Human lung tissue samples

Human lung tissues were provided by the LifeLink Foundation, a Tampa-based nonprofit corporation operating federally certified organ procurement organizations across Florida, Georgia, and Puerto Rico. An MTA has been executed enabling transfer of non-transplantable human organs or tissues to the labs at the University of South Florida Morsani College of Medicine for translational research. While experiments in tissues from decedents do not meet the NIH definition of human subject research, donors were de-identified according to HIPAA/IRB standards, with their age, sex, medical history, diagnosis, and cause of death recorded. Organs were recovered by designated transplant surgeons, processed according to the standard transplant protocols, and transported via authorized medical carriers. Tissue samples were collected from lungs, preserved in cryogenic molds using Tissue Plus® O.C.T. compound (Fisher HealthCare, Waltham, MA; catalog no. 4585), snap frozen in liquid nitrogen, and stored at –80 °C for future experimentation. Tissue sections were cut to 1-μm thickness for immunofluorescence analysis and SA-β-Gal staining.

Animal studies

All experimental animal protocols were approved by the Institutional Animal Care and Use Committee at the University of South Florida and were conducted in accordance with the Guide for Care and Use of Laboratory Animals. Male C57BL/6 J mice purchased from the Jackson Laboratory (Bar Harbor, ME) were used in this project at the ages of 3 (young) and 19 (old) months old.

Perfusion and mouse tissue collection

Mice were anesthetized with urethane and cardially perfused with PBS, followed by 4% paraformaldehyde (PFA). The collected tissues were fixed in 4% PFA for 24 h, washed with PBS, and transferred to 70% ethanol until tissue processing. The tissue was then embedded in paraffin and sectioned (4 μm thick) using a microtome. For “fresh-frozen” preparations, animals were cardially perfused with PBS. Then, the lungs were dissected, embedded in Tissue Plus® O.C.T. compound, snap frozen in liquid nitrogen, and sectioned (15 μm thick) using a cryostat (Leica CM1950).

Immunofluorescence of senescent markers in lung tissue

Paraffin-embedded mouse lung sections were deparaffinized and subjected to appropriate antigen retrieval at 95 °C for 20 min. After permeabilization and blocking, the slides were incubated with primary antibodies p21 (1:50; Santa Cruz Biotechnology, Dallas, TX; catalog no. sc-6246), Ki-67 (1:20; Abcam, Waltham, MA; catalog no. ab16667), Lamin B1 (1:100; Abcam; catalog no. ab16048), or γH2AX (1:50; Abcam; catalog no. ab81299) overnight at 4 °C, washed, and then incubated with secondary antibodies for 1 h at room temperature. Slides were incubated with DAPI (1:2000; Thermo Scientific, Waltham, MA; catalog no. 62248) for 20 min at room temperature, and cover slipped using ProLong™ diamond antifade mountant (Invitrogen, Waltham, MA; catalog no. P36962). Images were captured using a Leica SP8 confocal microscope (Leica Microsystems, Weltzar, Germany).

Snap-frozen human and mouse lung sections were fixed in ice-cold methanol or 4% PFA for 15 min, washed with PBS, permeabilized, blocked, and incubated with primary antibodies: p21 (1:50; Abcam; catalog no.109520 or 1:50; Santa Cruz Biotechnology for human and mouse, respectively), ZO-1 (1:100; Invitrogen; catalog no. 40–2200), VE-cadherin (1:50; Cell Signaling Technology; catalog no. D87F2 or 1:100, BD Pharmingen; catalog no. 555289, for human and mouse, respectively), Ki-67, Lamin B1, or γH2AX overnight at 4 °C. Slides were then washed and incubated with secondary Alexa Fluor® 488 antibodies (1:500; Invitrogen; catalog no. A21206; A21202 and A21208) for 1 h at room temperature. Slides were rinsed, stained with DAPI, cover slipped, and imaged using a confocal microscope. Antibody dilutions were chosen based on previous series of titration experiments showing the best staining with minimum background fluorescence.

SA-β-Gal staining in lung tissue

SA-β-Gal staining in lung tissue samples was performed as previously described [17]. Briefly, the sample slides with frozen tissue were washed twice with cold PBS, fixed with 2% formaldehyde + 0.2% glutaraldehyde in PBS on ice, and rinsed shortly in cold ultrapure water. The slides were incubated overnight at 37 °C in a non-CO2 incubator with fresh SA-β-Gal staining solution containing X-gal (20 mg/mL, pH 6.0; ThermoFisher; catalog no. 10113253) in dimethylformamide (1 mg/mL; MilliporeSigma; catalog no. 270547), citric acid/sodium phosphate buffer (40 mM, pH 6.0; Sigma-Aldrich; catalog no. C1909 and Fisher Scientific; catalog no. S375-500, respectively), potassium ferrocyanide (5 mM; Acros Organics; catalog no. 211095000), potassium ferricyanide (5 mM; Electron Microscopy Sciences; catalog no. 20150), sodium chloride (150 mM; Fisher Scientific; catalog no. S271-3), and magnesium chloride (2 mM; Fisher Scientific; catalog no. M35-500). Upon successful staining (deep blue color development in the tissue), slides were washed with PBS and cover slipped using mounting medium. Imaging was performed using an Olympus VS120 slide scanner.

Endothelial cell culture and senescence induction in vitro

Mouse primary lung microvascular ECs (Cell Biologics; catalog no. C57-6011) were cultured in recommended medium (Cell Biologics; catalog no. M1168) and used at passages 5–7. In vitro senescence induction was carried out using two senescent inducers: SAHA and doxorubicin [17]. In brief, ECs were treated with SAHA (4 μM; Tocris; catalog no. 4652) for 8 days (drug was refreshed daily) and control (non-senescent) cells were cultured in parallel and treated with identical volumes of vehicle DMSO. Another group of ECs received doxorubicin (250 nM; Tocris; catalog no. 2252), and their control cells were treated with equal volumes of vehicle (sterile water) for 24 h, followed by a washout period for 7 days.

Double staining for SA-β-Gal and EdU

Co-staining of SA-β-Gal and EdU was performed in cultured ECs as previously described [17]. Briefly, mouse primary lung microvascular ECs plated on 4-well cell culture chamber slides (Nest Scientific; catalog no. 230104) were treated with two senescent inducers (SAHA and doxorubicin) as mentioned above. The cells were then cultured in medium containing EdU (10 μM; Lumiprobe; catalog no. 10540) for 24 h. After washing with PBS, cells were fixed in 2% formaldehyde + 0.2% glutaraldehyde in PBS at room temperature, rinsed with PBS, and incubated in a non-CO2 incubator at 37 °C for 16 h with fresh SA-β-Gal staining solution, as described above. Upon successful staining, cells were then rinsed in PBS. After fixation and permeabilization, the cells were stained for 30 min in a dark at room temperature with fresh EdU staining solution containing CuSO4 (2 mM; Sigma-Aldrich; catalog no. 209198), sulfo-Cy3-azide (4 μM; Lumiprobe; catalog no. D1330), and sodium ascorbate (20 mg/mL; Sigma-Aldrich; catalog no. A4034) in PBS, washed with PBS, incubated with DAPI, and cover slipped using mounting medium. A Leica SP8 confocal microscope was used for imaging.

Immunocytochemistry of senescent markers

After senescence induction, mouse lung ECs were fixed, permeabilized, blocked, and incubated with primary antibodies p21 (1:50; Abcam; catalog no. ab188224), Ki-67 (1:20; ThermoFisher Scientific; catalog no. MA5-14,520), Lamin B1, ZO-1, claudin-5 (1:50; Invitrogen; catalog no. 35–2500), or VE-cadherin (1:1000; Abcam; catalog no. ab205336) overnight at 4 °C. After washing, the slides were incubated with secondary Alexa Fluor® 488 antibodies (1:500; Invitrogen; catalog no. A21206; A21202) for 1 h at room temperature, followed by DAPI staining for 20 min at room temperature. Slides were then mounted and imaged using confocal microscopy. F-actin staining was performed using Alexa Fluor® 568 Phalloidin (1:40; Invitrogen; catalog no. A12380).

Immunoblotting

Mouse lung tissue or lung microvascular ECs were lysed in 1 × RIPA lysis buffer (Millipore; catalog no. 20–188) containing protease and phosphatase inhibitors (Roche; catalog no. 11697498001; 04906845001). After centrifugation, the supernatant was collected, and protein quantitation was performed by Pierce BCA protein assay (ThermoFisher Scientific Inc.; catalog no. 23227). The lysates were then loaded onto 4–20% Tris–glycine gel (Bio-Rad; catalog no. 456–1094) and transferred onto a nitrocellulose membrane using Trans-Blot Turbo equipment. The membranes were stained with Revert™ 700 Total Protein Stain Kit (LI-COR Biosciences; catalog no. 926–11,016) and blocked in Intercept® (PBS) blocking buffer (LI-COR Biosciences; catalog no. 927–70,001) for 1 h at room temperature, probed with primary antibodies against p21 (1:1000), Lamin B1 (1:1000), ZO-1 (1:500), claudin-5 (1:250), ICAM-1 (1:250; Invitrogen; catalog no. MA5407), VE-cadherin (1:1000; Abcam; catalog no. ab205336), and β-actin (1:1000; LI-COR Biosciences; catalog no. 926–42,212; 926–42,210) overnight at 4 °C. After washing, membranes were probed with appropriate LI-COR secondary antibodies diluted in blocking buffer for 1 h at room temperature. The membranes were imaged using LI-COR Odyssey CLx system. The signal intensity of all protein bands was obtained by densitometry analysis and normalized to the total protein per lane (please see full-length Western blotting images; Supplemental Fig. S1-S7).

Mouse bone marrow neutrophil isolation

Mouse bone marrow neutrophils were isolated as described previously [18, 19]. In brief, femurs and tibias were cut at one end, placed into a 0.5-mL tube, nested into a 1.5-mL Eppendorf tube, and centrifuged at 10,000 g for 30 s [20]. The bone marrow cells were collected, and red blood cells were lysed with 20 mL of 0.2% NaCl for 20–30 s, followed by addition of 20 mL of 1.6% NaCl. The cell pellet was then filtered with a cell strainer, counted using a Luna cell counter, and resuspended in 1 mL of PBS. Afterwards, 3 mL of Histopaque 1119 was gently added in a 15-mL conical tube, followed by 3 mL of Histopaque 1077, and then cell suspension (1 mL) was added on top of Histopaque 1077. After centrifugation at 2000 rpm for 30 min at 25 °C without brake, neutrophils were collected.

Neutrophil adhesion assay

EC senescence was induced as mentioned above. Isolated bone marrow neutrophils (1 × 105) were applied on senescent ECs, incubated for 2 h at 37 °C, and rinsed with PBS to remove all non-adherent neutrophils. Slides were then fixed in 4% PFA, rinsed with PBS and imaged using a bright-field microscopy. Slides were then permeabilized, blocked, and labelled with primary antibodies p21 (1:50; Abcam) and MPO (1:40; R&D systems; catalog no. AF3667) overnight at 4 °C. For paraffin-embedded mouse lung tissues, incubation with a primary antibody specific for neutrophils (1:100; Cederlane; catalog no. CL8993AP, clone 7/4) was performed. Cells or tissues were then incubated with secondary Alexa Fluor® 488 antibody (1:500; Invitrogen; catalog no. A21206; A11055) and Alexa Fluor® 568 antibodies (1:500; Invitrogen; catalog no. A10042; A11057 and A78946) for 1 h at room temperature. After washing, slides were stained with DAPI and imaged using a Leica SP8 scanning confocal microscope.

Neutrophil chemotaxis assay

SAHA or doxorubicin-treated cells were seeded in the bottom chamber of gelatin-coated 24-well transwell plates (Costar, Corning, NY; catalog no. 3472) for 24 h. The following day, neutrophils (1 × 105) were added to the top chamber insert (3 μm pore size) and incubated for 2 h at 37 °C with or without LTB4 (100 nM; Tocris; catalog no. 2307) as the chemoattractant agent in the bottom chamber. The number of neutrophils that chemotactically migrated into the bottom chamber was counted using a hemocytometer. The experiment was performed using supernatant of senescent cells in the bottom chamber as well.

Neutrophil trans-endothelial migration assay

The assay was conducted according to our previously reported method with minor modifications [21]. Briefly, neutrophil trans-endothelial migration experiments were performed utilizing senescent and non-senescent ECs seeded on gelatin-coated 24-well transwell inserts of 3-μm pore size (Costar; catalog no. 3472) for 24 h. On the day of the experiment, neutrophils (1 × 105) were added to the top chamber, incubated for 2 h at 37 °C with or without LTB4 in the bottom chamber as the chemoattractant. A hemocytometer was used to count the number of neutrophils transmigrated into the bottom chamber.

Transwell permeability assay

To evaluate endothelial permeability, we utilized our previous described protocol with minor changes [22]. In brief, mouse primary lung microvascular ECs treated with two senescent inducers (SAHA and doxorubicin) were seeded (3 × 105) on 0.33 cm2 inserts with pore size of 0.4 μm (Costar; catalog no. 3470) for 24 h. On the day of the experiment, 10 mg/mL of fluorescein isothiocyanate (FITC)-bovine serum albumin (FITC-BSA; Sigma-Aldrich; catalog no. A-9771) and 1 mg/mL rhodamine 3-kD dextran (Invitrogen; catalog no. D3307) were added to the top chamber. The passage of the fluorescence tracers was measured by collecting media from the bottom chamber, using a fluorescence microplate reader (SpectraMax M3; Molecular Devices) and apparent permeability coefficient (Papp) of mouse primary lung microvascular EC monolayers were calculated as previously described [22].

Statistical analysis

All data are presented as mean ± standard error mean (SEM). The comparison of young and old groups was analyzed by unpaired Student’s t-test. The comparison of SAHA or doxorubicin-treated cells and non-senescent cells was analyzed by paired Student’s t-test. Multiple comparisons were performed by one-way ANOVA with Tukey’s multiple comparison tests. Statistical analyses and graphs were performed with GraphPad Prism version 9 software. A value of P < 0.05 was considered statistically significant.