation between extravascular microbead accumulation and the number of neutrophils per field of view (FOV). The bold line shows linear regression with the 95% confidence intervals indicated by light lines (n = 138 images from 20 different mice in 5 independent experiments). Data information: (A, B, D, E) Data are mean ± SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001. (A, B) Two-way ANOVA followed by Tukey’s post hoc test. (D) One-way ANOVA followed by Tukey’s post hoc test. (E) Pearson coefficient R² and P value (deviation from zero slope). Source data are available online for this figure.

Figure 6. IL-1β-stimulated progerin-expressing endothelial cells exhibit a pro-inflammatory phenotype in vivo.

Cre^- and Cre⁺ mice were subjected to systemic inflammation using a model of endotoxemia (LPS/PepG, 2 h) or locally inflamed with intrascrotal injection of IL-1β. (A) Volcano plot of the relative difference in gene expression in whole lung tissue of stimulated Cre^- and Cre⁺ mice assayed by NanoString (785 genes; n = 3 mice/group in 1 experiment). Significantly differentially expressed genes (FDR > 0.05) in Cre⁺ lung tissue is color coded as follows: Downregulated genes in dark blue (fold change, FC < −1) and light blue (−1 < FC < 0); upregulated genes in orange (0 < FC < 1) and red (FC > 1). The presented results are based on Fisher’s exact test with false discovery rate adjustment. See Methods for further analytical details. (B, C) Quantification of EC CXCL1 protein expression (MFI) in control and stimulated Cre^- and Cre⁺ mice as acquired by confocal microscopy in (B) lungs (n = 4–9 mice/group in 3 independent experiments) and (C) cremaster muscles (n = 3–5 mice/group in 2 independent experiments). (D) Representative confocal images of lung tissue sections of LPS/PepG-treated Cre^- and Cre⁺ animals immunostained for PECAM-1 (ECs) and CXCL1 with the signals exhibited in relation to progerin expressing ECs (tdTomato fluorescence). Scale bar: 30 µm. (E) CXCL1 expression (MFI) in progerin negative and positive IL-1β-stimulated cremasteric ECs (n = 5 mice/group in 2 independent experiments). Data information: (B, C) Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. Two-way ANOVA followed by Tukey’s post hoc test. (E) *P < 0.05. Two-tailed paired Student’s t-test. Source data are available online for this figure.

Figure 7. CXCL1 blockade in EC progerin expressing and aged mice normalizes dysregulated inflammatory responses.

(A–C) Cremaster microvasculature of Lyz2-EGFP-ki Cre^- and Cre⁺ mice were acutely inflamed (IL-1β) and mice were treated with intravenously administered control (IgG) or neutralizing anti-CXCL1 mAbs. Neutrophil behaviors were analyzed by confocal intravital microscopy. (A) Number of firmly adherent neutrophils per progerin negative or positive EC (n = 4–6 in 19 independent experiments). (B) Duration of neutrophil firm adhesion to progerin positive versus negative ECs in the same vessel segment (n = 4–6 in 19 independent experiments). (C) Cremaster microvascular leakage as quantified by fluorescent bead extravasation (n = 4–8 mice/group in 5 independent experiments). (D) Lung microvascular leakage in mice treated with i.p. LPS/PepG and i.v. IgG or anti-CXCL1 Ab with bead extravasation expressed as percentage coverage (n = 4 mice/group in 2 independent experiments). (E–H) Cremaster muscles of aged mice were acutely inflamed with IL-1β. (E) Representative confocal images of cremaster muscles of IL-1β-treated aged animals immunostained for PECAM-1 (ECs), p21 (senescent EC), MRP14 (neutrophils) and CXCL1. Scale bar: 10 µm. (F) Number of luminal neutrophils per p21^low or p21^high ECs in the same venular segment (n = 6 mice/group in 2 independent experiments). (G) CXCL1 expression (MFI) in IL-1β-stimulated cremasteric ECs of aged mice (n = 6 mice/group in 2 independent experiments). (H) Aged Lyz2-EGFP-ki mice stimulated with IL-1β were treated with i.v. control (IgG) or neutralizing anti-CXCL1 mAbs, and the number of firmly adherent neutrophils was quantified (n = 3 mice/group in 9 independent experiments). Data information: (A–D, H) Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001; ns: not significant. (A, B) Two-way ANOVA followed by Tukey’s post hoc test. (C, D, H) One-way ANOVA followed by Tukey’s post hoc test. (F, G) Two-tailed paired Student’s t-test. Source data are available online for this figure.

Figure 8. Progerin-expressing human ECs support increased neutrophil adhesion and activation.

(A) Representative confocal images of GFP-lamin A- or GFP-progerin-transduced HUVECs stained for PECAM-1, progerin, and DAPI. Scale bar: 5 µm. (B) Cell size of lamin A-GFP and progerin-GFP transduced HUVECs (GFP^-: non-transduced) (n = 4 in 2 independent experiments). (C) Analysis of secreted proteins from PBS (control) and IL-1β-stimulated GFP-lamin A and GFP-progerin-expressing HUVECs, as assayed using a multiplex antibody array (targeting 36 proteins). (D) CXCL8 levels in control (PBS) and IL-1β stimulated transduced HUVECs (n = 5 transduced cell cultures per group, analyzed by ELISA in 4 independent experiments). (E–G) Lamin A-GFP- and progerin-GFP-transduced HUVECs were stimulated with IL-1β and co-incubated with purified human neutrophils loaded with a ROS tracker. (E) Representative image of washed and fixed cells stained for PECAM-1, DAPI and ROS. The DAPI signal intensity for HUVEC nuclei was thresholded out using IMARIS software based on their dimmer fluorescence compared to neutrophil nuclei. Yellow arrows highlight ROS-positive neutrophils. Scale bars: 20 µm. (F) Number of neutrophils firmly adherent to non-transduced (GFP^-) and lamin A- or progerin-transduced (GFP⁺) HUVECs as quantified per ten cells for each data point (mean ± SEM, n = 5 in 5 independent experiments). (G) Number of ROS-positive neutrophils adherent to GFP-lamin A- or GFP-progerin-transduced HUVECs, expressed as a percentage of the total number of neutrophils adherent to the respective HUVEC transductants (n = 7 in 7 independent experiments). Data information: (B, D, F, G) Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001; ns: not significant. (B, D, F) Two-way ANOVA followed by Tukey’s post hoc test. (G) Two-tailed paired Student’s t-test. Source data are available online for this figure.

Figure EV1. Cellular and molecular characteristics of tdTmt-progerin-expressing endothelial cells.

(A, B) The percentage of tdTomato positive ECs in Cre^- and Cre⁺ mice was quantified in (A) lung ECs by flow cytometry (n = 4–21 mice/group in 4 independent experiments) and (B) cremaster microvasculature ECs by confocal microscopy (n = 4–11 mice/group in 4 independent experiments). (C) Representative immunoblot for progerin and Histone H2B (loading control) of flow cytometry sorted lung ECs from WT, Cre^- and Cre⁺. (D) Representative confocal images of cremaster microvasculature of acutely inflamed (IL-1β) Cre^- or Cre⁺ mice. Anti PECAM-1 mAb was injected i.s. and fixed tissues were immunostained for MRP14 (neutrophils) and progerin. Scale bar: 5 µm. (E–G) Lung ECs from Cre⁺ mice were analyzed by flow cytometry, and (E, F) representative plots presented according to tdTomato fluorescence intensity, depicting EC (E) size (FSC-B) and (F) granularity (SSC-A). (G) Surface expression of selected EC proteins quantified and expressed as RFI (n = 3–4 mice/group in 6 independent experiments). Data information: (A, B, G) Data are mean ± SEM. *P < 0.05; ns: not significant. (A) Two-tailed paired Student’s t-test. (G) One-way and (B) two-way ANOVA test followed by Tukey’s post hoc test.

Figure EV2. EC progerin expressing mice exhibit dysregulated luminal neutrophil crawling in IL-1β-stimulated cremasteric venules.

Cremaster microvasculature of Tie2-Cre;Lmna^LCS/LCS;Rosa26^tdTmt/+;Lyz2-EGFP-ki Cre^- or Cre⁺ mice were acutely inflamed (IL-1β) and analyzed by confocal IVM. (A, B) Crawling profiles of neutrophils on ECs (responses from 28 to 34 neutrophils are displayed). (C) Luminal neutrophil track straightness (straightness index; displacement/track length) (n = 6 mice/group in 12 independent experiments). (D, E) Cremaster muscles of Tie2-Cre;Lmna^LCS/LCS;Rosa26^+/+ Cre⁺ mice were acutely inflamed (IL-1β). (D) Fixed cremaster muscles were immunostained for progerin, MRP14 (neutrophils) and PECAM-1 (ECs), and (E) number of luminal neutrophils per progerin negative or positive EC in the same vessel segment was quantified (n = 4–7 mice/group in 4 independent experiments). Data information: (C) Data are mean ± SEM. *P < 0.05. Two-tailed unpaired Student’s t-test. (E) *P < 0.05. Two-tailed paired Student’s t-test.

Figure EV3. EC progerin-expressing mice exhibit enhanced microvascular permeability in IL-1β-stimulated cremaster muscles.

(A, B) Cremaster microvasculature of Tie2-Cre;Lmna^LCS/LCS;Rosa26^tdTmt/+ Cre^- or Cre⁺ mice were acutely inflamed with IL-1β or PBS (control) and analyzed by confocal microscopy. Anti-PECAM-1 mAb was injected i.s. and fluorescent beads or 70-kDA dextran were injected i.v. (A) Representative confocal images of control (PBS) and stimulated (IL-1β) post-capillary venules illustrating accumulation of extravasated beads in the Cre⁺ sample. White box is magnified and displayed in the right panel. Scale bar: 5 µm. (B) Time course of dextran accumulation in the perivascular region of a selected postcapillary venule (n = 4–7 mice/group in 18 independent experiments). (C) Quantification of microvascular leakage calculated as percentage coverage of extravasated fluorescent beads (percentage coverage) in Tie2-Cre;Lmna^LCS/LCS;Rosa26^+/+ Cre^- and Cre⁺ IL-1β-stimulated tissues (n = 4–7 mice/group in 4 independent experiments). (D) Vascular leakage in the cremaster muscle of Tie2-Cre;Lmna^LCS/LCS;Rosa26^tdTmt/+ Cre^- or Cre⁺ mice injected i.s. with PBS, or histamine (30 min) (n = 4–7 mice/group in 7 independent experiments). (E) Lungs from Cre^- and Cre⁺ mice treated i.p. with PBS (control) or LPS/PepG were analyzed by confocal microscopy for vascular leakage (extravasation of i.v. injected fluorescent microbeads). Representative confocal images of lung tissue sections immunostained for PECAM-1 (ECs) illustrate increased bead extravasation (white) in close apposition to tdTmt positive vessel segments of Cre⁺ mice. Scale bar: 30 µm. Data information: (C, D) Data are mean ± SEM. *P < 0.05; ns: not significant. (C) Two-tailed unpaired Student’s t-test and (D) one-way ANOVA followed by Tukey’s post hoc test.

Figure EV4. Molecular characteristics of inflamed tissues of EC progerin expressing mice.

(A–C) Cre^- and Cre⁺ mice were stimulated with i.p. PBS or LPS/PepG (2 h) after which tissue samples were analyzed by targeted NanoString transcriptomics. (A) Volcano plot of the relative difference in gene expression in whole lung tissue of control or stimulated Cre^- mice as assayed by NanoString (785 genes; n = 6 mice/group in 1 experiment). Significantly differentially expressed genes (FDR > 0.05) are color coded as follows: Downregulated genes in dark blue (fold change, FC < −1) and light blue (−1 < FC < 0); upregulated genes in orange (0 < FC < 1) and red (FC > 1). (B, C) Gene expression profiles in inflamed lung tissues of Cre⁺ mice, relative to Cre^-. (B) The 15 most highly upregulated (red) and highly downregulated (blue) genes in Cre⁺ mice are displayed (785 genes; n = 3 mice/group in 1 experiment). (C) Gene Ontology (GO) terms enrichment analysis of 785 differentially expressed genes (DEGs) in inflamed lungs of Cre⁺ mice (scored as −log₁₀ (p-value)). The presented results are based on Fisher’s exact test with false discovery rate adjustment. (D) Volcano plot of the relative difference in gene expression in whole lung tissue of unstimulated Cre+ vs Cre- mice as assayed by NanoString (785 genes; n = 3 mice/group in 1 experiment). (E) Representative confocal images of cremaster muscles treated with PBS (control) or IL-1β-stimulated cremaster muscles of Cre^- and Cre⁺ mice. Tissues were immunostained for PECAM-1 and CXCL1, with tdTomato fluorescence identifying progerin expressing ECs. Scale bar: 5 µm. (F–H) Surface expression of (F) ICAM-1, (G) VCAM-1, and (H) E-Selectin, presented as RFI, on progerin negative and positive lung ECs derived from Cre⁺ mice subjected to LPS/PepG stimulation (n = 4–6 mice/group in 6 independent experiments). Data information: (F–H) *P < 0.05; ns: not significant. Two-tailed paired Student’s t-test.

Figure EV5. Progerin expressing HUVECs exhibit cellular senescence and a pro-secretory phenotype.

HUVECs were transduced with GFP-tagged lamin A or GFP-tagged progerin expressing lentiviral constructs. (A) Representative immunoblot of control and transduced HUVECs probed for progerin, GFP, β-Actin, and Histone-H2B proteins. (B) Representative bright field image of transduced HUVECs assayed for SA-β-Gal activity (blue) and (C) its associated quantification (n = 4–5 in 5 independent experiments). Scale bar: 40 µm. (D, E) Non-transduced (GFP-) and transduced (GFP+) HUVECs (n = 5 in 5 independent experiments) were quantified for (D) nuclear shape abnormalities and (E) nuclear size. Data information: (C–E) Data are mean ± SEM. *P < 0.05; **P < 0.01, ***P < 0.001; ****P < 0.0001; ns: not significant. (C) One-way and (D, E) two-way ANOVA followed by Tukey’s post hoc test.